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Nitrogen and sulphur:
uptake, transport and remobilisation in canola genotypes at
vegetative stage and at maturity
By
Tatjana Balint
This thesis is presented for the degree of
Doctor of Philosophy
at
The University of Western Australia
School of Earth and Environment, Soil Science and Plant Nutrition
2011
i
Abstract
Canola (Brassica napus) is the major oilseed crop in Western Australia. The areas sown to
canola vary between 600,000 and 700,000 hectares per season, with the potential for an
increase by up to half a million hectares per season, with the seed production of up to
million tonnes per year. Canola production in Western Australia is constrained by poor soil
fertility. In Western Australia, soils are among the most ancient and therefore heavily
leached in the world, and have low availability of P, N and S.
Identification and breeding of genotypes that could use soil N and S as well as
fertilizer N and S more efficiently than standard genotypes would be a sustainable long-
term approach to dealing with (i) high N and S requirements in canola, and (ii) low N and S
availability in Western Australian soils.
In the study presented here, modern Australian canola germplasm was screened for
N and S efficiency during the vegetative stage. Group of genotypes either with high or low
N or S efficiency in the preliminary study were then screened for N or S efficiency at
maturity. The mechanisms involved in transport and remobilization of N- or S- containing
compounds were investigated via analyses of the xylem and phloem analyses following soil
and leaf labeling with stable isotopes 15
N and 34
S.
Nitrogen and S efficiency in Australian canola germplasm was successfully
differentiated in the vegetative growth stage in 84 canola genotypes. Nitrogen and S
efficiency ranking among six N- or S-efficient and six N- and S-inefficient from
preliminary screening study was verified in the additional experiment during the vegetative
stage.
Canola genotypes differed significantly in N efficiency at grain maturity. Genotypes
Wesway and 46C74 that were ranked as efficient in the vegetative stage remained efficient
at maturity, whereas genotype Surpass 603 CL was ranked inefficient during the vegetative
stage and efficient at maturity.
Canola genotypes significantly differed in S efficiency at maturity. However, there
was no correspondence between efficiency ranking in the vegetative stage and the maturity.
Genotypes Surpass 402 CL and 46C74 ranked as efficient in the vegetative stage, but not at
maturity. Genotype IB 1368 was ranked inefficient during vegetative stage and efficient at
ii
maturity. The yellow-coated genotypes IB 1337, IB 1363 and IB 1368 accumulated higher
amounts of S in seed than other genotypes.
There was little consistency in the N or S efficiency ranking from the vegetative
stage to physiological maturity in tested genotypes. Hence, vegetative-stage assessments
have poor predictive ability with respect to N or S efficiency at maturity and can not be
used as an efficient selection tool for N or S efficiency of canola cultivars.
The results from soil and leaf labelling studies using stable isotopes 15
N and 34
S
suggested that genotype Wesway was more efficient than Westar in taking up N, whereas
Surpass 402 CL was more efficient than Karoo in taking up S during the vegetative stage.
Amino acid content of xylem sap in genotypes differing in N and S efficiency was
identified in the vegetative stage. Genotype Wesway (one of N-use efficient genotypes),
had significantly higher concentrations of amino acids glutamine and asparagine (important
N-transporting compounds) at adequate N supply whereas Westar (one of N-use inefficient
genotypes) had significantly higher concentrations of glutamine and asparagine under
deficient N supply. In the S study, genotypes Surpass 402 CL and IB 1368 (S-use efficient),
and Karoo and Surpass 300 TT (S-use inefficient) also differed in concentrations of
methionine (S-transporting compound).
Amino acid content of phloem sap in chosen genotypes was identified at maturity.
Inefficient genotype Westar had higher amino acid concentrations than Wesway at deficient
N supply, suggesting negative correlation between NUE rating of genotypes and amino
acid concentration in the phloem sap. In the S study, genotype Karoo (S-use inefficient) had
a higher concentration of methionine under adequate S supply and of glutamate under
deficient S supply compared to Surpass 402 CL (S-use efficient genotype).
In conclusion, differences in N and S efficiency in large canola germplasm were
established in the vegetative stage as well as at maturity, with poor correspondence between
the two rankings during plant cycle. The study presented here improved understanding of
the mechanisms underlying N and S efficiency in canola genotypes by identifying
differential abundance of N-(glutamine and asparagine) and S-(methionine and the
glutathione precursor glutamate) transporting amino acids in the xylem sap (vegetative
stage) and in the phloem sap (maturity stage). For better understanding of oil and protein
accumulation in seeds in canola genotypes differing in N or S efficiency, work on embryos
at various development stages would be required. Further work is recommended to
elucidate links between yellow seed coats and increased S accumulation in seeds.
iii
Acknowledgements
There are many people I would like to thank for their assistance in completing this thesis:
Firstly I would like to thank Winthrop Professor Zed Rengel for his supervision during the
past few years. He gave me opportunity to explore my own ideas and hence develop my
own project. However, he was always available for advice, guidance and encouragement.
I would also like to thank Lorraine Osborne as well as Paul Damon for their practical help;
their efforts in managing the lab were greatly appreciated.
Thanks also to Michael Smirk for his advice and guidance with several chemical reactions.
Many thanks to Lydia Bednarek for Isotope Ratio Mass Spectrometry (IRMS) analyses as
well as Greg Cawthray for assistance with the Gas Liquid Chromatography (GC-FID)
analyses.
Many thanks to fellow colleagues from Soil Science and Plant Nutrition, CLIMA and
School of Animal Science (Olga Babourina, Heather Clarke, Oonagh Byrne, Zoey Durmic)
and many others for their social support during lunch times, coffee breaks and after work.
I would also like to thank my family for their love and never ending support during my
study.
iv
List of abbreviations
AMT genes Ammonium transporters gene family
ATF Amino acids transporters family
ANOVA Analysis of variance
Asn Asparagine
Cys Cysteine
DW Dry weight
E Efficient
Gln Glutamine
Glu Glutamate
Gly Glycine
HATS High-affinity transport system
HI Harvest index
I Inefficient
LATS Low-affinity transport system
M Medium efficient
Met Methionine
N Nitrogen
NH4+
Amonium
NHI Nitrogen harvest index
NO3- Nitrate
NRT Nitrate transporter gene
NUE Nitrogen-use efficiency
S Sulphur
SUE Sulphur-use efficiency
VSP Vegetative storage protein
v
Table of contents
Thesis abstract………………….............................................................................................i
Acknowledgments……………………………………………………….….…………...…iii
List of abbreviations………………………………………………………..…………..…..iv
Table of contents…………………………………………………………..……………..…v
List of figures………………………………………………………………..…………..….x
List of tables……………………………………………………………………….……...xvi
Chapter 1 Introduction and organisation of the thesis…………………….…….….......1
1.1. Introduction…………………………………………………….…….…....…1
1.2. Structure and organisation of the thesis………………………..……..….…..3
1.3. References..…………………………………………………………….……4
Chapter 2 Literature review……………………………………………..…….…..…....7
2.1 Canola ………………………………………………………..…….…..…...7
2.1.1 Canola industry in Australia…………………………..…….…...….7
2.1.2 The quality of Australian canola………………….……..……...…...8
2.2 Nutrient efficiency……………………………………………………....……..……8
2.2.1 Definition and screening methodologies for nutrient efficiency..…..8
2.3 Nitrogen ………………………………………………………………...…10
2.3.1 Genotypic differences in N efficiency in crops………………………...........10
2.3.2 Nitrogen-use efficiency (NUE)…………………………….…...….11
2.3.3 Nitrogen uptake (efficiency) and assimilation……………..……....11
2.3.4 Nitrogen assimilation ……………………………………………...13
2.3.5 Nitrogen transporters systems……………………………………...14
2.3.6 Nitrogen harvest index ………………………………………….…15
2.3.7 The biochemical mechanisms and genetics of N remobilisation ....17
2.4 Sulphur……………………………………………………………..…...…18
2.4.1 Sulphur uptake from soil……………………….…………………..19
2.4.2 Sulphur assimilation………………………………………………..20
2.4.3 Sulphur remobilization ………………………………………….....22
2.4.4 Genotypic differences in sulphur uptake and remobilisation in
canola………………………………..........................……..………24
vi
2.5 Nitrogen and sulphur interaction in canola ……………………...…......…24
2.6 Conclusions and opportunities for further studies ……………..…….….. 24
2.7 References……………………………………………………...…………..27
Chapter 3 Australian canola germplasm differs in nitrogen and sulphur efficiency….41
3.1 Abstract………………………………………………………………….…………41
3.2 Introduction……………………………………………………………..………….41
3.3 Materials and methods…………………………………………………..……..…..43
3.4 Results………………………………………………………………….…..……....46
3.4.1 Growth at low nitrogen or sulphur supply…………………….…...46
3.4.2 Concentration of nitrogen and sulphur in canola shoots…………..49
3.4.3 Growth at low relative to adequate nitrogen or sulphur supply …...51
3.4.4. Nitrogen and sulphur utilization efficiency………………………..53
3.4.5 Nitrogen and sulphur efficiency based on all three criteria combined
…………………………………………………………………………..…53
3.5 Discussion…………………………………………………………………..……..53
3.6 Conclusions………………………………………………………………………..57
3.7 Acknowledgements……………………...……………………………………...…57
3.8 References………………………………………………………………..………..58
Chapter 4 Nitrogen efficiency of canola genotypes varies between vegetative stage
and grain maturity……………………………….……………………...….61
4.1 Abstract………………………………………………………..…………...61
4.2 Introduction……………………………………………………….….….…61
4.3 Materials and methods…………………………………………….…….....63
4.4 Results and discussion……………………………………………….….....65
4.5 Vegetative stage……………………………………………………..……..65
4.5.1 Effect of N supply on dry matter production……………..……..……66
4.5.2 Concentration of N in canola shoots ……………………….….……..67
4.5.3 Dry matter production at deficient relative to adequate N supply...…68
4.5.4 Nitrogen utilisation efficiency…………………….…………….…....68
4.5.5 Nitrogen efficiency based on all three criteria combined………....….70
4.5.6 Conclusion………………………………………………………....…70
4.6 Grain maturity…………………………………………………………….70
4.6.1 Effect of N supply on growth………………………………………...70
vii
4.6.2 Nitrogen concentration in dropped leaves, stems, siliques and seeds..73
4.6.3 Growth at deficient relative to sufficient N supply……….…………..75
4.6.4 Nitrogen utilisation efficiency ………………………………………75
4.6.5 Harvest index and N harvest index…………………………….…….77
4.6.6 Oil and protein concentration in seed………………….……………..78
4.7 Conclusion…………………………………………………….…….…….80
4.8 Acknowledgements…………………………………………..…….……..80
4.9 References………………………………………………………….….….82
Chapter 5 Differential sulphur efficiency in canola genotypes at vegetative and grain
maturity stage……………………………………………………………………….…..….85
5.1 Abstract…………………………………………………………….……..85
5.2 Introduction……………………………………………………………….85
5.3 Materials and methods…………………………………………...……….86
5.4 Results………………………………………………………………...…..88
5.4.1 Vegetative stage……………………………………………………..88
5.4.2 Grain maturity stage……………………………………..…………..91
5.5 Discussion……………………………………………………...……..…..99
5.6 Conclusion………………………………………………………...…….101
5.7 Acknowledgments…………………………………………..…………..101
5.8 References………………………………………………………….……103
Chapter 6 Nitrogen and sulphur uptake and remobilisation in canola genotypes with
varied N- and S-use efficiency differ at vegetative and maturity stages……………….…105
6.1 Abstract……………………………………………………………..…...105
6.2 Introduction……………………………………………………………...105
6.3 Materials and methods…………………………………………..………107
6.3.1 Uptake experiments (soil labelling)……………………….….……107
A) Nitrogen uptake ……………………………………………...…107
B) Sulphur uptake……………………………………………….…110
6.3.2 Remobilisation experiments (leaf labelling)…………………….…113
A) Nitrogen remobilisation ………………………………….…….111
B) Sulphur remobilisation …………………………………………105
6.3.3 Statistics………………………………………………………...….114
6.4 Results…………………………………………………….……………..114
viii
6.4.1 Uptake experiments (soil labelling)…………………………..……114
A) Nitrogen uptake ………………………………………….……..114
B) Sulphur uptake …………………………………………………118
6.4.2 Remobilisation experiments (leaf labelling)……………….………122
A) Nitrogen remobilisation ………………………………………..122
B) Sulphur remobilisation …………………………………...…….125
6.5 Discussion……………………………………………………………….128
6.5.1 Vegetative stage (uptake and remobilisation study)………….……128
A) Nitrogen……………………………………………….………..128
B) Sulphur……………………………………………………...…..132
6.5.2 Maturity stage (uptake and remobilisation study)……………….…130
A) Nitrogen…………………………………………………….…..130
B) Sulphur……………………………………………...…………..132
6.6 Conclusions………………………………………………….…………..134
6.7 Acknowledgment…………………………………………….………….134
6.8 References……………………………………………………………….135
Chapter 7 Amino acid composition of xylem and phloem sap varies in canola
genotypes differing in nitrogen- and sulphur-use efficiency……………………….…….140
7.1 Abstract……………………………………………………………….…..140
7.2 Introduction…………………………………………………………….…141
7.3 Materials and methods…………………………………………...……….142
7.3.1 Xylem sap sampling and analyses…………………….………….143
7.3.2 Phloem sap sampling and analyses………………………….……144
7.4 Results……………………………………………………………...……..145
7.4.1 Vegetative stage…………………………………………………....145
A) N experiment……………………………………………...…….145
B) S experiment …………………………………………..………..149
7.4.2 Maturity stage………………………………………………………151
A) Nitrogen and amino acid concentrations in the phloem sap…..152
B) Sulphur and amino acid concentrations in the phloem sap……153
7.5 Discussion ………………………………………………………..………154
7.5.1 Nitrogen and S in the xylem and phloem sap…………………….154
7.5.2 Amino acid composition of the xylem and phloem sap of different
ix
genotypes as influenced by N or S supply………..…………….……….155
7.6 Conclusions…………………………………………………...…………..158
7.7 Acknowledgment………………………………………………..………..158
7.8 References…………………………………………………………...……159
Chapter 8 General discussion and conclusions………………………………………164
8.1 N and S remobilisation in canola genotypes………………………...……168
8.2 Suggestions for further work………………………………………..…….170
8.3 References……………………………………………………..………….172
x
List of Figures
Figure 2.1 Canola (Source: ''Koehler's Medicinal-Plants'')………...……………..……7
Figure 2.2 Nitrogen assimilation in roots (Oaks 1992). 1. Nitrogenase (legumes only, in
association with nitrogen-fixing bacteria) 2. Nitrate uptake system 3.
Nitrate reductase (NR) 4. Nitrite reductase (NiR) 5. Glutamine synthetase (GS) 6.
Glutamate synthetase (GOGAT) 7. Glutamate dehydrogenase (GDH) 8.
Phosphoenolpyruvate carboxylase (PEPcase) 9. Ferredoxin pyridine
nucleotide reductase 10. Asparagine synthetase 11. Glutamate-oxaloacetate
aminotransferase. …………………….……………………………..….....12
Figure 2.3 Schematic presentation of the known localisation of nitrate transporters NRT1,
NRT2 and ammonium transporter AMT genes in Arabidopsis. Two nitrate
transport systems have been shown to coexist in plants and to act co-
ordinately to take up nitrate from the soil solution and distribute nitrate within the whole
plant. The role of each ammonium transporter has been shown by the study of
single, double, triple and quadruple mutants (Masclaux-Daubresse et al.
2009)………………………………………………………………………...…15
Figure 2.4 A phylogenetic tree of the Arabidopsis sulphate transporter gene family (Page
1996). ………………………………………………………………………….19
Figure 2.5 Outline of sulphate assimilation and cysteine metabolism in plants
(Lewandowska and Sirko 2008). Enzymes involved in sulphate and sulphite
reduction are present only in plastids, while SAT and OAS-TL are present in
plastids, mitochondria and cytosol. Enzymes involved in GSH synthesis are
present in chloroplasts and in extra chloroplast fractions. The first two steps of
methionine synthesis proceed only in plastids, while the third step only in
cytosol because of strictly cytosolic location of MS. APSK, APS kinase; APR,
APS reductase; APS, adenosine 5’-phosphosulphate;ATPS, ATP sulphurylase;
CBL, cystathionine β-lyase; CGS, cystathionine γ-synthase; GCL, glutamino-
cysteine lyase; GSHS, glutathione synthetase; HCys, homocysteine; MMT, S-
adenosylmethionine: l-methionine S-methyltransferase; MS, methionine
synthase; OAS, O-acetylserine; OAS-TL, O-acetylserine (thiol)-lyase; PAPS,
adenosine 3’-phosohate 5’-phosphosulfate; SAM, S-adenosylmethionine;
xi
SAMS, SAM synthetase; SAT, serine acetyltransferase; SMM, S-
methylmethionine; SiR, sulphite reductase; SOX, sulphite oxidase; SOT,
sulphotransferase; ST, sulphate transporter. ………………………………..…21
Figure 3.A. Pedigrees of Australian canola varieties 1970–2004 (In Cowling 2007)……45
Figure 3.1 Shoot dry weight of 84 canola genotypes grown at low nitrogen supply (16 mg
N/kg soil) for 38 days. Genotypes are ranked in the order of decreasing nitrogen
efficiency (E, efficient; M, medium; I, inefficient). The boundaries for the
medium efficiency interval were formulated by subtracting or adding the value
of one standard error from the median point of the efficiency criterion. ….…48
Figure 3.2 Shoot dry weight of 84 canola genotypes grown at low (0 mg added) sulphur
supply in Lancelin soil for 38 days. Efficiency intervals (E, efficient; M,
medium; I, inefficient) were constructed as explained in Figure 3.1. ….……..48
Figure 3.3. Ranking of 84 canola genotypes based on the ratio between shoot dry weight at
low (16 mg/kg) and adequate nitrogen supply (200 mg/kg soil) after 38 days of
growth. Efficiency intervals (E, efficient; M, medium; I, inefficient) were
constructed as explained in Figure 3.1. …………………….…………………52
Figure 3.4 Ranking of 84 canola genotypes based on the ratio between shoot dry weight at
low (0 mg) and adequate sulphur supply (54 mg/kg soil) after 38 days of
growth. Efficiency intervals (E, efficient; M, medium; I, inefficient) were
constructed as explained in Figure 3.………………………….………………52
Figure 3.5 Nitrogen utilisation efficiency (calculated as the amount of shoot dry weight
(DW) produced per unit of nitrogen accumulated in shoots) of 38-day-old
canola genotypes grown at low nitrogen supply (16 mg/kg soil). Efficiency
intervals (E, efficient; M, medium; I, inefficient) were constructed as explained
in Figure 3.1. ……………………………………………….…………………55
Figure 3.6 Sulphur utilisation efficiency (calculated as the amount of shoot dry weight
(DW) produced per unit of sulphur accumulated in shoots) of 38-day-old canola
genotypes grown at low (0 mg) sulphur supply in Lancelin soil. Efficiency
intervals (E, efficient; M, medium; I, inefficient) were constructed as explained
in Figure 3.1. …………………………………………………………………..55
Figure 3.7 First screening experiment. ……………………………………………………57
Figure 4.1 Shoot dry weight of 12 canola genotypes grown at deficient nitrogen supply (51
mg N/kg soil) for 42 days. Genotypes are ranked in the order of decreasing
xii
nitrogen efficiency (E, efficient; M, medium; I, inefficient). The boundaries for
the medium efficiency interval were created by subtracting or adding the value
of one standard error to the median point of the efficiency criterion. Black bars
represent the most (46C74) and least efficient genotypes (Surpass 300 TT)
based on the combination of three N efficiency criteria in the vegetative
stage……………………………………………………………………………66
Figure 4.2 Ranking of 12 canola genotypes based on the ratio between shoot dry weight at
deficient (51 mg N/kg soil) and adequate N supply (140 mg N/kg soil) after 42
days of growth. For efficiency intervals (E, efficient; M, medium; I, inefficient)
and genotypes singled out with black bars see Figure 4.1. ……………………68
Figure 4.3 Nitrogen utilisation efficiency [calculated as the amount of shoot dry weight
(DW) produced per unit of nitrogen in shoots) of 42-day-old canola genotypes
grown at deficient nitrogen supply (51 mg N/kg soil). For efficiency intervals
(E, efficient; M, medium; I, inefficient) and genotypes singled out with black
bars see Figure 4.1. ……………………………………………….……………69
Figure 4.4 Seed dry weight (A) and ranking of 12 canola genotypes based on the ratio (B)
between total seed dry weigh at deficient (180 mg N/kg soil) and adequate
nitrogen supply (400 mg N/kg soil) at grain maturity. The boundaries for the
medium efficiency interval were formulated by subtracting or adding the value
of one standard error to the median point of the efficiency criterion. Black bars
represent the most (Wesway) and least efficient genotypes (Westar) based on
the combination of N efficiency criteria at maturity. ………………….………72
Figure 4.5 Nitrogen content of stems, leaves, siliques and seed of 12 canola genotypes
grown at deficient nitrogen supply (180 mg N/kg soil) at grain maturity. ……75
Figure 4.6 Nitrogen utilisation efficiency of seeds [calculated as the amount of seed dry
weight (DW) produced per unit of nitrogen in seeds] of canola plants grown at
deficient nitrogen supply (180 mg/kg soil). For efficiency intervals (E, efficient;
M, medium; I, inefficient) and genotypes singled out with black bars see Figure
4.1. …………………………………………………………………….………76
Figure 4.7 Harvest index (A) [(seed DW/above-ground DW) x 100%] and nitrogen harvest
index (B) [(seed N content/above-ground N content) x 100%] of 12 canola
genotypes grown at deficient nitrogen supply. For efficiency intervals (E,
xiii
efficient; M, medium; I, inefficient) and genotypes singled out with black bars
see Figure 4.1. …………………………..…..………………..…………….….78
Figure 4.8 Oil (A) and protein (B) concentration in seeds of 12 canola genotypes grown at
deficient nitrogen supply (180 mg N/kg soil). For efficiency intervals (E,
efficient; M, medium; I, inefficient) and genotypes singled out with black bars
see Figure 4.1. ………………………………..……………..…………………79
Figure 4.9 Example of nitrogen deficiency symptoms in canola shoot at vegetative stage
(pot on the right hand side)…………………..……………..………………….81
Figure 5.1 Shoot dry weight (A), relative shoot dry weight (adequate S supply = 100 %, B),
and S utilisation efficiency (C) of 12 canola genotypes grown at deficient S
supply (3 mg S/kg soil) for 48 days. Genotypes are ranked in the order of
decreasing S efficiency (E, efficient; M, medium; I, inefficient). The boundaries
for the medium efficiency interval were formulated by subtracting or adding the
value of one standard error from the median point of the efficiency criterion.
Black bars represent the most (Surpass 402 CL) and the least efficient genotypes
(IB 1368) based on the combination of the three efficiency criteria in the
vegetative stage. ……………………………………………………………….90
Figure 5.2 Seed dry weight (A) and relative seed yield (adequate S supply = 100 %, B) of
12 canola genotypes grown at deficient S supply (6 mg S/kg soil) and sufficient
S supply (87 mg S/kg soil) at maturity. The boundaries for the medium
efficiency interval were formulated by subtracting or adding the value of one
standard error from the median point of the efficiency criterion. Black bars
represent the most (IB 1368) and the least efficient genotypes (Karoo) based on
the combination of three efficiency criteria at the maturity stage….…………93
Figure 5.3 Sulphur content of stems, siliques and seed of 12 canola genotypes grown at
deficient S supply (6 mg S/kg soil) at maturity…….………………..…………95
Figure 5.4 Sulphur utilisation efficiency of seeds (seed dry weight produced per unit of S
accumulated in shoots) of 12 canola genotypes grown at deficient S supply (6
mg S/kg soil). For efficiency intervals (E, efficient; M, medium; I, inefficient)
and genotypes singled out with black bars see Figure 5.2. ……………..….….96
Figure 5.5 Harvest index (A) [(seed DW/shoot DW) x 100%] and S harvest index (B)
[(seed S content/shoot S content)] of 12 canola genotypes grown under deficient
xiv
S supply (6 mg S/kg soil). For efficiency intervals (E, efficient; M, medium; I,
inefficient) and genotypes singled out with black bars see Figure 5.2. …..…97
Figure 5.6 Oil (A) and protein (B) concentrations in seeds of 10 canola genotypes grown at
deficient S supply (6 mg S/kg soil). For efficiency intervals (E, efficient; M,
medium; I, inefficient) and genotypes singled out with black bars see Figure 5.2.
……………………………………………………………………………..…...98
Figure 5.7 Sulphur deficiency symptoms in canola plants at vegetative stage (1st pot from
the left- canola plant grown at adequate S supply)………..………………..102
Figure 6.1 Nitrogen concentration in shoots (stems + leaves) in the vegetative stage (A) and
different plant tissues at maturity (B) in canola genotypes grown at adequate and
deficient N supply (soil labelling). The error bars represent ±SE. ……..…….115
Figure 6.2 15
N atom excess in plant tissues of canola genotypes grown to maturity stage –
(soil labelling). The error bars represent ±SE. ……………………..…….…..117
Figure 6.3 Sulphur concentration in shoots (A) and 34
S atom excess in shoots (B) of canola
genotypes grown under adequate and deficient S supply in the vegetative stage
(soil labelling). The error bars represent ±SE. ……………………….………119
Figure 6.4 S concentration (A) and 34
S atom excess in plant tissues at adequate S (B) and
deficient S (C) supply of canola genotypes grown to maturity (soil labelling).
The error bars represent ±SE (A) or +SE (B and C). ………….…….…..121-122
Figure 6.5 15
N atom excess in plant tissues 6 days (2nd
harvest) after labelling started at
vegetative stage (A) and 2 days (1st harvest) and 6 days (2
nd harvest) after
labelling started at maturity (B) in canola genotypes grown under deficient N
supply – (leaf labelling). The error bars represent +SE. …………….…...…..124
Figure 6.6 34
S atom excess in leaves 2 days (1st harvest) and 6 days (2
nd harvest) after
labelling started in the vegetative stage (A) and 34
S atom excess in plant tissues
6 days (2nd
harvest) after labelling started at maturity (B) in canola genotypes
grown under deficient S supply (leaf labeling). The error bars represent ±SE (A)
or + SE (B). ………………………………………………………..……126, 127
Figure 7.1 Nitrogen concentration in the xylem sap in four canola genotypes grown at
adequate and deficient N supply at vegetative stage. …………………….…146
Figure 7.2 Amino acid concentration in the xylem sap in four canola genotypes grown
under adequate and deficient N supply at vegetative stage Only amino acids for
which the interaction genotype x N treatment was significant were shown.....148
xv
Figure 7.3 Sulphur concentration in the xylem sap in four canola genotypes grown at
adequate and deficient S supply at vegetative stage. ……………….…..…....149
Figure 7.4 Amino acid concentration in the xylem sap in four canola genotypes grown
under adequate and deficient S supply at vegetative stage. Only amino acids
relevant for S transport, and those for which the interaction genotype x S
treatment was significant were shown (Met, Glu and Cys). …………………151
Figure 7.5 Amino acid concentration in the phloem sap in two canola genotypes grown at
adequate and deficient N supply at maturity. ……………….………………..152
Figure 7.6 Amino acid concentration of in the phloem sap in two canola genotypes grown
at adequate and deficient S supply at maturity. Only amino acids relevant for S
transport, and those for which the interaction genotype x S treatment was
significant were shown (Met and Glu). ………….…………………..…….…153
Figure 7.A Phloem sap collection [(silique petioles immersed in 250 µL Eppendorf tubes
containing 200 µL of 20 mM ethylenediaminetetraacetate (EDTA) solution (pH
6.5)]…………………………………………………….….………………….158
xvi
List of tables
Table 3.1. Analyses of variance for dry weight, nitrogen and sulphur concentration and
content in shoots and nitrogen and sulphur utilisation efficiency in canola
genotypes.………………………………………………………………….….50
Table 3.2. Ranges of dry weight and nitrogen and sulphur content in shoots of canola
genotypes grown at adequate (control) and deficient (low) nitrogen and sulphur
supply. …………………………………………………………………………49
Table 4.1. Analyses of variance for dry weight, N concentration and content in shoot, and N
utilisation efficiency of canola genotypes in the vegetative stage. ……………67
Table 4.2. Analyses of variance for dry weight, N concentration and N uptake of stems,
siliques and dropped leaves of canola genotypes at maturity………………….71
Table 4.3. Analyses of variance for seed dry weight, N concentration and content in seed
and N utilisation efficiency of canola genotypes at maturity.……….………...77
Table 5.1. Sulphur supply at vegetative and grain maturity stage. …………….……….…87
Table 5.2. Analysis of variance for dry weight, S concentration in shoots, shoot S content,
and S utilisation efficiency of 12 canola genotypes at the vegetative stage. .…89
Table 5.3. Analysis of variance for dry weight, S concentration and S content, and S
utilisation efficiency of stems, siliques and seeds of 12 canola genotypes at
maturity. Analysis of variance for oil and protein concentration in seeds was for
10 canola genotypes (remaining two genotypes did not yield any seed). .….…92
Table 6.1. Supplies of N as 15
N-urea (~10 atom % enrichment) (A) and supplies of S as
K234
SO4 (~20 atom % enrichment) (B) to canola plants in the experiments (the
amounts for mature stages include those applied in the vegetative stages). …109
Table 6.2. Analyses of variance for shoot dry weight and N parameters in the soil-labelling
experiments for plants in the vegetative stage (A) and at maturity (B)...….…116
Table 6.3. Analyses of variance for dry weight and S nutrient parameters in soil labelling
experiments in the vegetative stage (A) and at maturity (B).……….………..120
Table 6.4. Analyses of variance for N concentration and 15
N atom excess in plant tissues 2
days (1st harvest) and 6 days (2
nd harvest) after leaf labelling commenced
(maturity stage). …………………………………………….…………..……123
xvii
Table 6.5. Analyses of variance for S concentration and 34
S atom excess in leaves and
stems 2 days (1st harvest) and 6 days (2
nd harvest) after leaf labelling
commenced at vegetative stage (A) and for leaves, stems, siliques with seeds at
maturity (B)…………………………………………….………………..125-126
Table 7.1. Analyses of variance for N and S concentration in xylem and phloem sap in
genotypes differing in N- or S-use efficiency. ….……………………...…….146
Table 7.2. Analyses of variance for amino acid concentrations in the xylem sap (46 days
after sowing, flowering stage) in genotypes differing in N-use efficiency (N
experiment)……………………….………………………..…………………147
Table 7.3. Analyses of variance for amino acid concentrations in xylem sap (46 days after
sowing, flowering stage) in genotypes differing in S-use efficiency (S
experiment)…………………..………………………………………………150
xviii
List of publications
Publications arising from this thesis:
1. Balint T, Rengel Z, Allen D (2008) Australian canola germplasm differs in
nitrogen and sulfur efficiency. Australian Journal of Agricultural Research 59,
167-174 (Chapter 3).
2. Balint T, Rengel Z (2008) Nitrogen efficiency of canola genotypes varies between
vegetative stage and grain maturity. Euphytica 164, 421-432 (Chapter 4).
3. Balint T, Rengel Z (2009) Differential sulphur efficiency in canola genotypes at
vegetative and grain maturity stage. Crop & Pasture Science 60, 262-270 (Chapter
5).
4. Balint T, Rengel Z (2011b) Nitrogen and sulfur uptake and remobilisation in canola
genotypes with varied N- and S-use efficiency differ at vegetative and maturity
stages. Crop & Pasture Science 62, 299-312 (Chapter 6).
5. Balint T, Rengel Z (2011a) Amino acid composition of xylem and phloem sap
varies in canola genotypes differing in nitrogen- and sulphur-use efficiency. Crop &
Pasture Science 62, 198-207 (Chapter 7).
1
Chapter 1 Introduction and organisation of the thesis 1
2
3
1.1 Introduction 4
5
Canola (Brassica napus) is the major oilseed crop in Western Australia. The areas sown to 6
canola in 2009/10 reached 745,000 hectares with total production of 986,000 tonnes, while 7
the estimates for 2010/11 season are 1,030,000 tonnes from 860,000 ha (Seberry et al. 8
2010). In the long term, Western Australia has the potential to extend the canola industry up 9
to 1,000,000 hectares with the seed production of up to 1,300,000 tonnes per year. 10
One of the main constrains for a further increase in canola production in Western 11
Australia is poor soil fertility. Soils in Western Australia are predominantly sandy in texture, 12
moderately acidic, and with low availability of N, P and S (Moore 1998). The selection and 13
breeding of canola crops (Selasbury et al. 1999; Cowling 2007) resulted in genotypes that are 14
responsive to high fertilizer applications. In contrast, the traits necessary for growth under 15
limited nutrient supply are likely to have been suppressed or eliminated, resulting in 16
genotypes with relatively poor N- and S-use efficiency. Hence, one of the foci of the future 17
development of Australian canola cultivars should become improvement of N-use efficiency 18
as suggested for European canola cultivars (Mollers and Schierholt 2002). In addition, 19
identification and breeding of genotypes that could use native soil S as well as fertilizer S 20
more efficiently than standard genotypes would be an environmentally-friendly long-term 21
approach to dealing with both low S availability in Western Australian soils and high S 22
requirements of canola. 23
Regarding Australian canola germplasm, some initial work characterising N 24
efficiency has been reported (Yau and Thurling 1987), but no data could be found for modern 25
Australian germplasm. Differences in N remobilization could contribute to genotypic 26
differences in N-use efficiency (eg. in barley, Mickelson et al. 2003), particularly in canola 27
because most of N used for grain filling in canola was derived from mobilisation of N stored 28
in vegetative tissue (Rossato et al. 2001). Glutamine represents the major form of N 29
transported in canola (Mollers et al. 1996). On the other hand, amino acid asparagine is the 30
more efficient form of N transport compared to glutamine (Seiffert et al. 1999). However, no 31
report could be found about the extent to witch canola genotypes differing in N-use 32
2
efficiency would differ in the proportion of these two amino acids in their xylem and phloem 1
saps. 2
Canola crop is very sensitive to shortage of S in soils. Western Australia has some of 3
the most ancient soils in the world that are deficient in S and prone to leaching losses of S 4
(Robson et al. 1995). Canola responds well to S fertilization on these types of soils by 5
increasing seed yield, as well as oil and protein content (Hocking et al. 1996; McGrath and 6
Zhao 1996; Ahmed et al. 1998; Jackson 2000). It has been well documented (Hawkesford 7
and DeKok 2006) that sulphate is the most common S form for uptake and transport in 8
plants. Its transport from roots to shoots can be dependent on/regulated via the amino acid 9
cysteine (sulphate is reduced to sulphide before incorporation into cysteine). On the other 10
hand, the thiol tripeptides [glutathione (Glu-Cys-Gly) and homoglutathione (Glu-Cys-Ala)] 11
were reported to play an important role in transporting S in soybean and wheat (Anderson 12
and Fitzgerald 2001). In contrast, the role of amino acids and other S-containing compounds 13
in uptake and transport of S into various organs in canola has not been reported yet. 14
Although the knowledge in both N- and S-use efficiency is improving fast (see 15
literature review), some important questions remain to be answered (eg. in what and/or why 16
genotypes with differential N- and S-use efficiency differ at vegetative and maturity stage). 17
18
19
The objectives of this study are therefore to: 20
21
Screen Australian canola germplasm for N- and S–use efficiency at 22
vegetative stage. 23
Characterie consistency of N- and/or S-use efficiency from the vegetative 24
stage to physiological maturity stage. 25
Elucidate differences in N and S remobilisation in canola genotypes differing 26
in N-and S-use efficiency. 27
Determine the relative importance of different N and S-containing 28
compounds transported in xylem and phloem in genotypes differing in N- 29
and S-use efficiency. 30
31
32
3
1.2 Structure and organisation of the thesis 1
2
The second chapter is a review of the literature providing a reader with the basic 3
background as well as the latest updates on N and S transport, remobilization and efficiency 4
in canola. Each chapter is constructed around one or combination of these topics. 5
Consequently, some of the topics are examined in more than one chapter. Most chapters are 6
based on publish papers and therefore formats may differ slightly between chapters. 7
8
Chapter 3: Title: Australian canola germplasm differs in nitrogen and sulphur efficiency 9
10
Topics: i) Genotypic differences, ii) N- and S-use efficiency in the 11
vegetative stage 12
13
Chapter 4: Title: Nitrogen efficiency of canola genotypes varies between vegetative 14
stage and grain maturity 15
16
Topics: i) N-use-efficiency of selected genotypes in the vegetative stage, ii) 17
N-use efficiency at grain maturity, iii) Grain quality (oil and protein 18
concentration in seed) 19
20
Chapter 5: Title: Differential sulphur efficiency in canola genotypes at vegetative and 21
grain maturity stage 22
23
Topics: i) S-use efficiency of selected genotypes in the vegetative stage, ii) 24
S-use efficiency at grain maturity, iii) Grain quality (oil and protein 25
concentration in seed) 26
27
Chapter 6: Title: Nitrogen and sulphur uptake and remobilisation in canola genotypes 28
with varied N- and S-use efficiency differ at vegetative and maturity stages 29
30
4
Topics: i) N and S uptake (via soil labelling) at vegetative and maturity 1
stages, ii) N and S remobilisation (via leaf labelling) at vegetative and 2
maturity stages 3
4
Chapter 7: Title: Amino acid composition of xylem and phloem sap varies in canola 5
genotypes differing in N- and S-use efficiency 6
7
Topics: i) N and S transport (vegetative stage). Amino acid assays in xylem 8
sap in canola shoots, ii) N and S transport (maturity stage). Amino acids 9
assays in phloem sap (silique petioles) at maturity. 10
11
Chapter 8: General discussion with conclusions 12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
1.3 References 32
5
1
2
Ahmad A, Abraham G, Gandotra N, Abrol YP, Abdin MZ (1998) Interactive effect of 3
nitrogen and sulphur on growth and yield of rapeseed– mustard (Brassica juncea 4
L.) (Zern. and Coss. And Brassica compestris L.) genotypes. Journal of Agronomy 5
and Crop Science 181, 193-199. 6
Anderson JW, Fitzgerald MA (2001) Physiological and metabolic origin of sulphur for the 7
synthesis of seed storage proteins. Journal of Plant Physiology 158, 447-456. 8
Cowling AW (2007) Genetic diversity in Australian canola and implications for crop 9
breeding for changing future environments. Field Crops Research 104, 103-111. 10
Hawkesford MJ, DeKok LJ (2006) Managing sulphur metabolism in plants. Plant Cell and11
Environment 29, 382-395. 12
Hocking PJ, Pinkerton A, Good A (1996) Recovery of field-grown canola from sulphur 13
deficiency. Australian Journal of Experimental Agriculture 36, 79-85. 14
Jackson GD (2000) Effects of nitrogen and sulfur on canola yield and nutrient uptake. 15
Agronomy Journal 92, 644-649. 16
Mickelson S, See D, Meyer FD, Garner JP, Foster CR, Blake TK, Fischer AM (2003) 17
Mapping of QTL associated with nitrogen storage and remobilization in barley 18
(Hordeum vulgare L.) leaves. Journal of Experimental Botany 54, 801-812. 19
McGrath SP, Zhao FJ (1996) Sulphur uptake, yield responses and the interaction between 20
nitrogen and sulphur in winter oilseed rape (Brassica napus). Journal of 21
Agricultural Science 126, 53-62. 22
Mollers C, Bolune T, Wallbraun M, Lohaus G (1996) Protein content and amino acid 23
composition of the seeds and the phloem sap of one Brassica carinata and different 24
B. napus L. genotypes. In Proceedings of the Symposium Breeding for Oil and 25
Protein Crops, Zaporozhye, Ukraina. pp.148-154. 26
Mollers C, Schierholt A (2002) Genetic variation of palmitate and oil content in a winter 27
oilseed rape doubled haploid population segregating for oleate content. Crop 28
Science 42,379-384. 29
Moore G (1998) Soil guide: a handbook for understanding and managing agricultural soils. 30
Bull. 4243. Agriculture Western Australia, Perth, Western Australia, Australia 31
Robson AD, Osborne LD, Snowball K, Simmons WJ (1995) Assessing sulfur status in 32
lupins and wheat. Australian Journal of Agricultural Research 35, 79-86. 33
6
Rossato L, Laine P, Ourry A (2001) Nitrogen storage and remobilization in Brassica napus 1
L. during the growth cycle: nitrogen fluxes within the plant and changes in 2
soluble protein patterns. Journal of Experimental Botany 52, 1655-1663. 3
Salisbury PA, Wratten N (1999) Brassica napus breeding. In: Salisbury PA, Potter TD, 4
McDonald G, Green AG (Eds.), Canola in Australia: The first 30 years. Organising 5
committee of the 10th international rapeseed congress. pp. 29-35. 6
Seiffert B, Ecke W, Lohaus G, Wallbraun M, Zhou Z, Mollers C (1999) Strategies for the 7
investigation of N-efficiency in oilseed rape. In Plant Nutrition – Molecular 8
Biology and Genetics, Gissel-Nielsen G and Jensen A (Eds.). 425-432. Kluwer 9
Academic Publishers, Dordrecht, Netherlands. 10
Yau SK, Thurling N (1987) Variation in nitrogen response among spring rape (Brassica 11
napus) cultivars and its relationship to nitrogen uptake and utilization. Field 12
Crops Research 16, 139-155. 13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
7
Chapter 2 Literature review 1
2
2.1 Canola 3
4
The term canola (‘double-low’ oil seed rape) (Brassica napus), was registered and adopted 5
in Canada in 1979 to describe the oil (seeds, plants) obtained from the cultivars Brassica 6
napus and Brassica campestris (now Brassica rapa) containing less than 2 % (w/w) erucic 7
acid in the oil and less than 30 μmol/g glucosinolates in the air-dried, oil-free meal (AU 8
Government, 2008). (Figure 2.1) 9
10
11 Figure 2.1. Canola (Source: ''Koehler's Medicinal-Plants'') 12
13
2.1.1 Canola industry in Australia 14
15
Rapeseed was first trialed in Australia in the early 1960’s, and the first commercial 16
seed, of the variety Target, was imported from Canada in 1967. Rapeseed was for the first 17
time grown commercially in Australia 1969. Since then, rapeseed (or canola from 1979) 18
(Figure 2.1) has become the main oilseed crop, grown on over 1.4 million hectares annually, 19
with the average seed production of 1.24 million tones per year (for the period between 20
2002 and 2008) (Seberry et al. 2009). The major canola-producing state in Australia is 21
8
Western Australia (WA). The areas sown to canola in 2009/10 reached 745,000 hectares 1
with total production of 986,000 tonnes, while the estimates for 2010/11 season are 2
1,030,000 tonnes from 860,000 ha (Seberry et al. 2010). 3
Canola production in Australia is constrained by poor soil fertility. Australia has 4
some of the most infertile soils in the world, as a consequence of the antiquity of the 5
continent and extensive weathering of soils. Many agricultural soils are deficient in 6
macronutrients and micronutrients, particularly Western Australian soils; these soils are 7
among the most ancient in the world, and are deficient in phosphorus and nitrogen (Moore, 8
1998) and prone to sulphur leaching and deficiency (Robson et al. 1995). The recent large 9
expansion of canola production in Australia (and especially in Western Australia) has 10
depended in part on overcoming many regional soil nutritional problems by using the 11
adequate crop management, particularly plant nutrition. 12
13
2.1.2 The quality of Australian canola 14
15
The average oil concentration of the Australian canola is variable and the figure for 16
the 2009 canola harvest (as an example) came to 41.6 % (w/w). Oil concentration ranged 17
from a low of 35.9 % (w/w) at Red Bend in Central NSW to a high of 47.7 % (w/w) at 18
Cummins in the Eyre Peninsula region of South Australia (Seberry et al. 2010). The oil 19
concentration of Western Australian canola is variable too and can range from less than 35 20
% (w/w) to more than 45 % (w/w) depending on the season and the growing area: e.g. in 21
2006, oil concentration ranged from as low as 35.8 % (w/w) in Mingenew to 44.3 % (w/w) 22
in the Ravensthorpe area (Seberry et al. 2006) whereas the oil concentration of the 2009 23
canola season ranged from 39.0 % (w/w) in Koorda to 42.7 % (w/w) in Geraldton (Seberry 24
et al. 2009). 25
It is important to note that the international market requires the ‘standard level’ of 42 26
% (w/w) oil. Hence, oil concentration in Australian canola needs to be increased in a 27
consistent manner to ensure competitiveness on the international market. 28
29
2.2 Nutrient efficiency 30
31
2.2.1 Definition and screening methodologies for nutrient efficiency 32
9
1
The definitions of nutrient efficiency vary greatly. Most definitions share the 2
concept that efficiency is the ability of a system to convert inputs into desired outputs, or to 3
minimize the conversion of inputs into waste (Lynch 1998). In general, approaches can be 4
divided into those emphasizing productivity and those emphasizing the internal nutrient 5
requirement of the plant (Gourley et al. 1994). Hence, the nutrient-efficient plants are those 6
that produce higher yields per unit of nutrient (applied or absorbed) than other plants 7
(standards) under similar agroecological conditions (Fageria et al. 2008). Additionally, 8
nutrient efficiency as a total plant biomass produced per unit nutrient absorbed (often called 9
the ‘nutrient efficiency ration’) was used to describe the internal nutrient requirement 10
(Gourley et al. 1994). Further, the nutrient efficiency could be split into two major 11
processes: uptake efficiency (the ability of a plant to absorb the nutrient from soil) and the 12
utilisation efficiency (the ability of a plant to utilise absorbed nutrient for dry matter 13
production). Nitrogen-use efficiency for example can be divided into: (i) assimilation 14
efficiency involving nitrogen (N) uptake and assimilation, and (ii) utilisation efficiency 15
involving N remobilisation (Kant et al. 2011). The variation in nutrient efficiency in crops 16
(defined as grain production per unit of nutrient available in the soil), at sufficient nutrient 17
supply in the field, is generally due to a difference in nutrient uptake efficiency, whereas 18
variation at a deficient nutrient supply is generally due to a difference in utilisation of 19
accumulated nutrients (Moll et al. 1982). 20
Choosing appropriate methodology is an important step in screening genotypes for 21
nutrient efficiency (Fageria 1992). Sceening can be done in controlled/glasshouse 22
environment (using soil or solution as growth medium) or under field conditions. The 23
response to nutrient deficiency stress can differ (to some degree) between field and 24
glasshouse environments (e.g. see works on potassium screening in wheat by Woodend and 25
Glass 1993; or barley by Damon and Rengel 2007) as plants under the field conditions may 26
be exposed to various abiotic stresses from which they are protected in the controlled 27
environment, including drought, high temperature and frost. Hence, both methodologies 28
include a number of advantages as well as disadvantages that should be taken into account 29
when considering results. 30
31
2.3 Nitrogen 32
10
1
2.3.1 Genotypic differences in N efficiency in crops 2
3
Genotypic differences in nutrient efficiency in various crops have been documented 4
by many authors: e.g. differences in N efficiency in wheat (Triticum aestivum) (Fageria and 5
Baligar 2005) and maize (Zea mays) (Gallais and Hirel 2003). Genetic variability for grain 6
yield at the low N supply (with significant genotype x N supply interaction) was reported in 7
wheat (Le Gouis et al. 2000). Differences in S (Ahmad et al. 2005) or N efficiency in 8
mustard (Brassica juncea) (Ahmad et al. 2008) and N efficiency in canola (Brassica napus) 9
(Yau and Thurling 1987, Kessel and Becker 1999, Svecnjak and Rengel 2006) have also 10
been found. Most of these authors reported that genotypic variations in utilisation efficiency 11
were more distinguishable under a limited than under an adequate nutrient supply (as 12
discussed earlier). Hence, the ‘efficient genotypes’ were most likely employing one or more 13
‘genotype-specific mechanisms’ under limited N supply, operating at one or several 14
different levels of plant organization ie. metabolic, physiological, structural or 15
developmental (cf. Rengel 2001). Nutrient efficiency among genotypes is still poorly 16
understood, but it is likely that more than one mechanism is responsible for a given level of 17
efficiency in a particular genotype (cf. Rengel 1999). 18
The selection and breeding of crops resulted in genotypes that are responsive to high 19
fertilizer applications (Lynch 1998). In contrast, the traits necessary for growth under 20
limited nutrient supply are likely to have been suppressed or eliminated, resulting in 21
genotypes with relatively poor nutrient-use efficiency (Lynch 1998). 22
In a study on N efficiency of 70 German canola genotypes from four different types 23
(lines, hybrids, and two resyntheses groups), significant differences were recorded, with the 24
N harvest index being the most important efficiency criterion (Kessel and Becker 1999). 25
Regarding Australian canola germplasm, some initial work characterising genotypic 26
differences in N efficiency has been reported (Yau and Thurling 1987). More recent work 27
on N efficiency in canola was reported for four Australian commercial canola cultivars 28
(Svecnjak and Rengel 2006): despite the similar total N uptake per plant in four tested 29
cultivars, significant differences existed in N-use efficiency because the more N-efficient 30
cultivars produced larger plant biomass with lower N concentrations in plant parts (except 31
roots) compared to less N-efficient cultivars. 32
11
1
2.3.2 Nitrogen-use efficiency (NUE) 2
3
For most plant species, NUE is related to how plants extract inorganic N from the 4
soil, assimilate nitrate and ammonium, and recycle organic N, and can be expressed as a 5
ratio of output (total plant N, grain N, biomass yield, grain yield) and input (total N, soil N 6
or N-fertilizer applied) (Masclaux-Daubresse et al. 2010). 7
However, canola has high demands for N (Holmes 1980); compared to cereal crops, the 8
requirement for N per unit of yield is higher (Hocking and Strapper 2001). Therefore, high 9
rates of N fertilizer are usually applied to canola in order to obtain maximum seed yields. 10
However, the ratio of plant N content to the N applied was at best 50–60 % whatever the 11
level of N fertilizer applied (Schjoerring et al. 1995; Hocking et al. 1997; Chamorro et al. 12
2002, Smith et al. 2010). 13
Further, canola has high N uptake efficiency from soil (Rossato et al. 2001), and thus 14
high capacity to accumulate large quantities of N in the vegetative parts at the beginning of 15
flowering. Hence, the contribution of fertilizer-derived N to total N content is higher in the 16
vegetative than the reproductive parts (Schjoerring et al. 1995; Rossato et al. 2001). The 17
yield of canola is usually half that of wheat (due to the oil production in seed), with N 18
content in canola seed ~30 % higher (3 % w/w in canola and 2 % w/w in wheat on average), 19
meaning that a large proportion of N accumulated in the vegetative organs at the beginning 20
of flowering does not get remobilised or used for seed production (Hirel et al. 2007). One of 21
the causes for N not being well used in seed production is a large quantity of N being lost in 22
early falling leaves (Malagoli et al. 2005); hence, despite the high N uptake efficiency from 23
soil, canola has relatively low N utilisation efficiency (Rossato et al. 2001). Interestingly, 24
the field experiment carried out to evaluate N efficiency among 16 winter varieties of 25
oilseed rape showed that the contribution of N utilisation efficiency to N efficiency was 26
higher than N absorption (uptake) efficiency (Zhang et al. 2009). 27
28
2.3.3 Nitrogen uptake (efficiency) and assimilation 29
30
Plants absorb nutrients mainly from the soil solution. Soil N is available to plants as 31
either nitrate (NO3-) or ammonium (NH4
+) and, to a lesser extent, organic N forms (e.g. 32
12
amino acids). Nitrogen assimilation requires the reduction of nitrate to ammonium, 1
followed by ammonium assimilation into amino acids. Plants supplied by ammonium 2
(NH4+) as the sole N source experienced ammonium toxicity (Zhang and Rengel 2003). 3
Nitrate is known to alleviate NH4+
toxicity in canola roots, most likely by affecting the low-4
affinity NH4+ influx system, with NH4
+ transport being inversely linked to Ca
+ net flux 5
(Babourina et al. 2007). In relation to NH4+ uptake system being less efficient than NO3
- as 6
proposed by Gansel et al. (2001), Schjoerring et al. (2002) re-evaluated the role of NH4+ 7
transport in the xylem, showing that NH4+ translocation in the xylem does indeed occur, as 8
significant concentrations of NH4+ (exceeding 1 mM) were measured in xylem sap of 9
oilseed rape plants growing with NO3- as the sole N source. When NO3
- was replaced by 10
NH4+, the xylem sap NH4
+ concentrations increased with increasing external concentrations 11
and with time of exposure to NH4+. However, the reported amount represented less than 11 12
% of N in the xylem sap. 13
The nitrate (NO3-) is the most common (Laine et al. 1994)/preferred (Pathak et al. 14
2008) source of inorganic N used by higher plants. It is generally accepted now that nitrate 15
(NO3-) is taken up by active transport via the roots, distributed via the xylem and 16
assimilated by the sequential action of various enzymes. 17
18
19
Figure 2.2 Nitrogen assimilation in roots (Oaks 1992). 1. Nitrogenase (legumes only, in 20
association with nitrogen-fixing bacteria); 2. Nitrate uptake system; 3. Nitrate reductase 21
13
(NR); 4. Nitrite reductase (NiR); 5. Glutamine synthetase (GS); 6. Glutamate synthetase 1
(GOGAT); 7. Glutamate dehydrogenase (GDH); 8. Phosphoenolpyruvate carboxylase 2
(PEPcase); 9. Ferredoxin pyridine nucleotide reductase; 10. Asparagine synthetase; and 11. 3
Glutamate-oxaloacetate aminotransferase. 4
5
2.3.4 Nitrogen assimilation 6
7
Following nitrate reduction, nitrite is reduced to ammonium (in the chloroplasts) by the 8
second enzyme of the pathway, the nitrite reductase (NiR). Originating from nitrate 9
reduction, and also from amino acid recycling, ammonium is further assimilated in the 10
plastids/chloroplasts by the so-called glutamine synthetase (GS)/glutamate synthase 11
(GOGAT) cycle (Lea and Forde 1994). The glutamine synthetase fixes ammonium on a 12
glutamate molecule to form glutamine (Masclaux-Daubresse et al. 2010). Two forms of 13
glutamate synthase are present in plants: Fd-GOGAT uses ferredoxin, whereas NADH-14
GOGAT uses NADH as the electron donors (Vanoni et al. 2005). Fd-GOGAT is mainly 15
localized in the leaf chloroplasts, whereas NADH-GOGAT location is in plastids of non-16
photosynthetic tissues, such as roots (Masclaux-Daubresse et al. 2010). 17
In addition to the GS/GOGAT cycle, three enzymes play a role in ammonium 18
assimilation: the cytosolic asparagine synthetase (AS), the mitochondrial NADH-glutamate 19
dehydrogenase (GDH) and the carbamoylphosphate synthase (CPSase). Cytosolic 20
asparagine synthetase catalyses the ATP-dependent transfer of the amido group of 21
glutamine to a molecule of aspartate to generate glutamate and asparagine (Lam et al. 22
2003). Asparagine has a higher N/C ratio than glutamine and can be used as a long-range 23
transport and storage compound, especially in legumes (Lam et al. 2003). However, the 24
roles of cytosolic asparagine synthetase and the other asparagine synthetases, ASN2 and 25
ASN3, in N recycling and mobilisation is complex and not well understood (Masclaux-26
Daubresse et al. 2010). The mitochondrial NADH-glutamate dehydrogenase (GDH) 27
incorporates ammonium into glutamate while acting as ‘antistress enzymes’ in response to 28
high concentration of exogenous ammonium (Skopelitis et al. 2006) and can contributes to 29
the plant survival in dark conditions (Miyashita and Good 2008). The carbamoylphosphate 30
synthase (CPSase) forms carbamoylphosphate (a precursor of citrulline and arginine) in the 31
plastids (Lam et al. 2003). 32
It has been suggested that the internal pools of amino acids within plants may 33
indicate N status by providing a signal that can regulate nitrate uptake by the plant (Miller et 34
14
al. 2008). In Brassica napus, a positive correlation between alpha-aminobutyric acid in the 1
phloem and NO3- uptake was reported (Beuve et al. 2004). This result is unusual because a 2
negative feedback system is more common for most plants, although even in Brassica 3
napus treating the roots with amino acids (glutamine, glutamate and asparagine) decreased 4
transporter expression and NO3- uptake (Beuve et al. 2004). 5
Nitrogen uptake could be negatively regulated, in some cases totally inhibited, 6
during seed production in some plant species. For example, in canola, the transition between 7
the vegetative and reproductive phases is characterised by a drastic decrease of high-affinity 8
transport system (HATS) and HATS + LATS (low-affinity transport system) activities 9
(Malagoli et al. 2004). This change is correlated with a strong repression of gene 10
expression, as shown by undetectable concentration of nitrate transporter BnNRT2 11
messenger ribonucleic acids (mRNA), at the flowering stage (Beuve et al. 2004). 12
13
2.3.5 Nitrogen transporters systems 14
15
Two nitrate transport systems have been shown to coexist in plants and to act co-16
ordinately to take up nitrate from the soil solution and distribute it within the whole plant 17
(Tsay et al. 2007). Two gene families encoding nitrate transporters have been identified in 18
plants, nitrate transporters NRT1 and NRT2 (Lea and Azevedo 2006). It is generally 19
assumed that the NRT1 gene family mediates the root low-affinity transport system (LATS), 20
except the AtNRT1.1, which is a dual affinity transporter gene (Wang et al. 1998; Liu et al. 21
1999) as well as a nitrate sensor (Ho et al. 2009). Fifty three genes belonging to the NRT1 22
family were identified in Arabidopsis (Tsay et al. 2007). The high-affinity transport system 23
(HATS) relies on the activity of the NRT2 gene family and is active when the external 24
nitrate concentration is low (Masclaux-Daubresse et al. 2010). In Arabidopsis, the 25
AtNRT2.1, in interaction with an NAR2 protein is a major component of the HATS (Orsel 26
et al. 2006). 27
15
NRT2.1
NRT2.2
NAR2NRT1.2
NRT1.5
NRT1.1
NRT2.7
NRT1.6
NRT1.4
AMT1;2
AMT1;1
AMT1;3
AMT1;5NRT2.1
NRT2.2
NAR2
1
Figure 2.3 Schematic presentation of the known localisation of nitrate transporters NRT1, 2
NRT2 (in interaction with NAR2 protein) and ammonium transporter AMT genes in 3
Arabidopsis. Two nitrate transport systems have been shown to coexist in plants and to act 4
co-ordinately to take up nitrate from the soil solution and distribute nitrate within the whole 5
plant. The role of each ammonium transporter has been shown by the study of single, 6
double, triple and quadruple mutants (Masclaux-Daubresse et al. 2009). 7
8
Six genes belonging to the family of ammonium transporters (AMT) were found in 9
Arabidopsis (Gazzarini et al. 1999), 10 in rice (Sonoda et al. 2003) a species adapted to 10
ammonium nutrition (Yoshida 1981) and 14 in poplar (Couturier et al. 2007). The 11
lysine/histidine transporter (LHT1) belonging to the amino acids transporters (ATF) family, 12
is crucial for root uptake of acidic and neutral amino acids (Lee et al. 2007), whereas uptake 13
of cationic amino acids such as L-lysine or L-arginine is mediated by amino acid permease 14
5 (AAP5) (Svennerstam et al. 2008). 15
The onset of grain and/or seed filling was a critical phase for N uptake in cereals and 16
canola as well as for N2 fixation in legumes because both processes declined during the 17
grain/seed-filling stage in the studies with 15
N tracing (Salon et al. 2001). During the 18
grain/seed filling stage, N uptake and assimilation is generally insufficient to meet an 19
increased N demand of the grain/seeds (Salon et al. 2001). Instead, the progressive and 20
numerous remobilization steps, occurring successively in the different plant organs are 21
taking place to route N to the grains/seeds. 22
23
2.3.6 Nitrogen harvest index 24
25
16
Nitrogen remobilisation is fundamental for plant N economy as it controls a large part 1
of the N fluxes from source to sink organs. Nitrogen remobilisation processes mainly take 2
place in senescing organs, and it is now generally assumed that the main purpose of 3
senescence in plants is to remobilise and recycle nutrients (Diaz et al. 2008). It is well 4
known that chloroplasts are the main source of nutrients used during senescence, with 5
Rubisco (enzyme/protein) as a major source of N used for remobilisation. 6
The remobilisation of the N accumulated by the plants takes place usually after 7
flowering, and shoots and/or roots then start to be sources of N by providing amino acids 8
released from protein hydrolysis, for export to reproductive and storage organs ie. 9
grains/seeds (Masclaux et al. 2001). In Arabidopsis and canola, it was reported that N can 10
be remobilised throughout plant development from senescing leaves to expanding leaves at 11
the vegetative stage (Diaz et al. 2008) as well as from senescing leaves to seeds at the 12
reproductive stage (Malagoli et al. 2005; Diaz et al. 2008). However, the total N content of 13
leaves in wheat and canola lost before pod filling (2–2.5 % of dry weight; Rossato et al. 14
2001) is high, suggesting that the vegetative parts (leaves, stem, taproot) are not effective in 15
mobilising N to the pods during the reproductive period (Malagoli et al. 2005). The N 16
harvest index in canola is usually low because of the fall of N-rich leaves, mostly during 17
flowering, with up to 15% of the entire N content lost that way (Rossato et al. 2001). 18
However, the N remobilisation from the vegetative organs to the grains in wheat (Triticum 19
aestivum), has been shown to depend on the genotype (Barbottin et al. 2005), with 20
contribution of leaf N remobilisation to grain N content varying from 50 to 90 % depending 21
on the wheat cultivar (Masclaux et al. 2001). 22
The leaf senescence is not only essential for N mobilisation, it is also important for 23
yield because in many plant species the developing seeds receive a large amount of N via 24
remobilisation from other plant organs rather that via new N uptake from soil (Ortiz-Lopez 25
et al. 2000). These findings are consistent with Rossato et al. (2001) who suggested that 26
most of N used for seed filling in canola was derived from mobilisation of N stored in 27
vegetative tissue (shoots). 28
Besides being a developmental process, senescence can be prematurely induced by 29
environmental factors. As plants cannot move, senescence was one mechanism that they 30
have developed to cope with unfavourable environmental conditions (Leopold 1961). 31
Several resource limitations, such as water deficit or mineral N deficiency, can induce 32
premature leaf senescence (Diaz et al. 2008). Hence, the N remobilisation process is 33
17
favoured under limiting nitrate supply (Lemaitre et al. 2008) and can be even enhanced by 1
biotic and abiotic stresses (Lemaitre et al. 2008). 2
3
2.3.7 The biochemical mechanisms and genetics of N remobilisation 4
5
It is generally accepted that the phloem loading process is a key step for efficient N 6
remobilisation. During senescence in many species, the concentrations of asparagine and 7
glutamine increase in the phloem sap (Herrera-Rodriguez et al. 2006). Further, the 8
percentage of glutamine in the phloem exudates being higher than that in the leaf blade 9
indicated that both glutamine and asparagine may play a key role in remobilising N from 10
the senescing leaves (Masclaux-Daubresse et al. 2006). 11
The amino acids in canola were reported to be the major transport form of reduced 12
N, translocated predominately in the phloem (Reins et al. 1991). In the phloem exudate 13
from pea shoots, the predominant amino acid was asparagine (Urquhart and Joy 1981). The 14
predominant amino acid in the canola phloem is glutamine (Mollers et al. 1996). However, 15
the genotypes more efficient in remobilising N were proposed to contain greater amounts 16
and/or proportions of amino acid asparagine in their phloem saps; asparagines is more 17
efficient N transporter with an N:C ratio of 0.5 compared to 0.4 for glutamine (Seiffert et al. 18
1999). Further, in the study of phloem sap in Brassica napus, an efficient phloem loading 19
process was reported, with concentration of free amino acids in the phloem sap going up to 20
650 mM compared to 50-200 mM concentration measured in the phloem sap of most other 21
plant species (Tilsner et al. 2005). 22
Various gene families could be involved in N remobilisation from the source to sink 23
tissues. Members of the amino acid permease (AAP) family were suggested to be involved 24
in the phloem loading process (Koch et al. 2003). Expression of three AAP family members 25
was detected in Brassica leaves during senescence, and the BnAAP1 and BnAAP2 mRNA 26
levels increased in old leaves (Tilsner et al. 2005). The AtLHT1 gene, high-affinity 27
lysine⁄histidine transporter, plays a role in amino acid uptake in root and has a role in 28
supplying the leaf mesophyll cells by xylem-derived amino acids (Hirner et al. 2006). The 29
AtANT1 gene, of another amino acid transporter family, has affinity for aromatic and 30
neutral amino acids. In Arabidopsis, the expression of cysteine, aspartate and serine 31
protease-encoding genes was enhanced during senescence (Buchanan-Wollaston et al. 2003; 32
18
Guo et al. 2004). The ATG (AuTophaGy, also called APG, for AutoPhaGy) genes (8 and 7) 1
were also induced during leaf senescence (van der Graaff et al. 2006). Overall, the 2
Arabidopsis genome encodes 67 amino acid transporters that belong to 11 gene families 3
(Rentsch et al. 2007). 4
The small peptides should also be considered as an alternative form of transported N 5
(Stacey et al. 2002; Dietrich et al. 2004). The peptide and various amino acids transporters 6
were expressed in the seed during embryogenesis, possibly involved in loading of N 7
compounds into the endosperm⁄cotyledon (Rentsch et al. 2007; Tsay et al. 2007). In the 8
final step, the senescence-associated cysteine protease encoded by the SAG12 (Senescence 9
Associated Gene) gene has been detected in senescence-associated vacuoles (Otegui et al. 10
2005). 11
Plant N metabolism during senescence involves many enzymes, such as cytosolic 12
glutamine synthetase (GS1) encoded by a multigene family, glutamate dehydrogenase 13
(GDH), asparagine synthetase (AS) and transaminases. Enzymes other than AS, GS1 and 14
GDH certainly participate in the N management and recycling during senescence. The 15
concentration of glutamate and aspartate, the most abundant amino acids in young leaves of 16
Arabidopsis, decreased with senescence. In contrast, amino acids tyrosine, leucine, 17
isoleucine and gama-aminobutyric acid (GABA) accumulated strongly with ageing. GABA 18
accumulation during Arabidopsis leaf senescence was correlated with an increase in 19
glutamate decarboxylase GAD2 gene expression (Diaz et al. 2008). 20
21
2.4 Sulphur 22
23
Sulphur is one of four major plant nutrients because it is a component of numerous 24
important metabolic and structural compounds (Leustec et al. 2000). Sulphur compounds 25
are involved in responses to abiotic and biotic stresses and plant secondary metabolism 26
(Maruyama-Nakashita et al. 2006; Mugford et al. 2009). 27
Canola is sensitive to low S supply in soils, which suppresses the development of 28
reproductive organs and may lead to silique abortion and decreased seed quality (De Pascale 29
et al. 2008) and yield (McGrath and Zhao 1996). Canola responds well to S fertilisation on 30
soils deficient in S by increasing the seed yield as well as the oil and protein content 31
(Hocking et al. 1996; McGrath and Zhao 1996; Ahmad and Abdin 2000). However, canola 32
19
plants are considered to have relatively poor S efficiency when supplied with adequate S 1
(MacGrath and Zhao 1996). 2
3
2.4.1 Sulphur uptake from soil 4
5
Sulphate is the major form of inorganic S in soil and is absorbed by plant roots and 6
transported in an inorganic form to developing leaf tissues (Anderson 2005) by a family of 7
sulphate transporters (SULTR) (Rouached et al. 2008). Five different gene families encode 8
the plant sulphate transporters (Hawkesford et al. 2003; Buchner et al. 2004). In general, 9
these proteins are proton/sulphate co-transporters in plants (Smith et al. 1995; Buchner et al. 10
2004; Rouached et al. 2005). 11
12
13
Figure 2.4 A phylogenetic tree of the Arabidopsis sulphate transporter gene family (Page 14
1996). 15 16 17 The high affinity transporters all belong to group/family 1, whereas the low affinity 18
transporters form group/family 2 (Hawkesford et al. 2003). Transporters of group/family 4 19
were shown to be responsible for efflux of sulphate from the vacuoles (Kataoka et al. 20
2004a). Much less is known about the function of group 3 and group 5 transporters. 21
However, it is now accepted that sulphate uptake in root is mainly performed under the 22
control of AtSULTR1;2, the main high-affinity sulphate root membrane transporter 23
(HASulT) (El Kassis et al. 2007). AtSULTR1;2 is expressed in the root plasma membrane 24
20
and in the root apex (Shibagaki et al. 2002). Also, the AtSULTR1;1 has been identified as 1
an HASulT (Vidmar et al. 2000), but it is much less expressed in roots compared to 2
AtSULTR1;2 (Rouached et al. 2008). Accumulation of transcripts for both genes is 3
stimulated by sulphate limitation (–S) (Yoshimoto et al. 2002, 2007). A third member of the 4
HASulT group 1 transporters, the AtSULTR1;3, is expressed in the phloem and responsible 5
for the source-to-sink transport of sulphate (Yoshimoto et al. 2003). 6
Several low-affinity SO42–
transporters (LASulT), AtSULTR2;1 and AtSULTR2;2, 7
have been expressed in (or near) the xylem parenchyma cells (Takahashi et al. 1997, 2000) 8
and were involved in long-distance transport. Interestingly, SULTR2;1 has been shown to 9
interact with SULTR3;5 (member of the HASulT group) in yeast. However, none of the five 10
members of the AtSULTR3 sub-type has been demonstrated to complement a yeast mutant 11
defective in its sulphate transporter. Several members of the AtSULTR3 family have been 12
expressed in seeds at various stages of seed development and shown to be involved in 13
regulation of sulphate translocation within developing seeds in Arabidopsis (Zuber et al. 14
2010), and chickpeas (Cicer arietinum) (Tabe et al. 2003). The two members of the 15
Arabidopsis SULTR 4 family have been shown to be localised in the tonoplast: 16
AtSULTR4;1 and AtSULTR4;2, both suggested to be involved in vacuolar SO42–
17
remobilisation to the cytosol (Kataoka et al. 2004b) and to play a role in redox homeostasis 18
in seeds (Zuber et al. 2009). The SULTR4;2 expression is more responsive to SO42–
19
starvation in both roots and shoots in comparison to SULTR4;1. Interestingly, expression of 20
both SULTR4 transporters in canola (Brassica napus) leaves revealed a differential S-21
dependent expression pattern: BnSULTR4;2 was expressed more highly than BnSULTR4;1 22
in response to sulphate depletion (Parmar et al. 2007; Dubousset et al. 2009). In the same S-23
depletion conditions, canola leaf sulphate content significantly decreased in relation to up-24
regulation of BnSULTR4;1, confirming that both SULTR4 members play a role in vacuolar 25
sulphate remobilisation (Dubousset et al. 2009). 26
27
2.4.2 Sulphur assimilation 28
29 30
In the leaf tissues sulphate can be stored in its inorganic form, or can be further 31
‘activated’ (prior to reduction) to adenosine 5’-phosphosulphate (APS) by ATP 32
sulphurylase (ATPS) (Logan et al. 1996). APS is reduced to sulphite (in plastids), which is 33
21
further reduced to sulphide by sulphite reductase (SiR). Sulphide is then incorporated into 1
the amino acid skeleton of o-acetylserine (OAS) to form cysteine (reviewed in Kopriva 2
2006). In Arabidopsis, under adequate growth conditions, sulphate reduction takes place in 3
plastids, whereas synthesis of cysteine can take place in plastids, mitochondria and the 4
cytosol (Kopriva et al. 2009). APS, besides being reduced to sulphite, can also be 5
phosphorylated to 3’ phosphoadenosine 5’-phosphosulphate (PAPS), which is a donor of 6
active sulphate for many reactions in the secondary metabolism (Mugford et al. 2009). 7
8
9
10
Figure 2.5 Outline of sulphate assimilation and cysteine metabolism in plants (in 11
Lewandowska and Sirko 2008). Enzymes involved in sulphate and sulphite reduction are 12
present only in plastids, whereas SAT and OAS-TL are present in plastids, mitochondria 13
and cytosol. Enzymes involved in GSH synthesis are present in chloroplasts and in 14
extrachloroplast fractions. The first two steps of methionine synthesis proceed only in 15
plastids, and the third step only in cytosol because of strictly cytosolic location of MS. 16
APSK, APS kinase; APR, APS reductase; APS, adenosine 5’-phosphosulfate;ATPS, ATP 17
sulphurylase; CBL, cystathionine β-lyase; CGS, cystathionine γ-synthase; GCL, glutamino-18
cysteine lyase; GSHS, glutathione synthetase; HCys, homocysteine; MMT, S-19
adenosylmethionine L-methionine S-methyltransferase; MS, methionine synthase; OAS, O-20
acetylserine; OAS-TL, o-acetylserine (thiol)-lyase; PAPS, adenosine 3’-phoshate 5’-21
phosphosulphate; SAM, S-adenosylmethionine; SAMS, SAM synthetase; SAT, serine 22
22
acetyltransferase; SMM, S-methylmethionine; SiR, sulphite reductase; SOX, sulphite 1
oxidase; SOT, sulphotransferase; ST, sulphate transporter. 2
3
4
Plants do not accumulate large amounts of cysteine, but rather maintain a high flux 5
of that amino acid. Cysteine is a major precursor/donor of reduced S for the synthesis of 6
methionine (Met) and other S-containing metabolites i.e. tripeptide glutathione (GSH) and 7
its homologues e.g. homoglutathione (Hawkesford and Wray 2000; Leustek et al. 2000; 8
Kopriva and Rennenberg 2004; Kopriva 2006). Methionine is the precursor of S-adenosyl 9
methionine (SAM), which is the precursor of ethylene (Burstenbinder et al. 2007) and 10
polyamines, as well as nicotinamine that is important for Fe nutrition in plants (Zuchi et al. 11
2009). Some intermediate metabolites of the S metabolic pathway are precursors of a 12
number of vitamins and sulpholipids in chloroplasts. Glutathione plays an important role in 13
defence against biotic (Schlaeppi et al. 2008) and abiotic stresses (Xiang et al. 2001), 14
regulating stress-related gene expression (Maruyama-Nakashita et al. 2005). 15
Since Cys can be promptly converted in plants into GSH, it is unclear which of these 16
two metabolites takes part in the control of sulphate uptake and assimilation. Experiments 17
conducted with Brassica napus suggested that GSH rather than Cys was responsible for 18
such control because the concentration of GSH was higher than that of free Cys in the cells, 19
indicating GSH was used for storage of reduced S (in Lewandowska and Sirko 2008). 20
Following uptake, sulphate is either transiently stored in vacuoles or assimilated into 21
reduced S compounds. The S reduction pathway is well characterised and conserved among 22
different plant species. However, the major site of S reduction varies and might be 23
dependent on environmental conditions and plant species (Hawkesford and De Kok 2006; 24
Hell et al. 2010). Following translocation of sulphate in the xylem transpiration stream, S 25
reduction predominantly occurs in mature source leaves (Lappartient et al. 1999; Hopkins et 26
al. 2005), although in legumes, S reduction also takes place in other organs. 27
28
2.4.3 Sulphur remobilisation 29
30
Regardless of the location of primary S assimilation, sink translocation of reduced S 31
predominantly occurs via the phloem. It might be loaded into the phloem of the source leaf 32
minor veins following synthesis, or it may be transferred from the xylem to the phloem 33
23
along the translocation path when sulphur assimilation takes place in roots (Buchner et al. 1
2004). Glutathione (GSH) is the main form of reduced S transported (through phloem) and 2
stored in plants (Rennenberg et al. 1979; Leustek et al. 2000). 3
For a long time it has been postulated that GSH acts as an inter-organ signal (from 4
shoots to roots) for the S status (Lappartient et al. 1999; Herschbach et al. 2000). 5
Glutathione (GSH), cysteine (Cys) and methionine (Met) have been suggested to be long-6
distance transport forms of organic S (Lappartient et al. 1999; Davidian and Kopriva, 2010), 7
but in a study by Bourgis et al. (1999), it was shown that the amino acid S-methyl- Met 8
(SMM) is a key contributor to phloem S transport in wheat (Triticum aestivum), canola 9
(Brassica napus), legumes, and other plant species. In these plants, it is suggested that 10
SMM is synthesised from Met by the enzyme Met-methyltransferase (MMT) in the source 11
leaves and loaded into the phloem for transport to seeds (Bourgis et al. 1999; Lee et al. 12
2008). In the seed coat, SMM is recycled back to Met via homo-Cys methyltransferase 13
(HMT) (Gallardo et al. 2007), and amino acids are finally released into the seed apoplast, 14
from where they are taken up by the embryo/cotyledons for development and the storage 15
product accumulation (Tan et al. 2011). 16
Several studies in the past have demonstrated that SMM is important for plant S 17
metabolism, although it might not be essential for plant growth (Kocsis et al. 2003; Gallardo 18
et al. 2007; Lee et al. 2008). The benefit of having SMM as a long-distance S transport 19
compound in plants lies in the fact that SMM could be involved in regulations of plant S 20
and N metabolism (Tan et al. 2010). 21
In canola, leaves represent a major store of nutrients for growing tissues; this store 22
could be remobilised and potentially mediated by senescence processes. In the study by 23
Parmar et al. (2007), the old and maturing canola leaves were considered to export large 24
amounts of S to growing tissues via remobilisation of endogenous S in response to S 25
limitation. Significant S remobilisation from leaves to roots in low-S canola plants was also 26
observed during the vegetative stage (Abdallah et al. 2010). The main strategy of canola to 27
maintain its root growth rate under limited S supply is not by increasing S mobilisation 28
from leaves, but rather by re-orientating more of the S fluxes (uptake and remobilisation) to 29
the roots (Abdallah et al. 2010). 30
The leaves appearing during the rosette stage were playing a crucial role in recycling 31
foliar S compounds to sustain seed filling during the reproductive stage and therefore 32
contribute to the maintenance of the seed yield of canola. Under high S fertilisation 33
24
amounts, the Rubisco, chlorophyll and protein contents increased in fully expanded upper 1
leaves of Brassica juncea (mustard), which implies a better photosynthetic activity in 2
comparison with plants grown without S (Ahmad and Abdin 2000). There results are in 3
accordance with Dubousset et al. (2009), whereby redistribution of S compounds from older 4
to young developing leaves and roots in canola under limited S availability was 5
hypothesized to be related to maintenance of photosynthetic capacities of shoot tissues and 6
subsequent metabolic activities within the whole plant (i.e. including uptake processes in 7
the root). 8
9
2.4.4 Genotypic differences in sulphur uptake and remobilisation in canola 10
11
Genotypic differences in S efficiency during vegetative stage in mustard (Ahmad et al. 12
2005) and grain maturity in canola (Mahli et al. 2007) have been reported. In the study of S 13
uptake efficiency in mustard (Ahmad et al. 2005), differences among genotypes were 14
hypothesized to be in differential S uptake kinetics at the root-cell plasma membrane. The 15
authors suggested the existence of biphasic transporter systems in mustard, (i.e. a 16
combination of high- and low-affinity transporters). 17
It is likely that not only uptake and transport mechanisms, but also remobilisation 18
mechanisms play an important role in genotypic differences in S-use efficiency (Ahmad et 19
al. 2005). 20
21
2.5 Nitrogen and sulphur interaction in canola 22
23
The effects of S fertilisation on canola were closely related to N supply (Zhao et al. 24
1993, Karamanos et al. 2007) as S fertilisation enhanced N efficiency in canola, leading to 25
increased N assimilation into leaf proteins (Ahmad and Abdin 2000). The S remobilisation 26
from vegetative tissues was particularly prominent in the low-N canola plants (Sunarpi and 27
Anderson 1997). Significant N by S interactions for canola seed yield, oil and protein 28
concentration in seed occurred when four different rates of N (0-138 kg/ha) and S (0-34 29
kg/ha) fertilisers were applied to field grown canola i.e. the oil concentration in seed tended 30
to decrease as more N was applied and increased as more S was applied (Brennan and 31
Boland 2008). In the study by Fismes et al. (2000) in the field-grown canola, S deficiency 32
25
reduced N-use efficiency (NUE: ratio of harvested N to N fertilisation) and vice versa, N 1
deficiency also reduced S-use efficiency (SUE). In the case of transient S limitation at the 2
rosette stage, canola, which is considered to be a high S-requiring plant, is able to maintain 3
its growth by an optimisation of N uptake and by recycling of endogenous foliar S 4
compounds (particularly SO42–
) predominantly from mature leaves (Abdallah et al. 2010). 5
6
2.6 Conclusions and opportunities for further studies 7
8
The issues related to N- and S-use efficiency in canola gained a lot of interest in the 9
last few years given that high rates of both nutrients are generally applied in order to 10
maximize yields. However, only about 50 % of applied N and only 20 % of applied S 11
fertilizer are usually recovered in the harvested seeds. Hence, genotypes that could use soil 12
N and S as well as fertilizer-derived N and S more efficiently would be a sustainable long-13
term approach to dealing with (i) high N and S requirements in canola and (ii) N and S 14
deficiency in Western Australian soils. 15
Genetic variation in N efficiency of 46 Australian canola genotypes (Yau and Thurling 16
1987) and four Australian commercial canola cultivars (Svecnjak and Rengel 2006) was 17
identified, but the underlying mechanisms were not elucidated. Remobilisation of N was not 18
studied in the above-mentioned reports, but differences in N remobilisation could contribute 19
to genotypic differences in N-use efficiency (eg. in barley, Mickelson et al. 2003). 20
Genotypic differences in S efficiency/uptake efficiency during vegetative stage in 21
mustard (Mahli et al. 2007; Ahmad et al. 2005) have been reported. It is likely that not only 22
uptake and transport mechanisms (as discussed earlier), but also remobilisation mechanisms 23
could play an important role in genotypic differences in S-use efficiency. The S 24
remobilisation from vegetative tissues was particularly prominent in low-N canola plants 25
(Sunarpi and Anderson 1997). Significant S remobilisation from leaves to roots in low-S 26
plants was reported (Abdallah et al. 2010), but that physiological adaptation was observed 27
only during the vegetative growth. 28
Therefore, to gain more knowledge on related subjects, some of the objectives of this 29
project are: 30
26
1. To screen/identify the variation in N and S efficiency within a large number of 1
Australian canola genotypes (germplasm) in the vegetative growth stage (Chapter 2
3). 3
2. To evaluate whether there is consistency in N and/or S efficiency from the 4
vegetative stage to physiological maturity in chosen genotypes from preliminary 5
study (Chapters 4 and 5). 6
3. To elucidate the differences in N and S uptake and remobilsation in canola 7
genotypes differing in N and S-use efficiency using stable isotopes 34
S and 15
N 8
via soil and leaf (Chapter 6). 9
4. The final objective of this project was to characterise the differences in amino acid 10
content of xylem and phloem sap at the vegetative and maturity stages in canola 11
genotypes with differential N- and S-use efficiency (Chapter 7). 12
The information from this project will help in developing more efficient genotypes for low–13
input canola production on the low-N and low-S Australian soils. 14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
2.7 References 32
33
27
1
Abdallah M, Dubousset L, Meuriot F, Etienne P, Avice JC, Ourry A (2010) Effect of 2
mineral sulphur availability on nitrogen and sulphur uptake and remobilization 3
during the vegetative growth of Brassica napus L. Journal of Experimental 4
Botany 61, 2635-2646. 5
Ahmad A, Abdin NZ (2000) Photosynthesis and its related physiological variable in 6
the leaves of Brassica genotypes as influenced by sulphur fertilization. 7
Physiologia Plantarum 110, 144-149. 8
Ahmad A, Khan I, Nasar AA, Yash PA, Iqbal M (2005) Role of sulphate transporter 9
system in sulphur efficiency of mustard genotypes. Plant Science 169, 842-846. 10
Ahmad H, Islam M, Khan IA, Ali H, Rahman H, Inamullah (2008) Evaluation of advanced 11
rapeseed line HS-98 for yield attributes and biochemical characters. Pakistan 12
Journal of Botany 40, 1099-1101. 13
Anderson JW (2005) Regulation of sulfur distribution and redistribution in grain plants. In: 14
Saito K, DeKok LJ, Stulen I, Hawkesford MJ, Schnug E, Sirko A, Rennenberg H, 15
Eds. Sulfur transport and assimilation in plants in the post genomic era. Leiden, The 16
Netherlands: Backhuys Publishers, 23-31. 17
Australian Government (2008) Department of health and aging, Office of the Gene 18
Technology regulator. The biology of the Brassica Napus L. (canola) Database 19
Search http://www.ogtr.gov.au 20
Babourina O, Voltchanskii K, McGann B, Newman I, Rengel Z (2007) Nitrate supply 21
affects ammonium transport in canola roots. Journal of Experimental Botany,22
58, 651-658. 23
Barbottin A, Lecomte C, Bouchard C, Jeuffroy MH (2005) Nitrogen remobilization 24
during grain filling in wheat : genotypic and environmental effects. Crop Science 45, 25
1141-1150. 26
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Xiang C, Werner BL, Christensen EM, Oliver DJ (2001) The biological functions of 19
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Plant Physiology 126, 564-574. 21
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Philippines 22, 1067-1074. 26
Yoshimoto N, Inoue E, Saito K, Yamaya T, Takahashi H (2003) Phloem-localizing 27
sulfate transporter, Sultrl;3,- mediates re-distribution of sulfur from source to sink 28
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Yoshimoto N, Takahashi H, Smith FW, Yamaya T, Saito K (2002) Two distinct high-30
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Arabidopsis roots. Plant Journal 29, 465-473. 32
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Zhang ZH, Song HX, Liu Q, Rong XM, Peng JW, Xie GX, Zhang YP (2009) Study on 4
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23
24
25
26
27
28
29
30
Chapter 3 Australian canola germplasm differs in nitrogen 31
and sulphur efficiency 32
41
1
3.1 Abstract 2
3
Eighty-four canola genotypes, including current commercial Australian genotypes, some 4
older Australian genotypes, new breeding lines and several genotypes from China were 5
screened for nitrogen and sulphur efficiency in the early growth stage. Plants were grown in 6
glasshouse using virgin brown Lancelin soil (Uc4.22) supplied with basal nutrients; three 7
treatments were: (i) adequate nitrogen and sulphur, (ii) low nitrogen, and (iii) low sulphur. 8
Canola shoots were harvested at 38 days after sowing (with 5 unfolded leaves) when growth 9
reduction and nitrogen and sulphur deficiency symptoms were evident in most genotypes. 10
Three criteria were used to evaluate nitrogen or sulphur efficiency in canola 11
genotypes: 1) growth at low nitrogen or sulphur supply; 2) growth at low relative to 12
adequate nitrogen and sulphur supply, and 3) nitrogen or sulphur utilisation efficiency 13
expressed as shoot dry weight per unit of nitrogen or sulphur content in shoots. 14
Significant genotypic variation in growth and nitrogen or sulphur efficiency was 15
obtained in canola germplasm. Two genotypes were ranked efficient (Chikuzen and 46C74) 16
or inefficient (CBWA-005 and Beacon) in uptake and utilisation of nitrogen under all three 17
criteria. In terms of sulphur efficiency, genotype Argentina was ranked efficient and 18
CBWA-003 and IB 1363 were classified inefficient under all three criteria. Two canola 19
genotypes (Surpass 600 and 46C74) were both nitrogen- and sulphur-efficient in terms of 20
relative growth at low vs. adequate nutrition; their use in the breeding programs could be 21
considered. 22
23
3.2 Introduction 24
25
Canola (Brassica napus) is the major oilseed crop in Australia. The oil content of the 26
Australian canola is relatively high but variable, with the average of 39.6 % in 1997, 40.6 % 27
in 1999 and 39.4 % in 2000 (Carmody and Walton 2003). The international market requires 28
the ‘standard level’ of 42 % oil. Hence, oil content in Australian canola needs to be 29
increased in a consistent manner to ensure competitiveness on the international market. 30
Crop management, particularly plant nutrition and choice of nutrient-efficient genotypes, 31
can play an important role in modifying oil content in canola seed. 32
42
Canola has high demands for nitrogen (Holmes 1980). Therefore, high rates of nitrogen 1
fertilizer are usually applied to canola crops in order to obtain maximum seed yields, but a 2
contribution of fertilizer-derived nitrogen to total nitrogen content is higher in vegetative 3
than in reproductive shoot parts (Schjoerring et al. 1995). Canola has high nitrogen uptake 4
efficiency from soil, but relatively low nitrogen-utilisation efficiency (Rossato et al. 2001). 5
Moreover, nitrogen efficiency decreases with an increase in nitrogen supply (Maman et al. 6
1999). Thus, fine-tuned nitrogen nutrition together with identification of more efficient 7
genotypes could result in better use of nitrogen fertilizers (Mason and Brennan 1998). 8
In a study on nitrogen efficiency of seventy German canola genotypes from four 9
different types (lines, hybrids, and two resyntheses groups), significant differences were 10
recorded, with the nitrogen harvest index being the most important efficiency criterion 11
(Kessel and Becker 1999). Regarding Australian canola germplasm, some initial work 12
characterising nitrogen efficiency has been reported (Yau and Thurling 1987), but no data 13
could be found for modern Australian germplasm. 14
Sulphur is one of the major nutrients affecting growth and yield (Zhao et al. 1993, 15
Ahmad et al. 2000). Canola is particularly sensitive to low sulphur supply in soils, which 16
suppresses the development of reproductive organs and may lead to silique abortion and 17
decreased seed yield (McGrath and Zhao 1996). 18
Western Australia has some of the most ancient soils in the world that are prone to 19
sulphur leaching and deficiency (Robson et al. 1995). Canola responds well to sulphur 20
fertilization on these soils by increasing seed yield, as well as oil and protein content 21
(Hocking et al. 1996; McGrath and Zhao 1996; Ahmad and Abdin 2000). However, no 22
report could be found on potential differences among canola genotypes to take up and 23
utilise sulphur. 24
The effects of sulphur fertilization on canola were closely related to nitrogen supply 25
(Zhao et al. 1993). In addition, sulphur fertilization enhanced nitrogen efficiency in canola, 26
leading to increased nitrogen assimilation into leaf proteins (Ahmad and Abdin 2000). 27
However, the interactions between nitrogen and sulphur fertilization have not been tasted on 28
different canola genotypes. Hence, a large study was initiated to characterize the role of 29
differential nitrogen and sulphur efficiency in canola genotypes in improving seed yield and 30
oil content. The present is aimed at identifying the variation in nitrogen and sulphur 31
efficiency within a large number of Australian canola genotypes in the vegetative growth 32
43
stage. The subsequent publications will concentrate on differences in nitrogen and sulphur 1
efficiency influencing seed yield and oil content in selected canola genotypes. 2
3
3.3 Materials and methods 4
5
Eighty-four canola genotypes were tested for nitrogen and sulphur efficiency during 6
vegetative stages of plant growth. This group of genotypes comprised current commercial 7
Australian genotypes (including some older ones), new breeding lines and several 8
genotypes from China to increase genetic variability. The pedigrees of some choosen 9
Australian canola genotypes/varieties bred from 1970-2004 was included in Figure 3.A. 10
(Cowling 2007). 11
A brown sandy soil (Uc4.22; Northcote 1971) was collected from bushland site 15 12
km south-east of Lancelin (3156’S, 11520’E). Soil chemical characteristics of pH (H2O) 13
5.9, 2 % clay and 8 g/kg organic carbon with low levels of essential plant nutrients (N, K, P, 14
Mg, S, Zn, Cu) (Brennan et al. 1980) made this soil the standard one used for nutritional 15
studies at the University of Western Australia for more than three decades. 16
The soil was air dried and sieved to 2 mm. Basal nutrient and treatment salt 17
solutions were added to soil, followed by air drying, thorough mixing and filling into plastic 18
pots lined with plastic bags (1,000 g of air-dry soil/pot). Basal nutrients were added at the 19
following rates (mg/kg soil): 65.8 N; 87.6 K; 20.9 P; 54.2 S; 92.7 Na; 40.9 Ca; 179.4 Cl; 20
7.10 Mg; 3.25 Mn; 2.85 Zn; 0.62 Cu; 0.12 B; 0.09 Co; and 0.08 Mo (cf. Osborne and 21
Rengel 2002a, b). Nitrogen was supplied as NH4NO3 and sulphur as Na2SO4 (for detailed 22
nitrogen and sulphur application rates see Table 3.2). 23
The experiment was set up in a completely randomised block design with three 24
treatments: adequate nitrogen and sulphur (control), low nitrogen, and low sulphur, in two 25
replicates (Figure 3A). For control and low sulphur treatments, nitrogen was reapplied at 26
65.8 mg/kg soil after 15 and 29 days after sowing, while no re-application of nitrogen on 27
top of the basal rate was made in the low nitrogen treatment. Sulphur was used only as a 28
basal nutrient (54.2 mg/kg) in the adequate nitrogen and sulphur and the low nitrogen 29
treatments. The low sulphur treatment received no sulphur application. 30
44
One hundred canola genotypes were tested for germination. The germination rate in 1
eighty-four genotypes ranged from 65 to 90 %. Sixteen genotypes had the germination rate 2
below 20 % and were excluded from the further study. 3
The experiment was conducted in Perth, Western Australia (3158’S, 11549’E) in 4
an evaporatively cooled glasshouse in early spring with average day/night temperatures of 5
25/14 C. Canola genotypes were sown at seven seeds per pot and thinned to three plants 8 6
days after sowing (when seedlings with cotyledones completely unfolded). Pots were 7
watered with deionised water daily and weighted to field capacity (10 % w/w) every second 8
day. Insect pests were controlled as required. 9
Canola shoots were harvested by cutting the plants at the base of the stems, just 10
above the ground, at 38 days after sowing (shoots with 5 unfolded leaves) when growth 11
reductions as well as nitrogen or sulphur deficiency symptoms were evident in most 12
genotypes in the low nitrogen or low sulphur treatment. Shoot dry weight was determined 13
after drying in an oven at 65 C for 7 days. The nitrogen content was determined on a 4-14
channel Autoanalyzer system, using modifications of Technical procedures (Anon 1979). 15
After digestion with sulphuric acid and hydrogen peroxide (Yuen and Pollards 1954), 16
nitrogen was determined colourimetrically by the indophenol blue method. Sulphur was 17
determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES) after 18
digestion with a mixture of nitric and perchloric acids (McQuaker et al. 1979). 19
The nitrogen or sulphur efficiency was evaluated using the following three criteria: 20
1) growth at low nitrogen or sulphur supply; 2) growth at low nitrogen or sulphur supply 21
relative to growth at adequate nitrogen and sulphur supply; 3) nitrogen or sulphur utilisation 22
efficiency, expressed as shoot dry weight per unit of nitrogen or sulphur content in shoots. 23
The nitrogen and sulphur efficiency ranking (I, inefficient; M, medium efficient; E, 24
efficient) was calculated according to Rengel and Graham (1995); boundaries for the 25
medium efficiency interval were formulated by subtracting or adding the value of one 26
standard error from the median point of a particular efficiency criterion. The data were 27
analysed by 2-way ANOVA using GENSTAT 6.1. When genotype x S or / N interaction 28
was not significant, the treatment mean effects were presented. Least significant difference 29
(LSD 0.05) was used to determine significant differences between treatment means. For 30
each ANOVA, standard error of the mean (SEM), significance and LSD 0.05 were reported. 31
32
45
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Figure 3.A. Pedigrees of selected Australian canola varieties 1970–2004, as reported by Salisbury and Wratten (1999) and 26 IPAustralia (http://www.ipaustralia.gov.au/pbr/). Tower-TT (abbreviation TT) is an informal name for backcross progeny of Tower 27 exhibiting cytoplasmically-inherited tolerance to triazine herbicides, derived from B. rapa (Beversdorf et al., 1980). Varieties not 28 listed: hybrids, ClearfieldTM (imidazolinone tolerant) and those with uncertain pedigrees (e.g. Rivette, Ripper, Purler, Lantern, AG-29 Emblem, AV-Sapphire) (In Cowling WA 2007). 30
31
46
3.4 Results 1
2
The earliest symptoms of nitrogen deficiency under low nitrogen supply (16 mg N/kg of 3
soil) were reduced growth and change in the leaf colour from dark to pale green (chlorosis) 4
18 days after sowing (shoots with 2 unfolded leaves ). As plants grew older, deficiency 5
symptoms became more severe, with leaves developing purple colour on the upper surface. 6
Similar nitrogen deficiency symptoms in canola plants were described by Pluske and 7
Osborne (2001). The intensity of purple pigmentation varied among genotypes. On the other 8
hand, plants supplied with adequate nitrogen (200 mg N/kg soil) were large and healthy 9
green. 10
The earliest symptoms of sulphur deficiency in canola genotypes under low sulphur 11
supply were reduced growth and leaf thickening; these symptoms occurred 20 days after 12
sowing (shoots with 2 unfolded leaves). As the plants grew older, intensive purple 13
pigmentation developed on the underside of the leaves, with the leaf margins rolled 14
inwards, creating a tubular appearance. In contrast, the plants with adequate sulphur supply 15
(54 mg S/kg of soil) were healthy green and with large leaves. 16
17
3.4.1 Growth at low nitrogen or sulphur supply 18
19
Canola cultivars differed considerably in growth and nitrogen uptake parameters. Shoot dry 20
weight was influenced by nitrogen treatment, but also by differences among genotypes 21
(significant nitrogen x genotype interaction) (Table 3.1). Dry matter production under 22
adequate nitrogen supply varied for different genotypes and was approximately four times 23
higher than under limited nitrogen supply (Table 3.2). It is worth noting that nitrogen 24
application in the adequate nitrogen treatment was five times higher than application in the 25
low nitrogen treatment, indicating higher nitrogen fertilizer use efficiency at low compared 26
to adequate application rates (Table 3.2). 27
Dry matter production under limited nitrogen supply ranged between 150 and 480 28
mg/shoot (Figure 3.1). Eighteen canola genotypes (including Drum, Wesway, Wesbarker, 29
Rainbow and Charlton) had dry matter production well above 390 mg/shoot and were 30
identified as efficient under limited nitrogen supply. These genotypes had approximately 2 31
times higher dry matter production than genotypes Westar, Eyre and CBWA-005 that 32
47
produced less than 260 mg/shoot under limited nitrogen supply and were considered 1
inefficient. 2
The interaction genotype x sulphur treatment (Table 3.1) for dry matter production 3
indicated that the genotypes that produced largest shoots under adequate sulphur supply did 4
not necessarily have the largest shoots under low sulphur supply. For instance, genotype 5
Taparoo was one of the high performing ones under adequate sulphur supply (data not 6
shown), but was one of the worst performing ones under limited sulphur supply. In contrast, 7
genotype Surpass 600 showed average growth under adequate sulphur supply, but was one 8
of the highest producing genotypes under limited sulphur supply, making this genotype 9
interesting for further study. 10
11
12
13
48
0.1 0.2 0.3 0.4 0.5
BEACON CG HUA YOU 1
CBWA - 004IB 1377
CBWA - 005PIONEER 44C73
EYRE WESTAR GROUSE
BUGLE IB 1363GRACE
CBWA - 003HYDEN
SURPASS 501 TT SURPASS 603CL
IB 1368DUNKELDWESREO
RANGECG HUA YOU 12
LANTERNAG VIC SAPPHIRE
CG HUA YOU 3RIVETTE KAROO EUREKA
OUTBACK EMBLEM
IB 1487WESBROOKCBWA - 002
SURPASS 300 TTCG 821
MONTY BLN 326
998IB 1509
BLN 5CASTLE
SURPASS 402CLCG HUA YOU 8
OSCAR SURPASS 404CL
HYOLA 60991
RIPPER PINNACLE
BLN 201BRONGORO -
CG HUA SHUANG 3SURPASS 400
TOWERIB 1337
WESBELLOSCAR
SURPASS 600BLN 301
OMIBAROSSA
IB 1524BLN 70
MALUKABLN 320BLN 287BLN 322
46C74 BLN 336
RAINBOW CHARLTON
IWASHIRO45C75
CHIKUZENBLN 156BLN 313
WESBARKERDRUM
BLN 331BLN 279
ARGENTINATAPAROO
GENKAITOKIWA
WESWAY
Shoot dry weight (g/plant)
M EI
1
Figure 3.1 Figure 3.2. 2
3
Figure 3.1 Shoot dry weight of 84 canola genotypes grown at low nitrogen supply (16 mg 4
N/kg soil) for 38 days (shoots with 5 unfolded leaves). Genotypes are ranked in the order of 5
decreasing nitrogen efficiency (E, efficient; M, medium; I, inefficient). The boundaries for 6
the medium-efficiency interval were formulated by subtracting or adding the value of 1 7
standard error from the median point of the efficiency criterion. 8
49
1
Figure 3.2 Shoot dry weight of 84 canola genotypes grown at low (0 mg added) sulfur 2
supply in Lancelin soil for 38 days (shoots with 5 unfolded leaves). Efficiency intervals (E, 3
efficient; M, medium; I, inefficient) were constructed as explained in Figure 3.1. 4
5
Table 3.2. Ranges of dry weight and nitrogen and sulphur content in shoots of canola 6
genotypes grown at adequate (control) and deficient (low) nitrogen and sulphur supply 7
8
Treatments
N and S supply / kg soil
DW (g/shoot)
N content
(mg/shoot)
S content
(mg/shoot)
Control
200 mg N, 54 mg S
0.8 – 1.6
35.9 - 56.4
5.2 - 10.2
Low N 16 mg N, 54 mg S 0.15 – 0.48 1.8 - 6.3 -
Low S 200 mg N, 0 mg S 0.25 – 0.68 - 0.20 - 0.55
9
All the growth and sulphur uptake parameters were significantly affected by sulphur 10
treatments (Table 3.1). Genotypes differed considerably in plant biomass. Dry matter 11
production in genotypes supplied with adequate amount of sulphur (54 mg added S/kg soil) 12
ranged from 0.8-1.6 g/shoot, approximately three times higher than under limited sulphur 13
supply (Table 3.2). Dry matter production above 0.5 g/shoot (a point between the medium 14
efficient and efficient range) was recorded in 17 canola genotypes (Figure 3.2). For instance 15
genotypes Oscar, Surpass 600 and Lantern had dry matter production above 0.65 g/shoot, 16
which was significantly higher than genotypes Karoo, Range and CBWA-003 with dry 17
matter production below 0.31 g/shoot. Twenty one canola genotypes had dry matter 18
production below 0.38 g/shoot (a point between medium efficient and inefficient). 19
20
3.4.2 Concentration of nitrogen and sulphur in canola shoots 21
22
Nitrogen concentration in shoots was significantly affected by nitrogen treatment. It 23
was also affected by the interaction nitrogen treatment x genotype, indicating the existence 24
of genotype-specific traits (Table 3.1). Canola genotypes under adequate nitrogen supply 25
(200 mg N/kg soil) accumulated 35.9 to 56.4 mg N/shoot, and about 14 times less under 26
limited nitrogen supply (16 mg N/kg soil) (Table 3.2). Genotypes Surpass 400, Surpass 300 27
TT and Wesway accumulated significantly higher amounts of nitrogen per shoot (making 28
them more efficient) than genotypes Beacon, Westar and Range under limited nitrogen 29
50
supply (data not shown). Canola genotypes in the control treatment accumulated in shoots 1
equivalent of approximately 25 % of supplied nitrogen compared with low nitrogen 2
treatment where plants took up 62 % of supplied amount. 3
Significant sulphur treatment x genotype interaction for sulphur uptake efficiency was 4
recorded (Table 3.1), suggesting that genotypes that performed well under adequate supply 5
were not necessarily the best performing ones under limited sulphur supply. For example, 6
genotype Castle was superior to genotype BLN 287 under low sulphur supply, but not under 7
adequate sulphur supply, making genotype Castle valuable for further study (data not 8
shown). Genotypes supplied with adequate sulphur amount (54 mg added S/kg soil) 9
accumulated 5.2 to 10.2 mg S/shoot, utilising approximately 30 % of fertilizer added. On 10
the other hand, genotypes without sulphur application managed to accumulate 0.2 to 0.55 11
mg S/shoot (approximately 22 times less than sulphur-adequate plants) (Table 3.2) because 12
of low-plant available sulphur in virgin sandy Lancelin soil. 13
14
Table 3.1. Analyses of variance for dry weight, nitrogen and sulphur concentration and 15
content in shoots and nitrogen and sulphur utilisation efficiency in canola genotypes 16
Genotype
(G)
Nitrogen treatment
(N)
G x N
Shoot dry weight *** *** *
Nitrogen concentration in shoots *** *** **
Nitrogen content in shoots *** *** *
Nitrogen utilisation efficiency *** *** ***
Genotype
(G)
Sulphur treatment
(S)
G x S
Shoot dry weight *** *** **
Sulphur concentration in shoots *** *** **
Sulphur content in shoots *** *** ***
Sulphur utilisation efficiency *** *** ***
*, **, *** Significant at P=0.05, P= 0.01 and P=0.001, respectively. 17
18
19
Significant differences in sulphur accumulation in shoots were recorded among 20
genotypes under limited sulphur supply. For example, genotypes Surpass 300 TT, Range 21
51
and Pinnacle accumulated approximately three times higher amounts of sulphur per shoots 1
under limited supply than genotypes Chikuzen, Castle and Beacon (data not shown). 2
3
4
3.4.3 Growth at low relative to adequate nitrogen or sulphur supply 5
6
For a range of tested genotypes, growth at low nitrogen supply was 14 to 52 % of that at 7
adequate nitrogen supply. Genotypes with the relative yield higher than 36 % were rated 8
efficient (Figure 3.3). Hence, efficient genotypes, for instance 46C74, Hyola 60 and 9
Wesway, had smaller differences in dry matter production between adequate and low 10
nitrogen supply compared to inefficient genotypes (eg. Westar and Beacon) where 11
differences in growth between two nutrient supplies were 5- to 6- fold. 12
Relative growth under low sulphur supply ranged between 23 and 70 % (Figure 3.4). 13
Twenty genotypes were ranked as efficient and had relative yield above 48 % (eg. 14
genotypes Lantern, Surpass 600 and Pinnacle). Twelve genotypes had relative yield below 15
33 % and were recognised as inefficient (eg. Range, Karoo and IB 1363) (Figure 3.4). 16
These genotypes had greater differences between adequate and low sulphur treatments 17
compared with efficient genotypes. 18
19
20
21
52
0 10 20 30 40 50 60
CG HUA YOU 1BEACON
CBWA - 004WESTAR
CBWA - 005SURPASS 603CL
PIONEER 44C73BLN 301WESREO
BLN 5RANGEIB 1363
CG HUA YOU 12MONTY
WESBROOKSURPASS 501 TT
GROUSE HYDEN BUGLE
BLN 201998
EYRE OSCAR
LANTERN EMBLEM
IB 1377DUNKELD
OUTBACK SURPASS 402CL
CASTLE AG VIC SAPPHIRE
RIPPER CBWA - 003CBWA - 002
CG HUA YOU 3RIVETTE
IB 1368CHARLTON
BLN 156BLN 70
KAROO SURPASS 400
CG HUA SHUANG 3EUREKA
CG HUA YOU 8BLN 326GRACE BLN 287CG 821
SURPASS 404CLTAPAROO
BLN 313BLN 336BLN 320
RAINBOW ARGENTINA
45C75OMI991
IWASHIROWESBELL
BLN 322TOWER
BLN 331BRONGORO - 135/19
MALUKAIB 1487OSCARIB 1509
BLN 279HYOLA 60
46C74 CHIKUZEN
IB 1337SURPASS 300 TT
BAROSSAWESBARKER
SURPASS 600DRUM
PINNACLE GENKAI
WESWAYTOKIWA
Shoot DW at 16 mg N / Shoot DW at 200 mg N (%)
I M E
0 20 40 60 80
IB 1363RANGE
SURPASS 603CLBLN 301
TAPAROOIB 1337
CBWA - 003KAROO
CBWA - 005RIPPER
CG HUA SHUANG 3HYDEN BLN 320MONTY
PIONEER 44C73WESTAR
CBWA - 004OUTBACK
OSCAR RIVETTE
CHARLTONHYOLA 60
BLN 156BEACON CASTLE MALUKABLN 201
RAINBOW SURPASS 501 TT
CHIKUZENEUREKA
AG VIC SAPPHIRECBWA - 002
GROUSE IB 1509
BLN 331BUGLE 45C75
SURPASS 404CLCG HUA YOU 1
SURPASS 402CLGRACE BLN 336BLN 70
BLN 287DRUM
SURPASS 400 SURPASS 300 TT
Fig. 4.WESREO
IB 1487EMBLEM BLN 322
OMICG HUA YOU 12
BLN 5EYRE
BLN 279IB 1377
BLN 326CG HUA YOU 3
BLN 313CG HUA YOU 8
PINNACLE DUNKELD
BAROSSAIWASHIRO
46C74 ARGENTINA
CG 821998
WESBARKER991
WESBELLTOWER
LANTERN BRONGORO - 135/19
TOKIWAWESWAY
GENKAIIB 1368OSCAR
SURPASS 600
Shoot DW at 0 S / Shoot DW at 54 mg S (%)
I M E
1
Figure 3.3 Figure 3.4 2
3
Figure 3.3 Ranking of 84 canola genotypes based on the ratio between shoot dry weight at 4
low (16 mg/kg) and adequate nitrogen supply (200 mg/kg soil) after 38 days of growth 5
(shoots with 5 unfolded leaves). Efficiency intervals (E, efficient; M, medium; I, inefficient) 6
were constructed as explained in Figure 3.1. 7
8
53
Figure 3.4 Ranking of 84 canola genotypes based on the ratio between shoot dry weight at 1
low (0 mg) and adequate sulphur supply (54 mg/kg soil) after 38 days of growth (shoots 2
with 5 unfolded leaves). Efficiency intervals (E, efficient; M, medium; I, inefficient) were 3
constructed as explained in Figure 3.1. 4
5
6
7
3.4.4 Nitrogen and sulphur utilisation efficiency 8
9
This index reflects how efficient canola genotypes were in converting to organic matter 10
nitrogen and sulphur taken up. When considering nitrogen utilisation efficiency and 11
genotype x nitrogen treatment interactions, significant differences were recorded (Table 12
3.1), suggesting that genotypes that utilised nitrogen more efficiently under limited supply 13
were not necessarily the more efficient ones under adequate supply. For example, genotype 14
BLN 331 was superior to genotype Rainbow under low nitrogen but not under adequate 15
nitrogen supply (Figure 3.5), making genotype BLN 331 valuable for further study. The 16
nitrogen utilisation efficiency ranged between 64 and 129 g of shoot dry weight/mg shoot N 17
(Figure 3.5). Sixteen canola genotypes were rated as efficient and had nitrogen utilisation 18
efficiency index above 115 (Figure 3.5). 19
In the present study, the sulphur utilisation efficiency values ranged from 95 to 177 g 20
shoots dry weight/mg S in shoot, with 25 genotypes ranked as efficient with indices above 21
150. Twenty three genotypes were recognised as inefficient with indices lower than 126 22
(Figure 3.6). 23
24
3.4.5 Nitrogen and sulphur efficiency based on all three criteria combined 25
26
Only two genotypes were classified as efficient under all three criteria considering nitrogen 27
nutrition (Chikuzen and 46C74). Two genotypes were inefficient under all three criteria 28
(CBWA-005 and Beacon). Considering sulphur nutrition, genotype Argentina was the only 29
one ranked efficient under all three criteria, whereas two genotypes (CBWA-003 and IB 30
1363) were ranked inefficient under all three criteria. There was no genotype ranked 31
efficient or inefficient in both sulphur and nitrogen under all three criteria. 32
33
3.5 Discussion 34
54
1
Genotypic differences in nutrient efficiency in various plant crops have been documented 2
previously. For example, differences in phosphorus efficiency were reported in wheat 3
(Osborne and Rengel 2002a, b, c) and maize (Fageria and Baligar 1997), in sulphur 4
efficiency in mustard (Ahmad et al. 2005) and nitrogen efficiency in canola (Yau and 5
Thurling 1987, Kessel and Becker 1999, Svecnjak and Rengel 2006a, b). Increased 6
efficiency at which a nutrient is used within the plant may be due to the increasing rate at 7
which the nutrient is transported around the plant or compartmented and utilised within 8
cells (eg. Rengel and Graham 1995, Rengel and Hawkesford 1997). Further work on 9
selected canola genotypes is warranted to elucidate the mechanisms underlying differential 10
nitrogen and sulphur efficiency in canola germplasm. 11
Improvement in the nitrogen uptake and utilisation efficiency has occurred with 12
modern canola breeding. Two out of 84 canola genotypes from the study presented here 13
(Charlton and Range) released in late 1990s were among the best performing canola 14
genotypes. On the other hand, genotype Tower released in late 1970s performed poorly. 15
Glutamine represents the major form of nitrogen transported in canola (Mollers et al. 16
1996). However, the amino acid asparagine is the more efficient form of nitrogen transport 17
compared with glutamine (Seiffert et al. 1999). It could be expected that genotypes with 18
higher nitrogen utilisation efficiency use asparagine as a major transport form of nitrogen. 19
Also, the relative importance of nitrogen transport forms may vary with plant 20
developmental stage. The genotype such as Wesway was superior to genotype Surpass 603 21
CL in nitrogen uptake efficiency under low but not under adequate nitrogen supply (data not 22
shown), suggesting that at the minimum nitrogen concentration at which nitrogen uptake 23
starts, genotype Wesway may have a higher number and/or higher activity of transporters 24
compared with Surpass 603 CL. Further study involving use of stable isotopes 15
N will be 25
undertaken to study efficiency of uptake, and xylem and phloem transport in genotypes 26
differing in nitrogen efficiency. 27
28
55
0 50 100 150
CG HUA YOU 1SURPASS 300 TT
PINNACLE EYRE
CBWA - 004GRACE KAROO TOWERBUGLE
CBWA - 003BEACON
CBWA - 005CBWA - 002
SURPASS 600IB 1368
SURPASS 501 TTEUREKA
SURPASS 400 WESWAY
DRUMMONTY
OMIIB 1487IB 1377
RIVETTE IB 1337
BRONGORO - 135/19ARGENTINA
TOKIWABAROSSA
BLN 70BLN 279IB 1363CG 821
GROUSE HYDEN OSCAROSCAR
WESREOSURPASS 603CLSURPASS 404CL
GENKAIMALUKA
IB 1524BLN 156BLN 320BLN 326BLN 287
45C75LANTERN WESTAR
IB 1509PIONEER 44C73
WESBARKERDUNKELD
OUTBACK IWASHIRO
BLN 301991
SURPASS 402CLCASTLE EMBLEM
HYOLA 60RIPPER
AG VIC SAPPHIRECG HUA SHUANG 3
998WESBELL
46C74 CG HUA YOU 12
BLN 201BLN 5
WESBROOKBLN 322
CHIKUZENBLN 313
CG HUA YOU 3RAINBOW
BLN 336CG HUA YOU 8
BLN 331RANGE
TAPAROOCHARLTON
Nitrogen utilisation efficiency (g shoot DW / mg N in shoot)
I M E
0 50 100 150 200
IB 1337IB 1363IB 1377
SURPASS 300 TTIB 1487GRACE IB 1509
TOKIWACBWA - 003
BRONGORO -IB 1524
OUTBACK KAROO
OMIHYDEN
DRUMBLN 287
BAROSSABUGLE
BLN 326GENKAI
MALUKAOSCARRANGE
EUREKASURPASS 501 TT
CHIKUZENGROUSE
CBWA - 004991
CG HUA SHUANG 3CG HUA YOU 12
TAPAROOBLN 336
CBWA - 002BEACON
IB 1368SURPASS 404CL
WESTAR CBWA - 005
PINNACLE AG VIC SAPPHIRE
EYRE BLN 320BLN 322
SURPASS 603CLBLN 313
WESBARKERWESBROOK
TOWERBLN 301WESREO
LANTERN MONTY
SURPASS 400 OSCAR BLN 156BLN 70
BLN 331998
SURPASS 600ARGENTINA
EMBLEM CG 821
BLN 279IWASHIRO
PIONEER 44C73HYOLA 60
45C75RIVETTE
WESBELLCHARLTON
CG HUA YOU 1RAINBOW WESWAYDUNKELD
RIPPER 46C74
CG HUA YOU 8CASTLE
SURPASS 402CLBLN 201
CG HUA YOU 3BLN 5
Sulphur utilisation efficiency (g shoot DW / mg S in shoot)
I M E
1
Figure 3.5 Figure 3.6 2
3 Figure 3.5 Nitrogen utilisation efficiency (calculated as the amount of shoot dry weight 4
(DW) produced per unit of nitrogen accumulated in shoots) of 38-day-old canola genotypes 5
(shoots with 5 unfolded leaves) grown at low nitrogen supply (16 mg/kg soil). Efficiency 6
intervals (E, efficient; M, medium; I, inefficient) were constructed as explained in Figure 7
3.1. 8
56
Figure 3.6 Sulphur utilisation efficiency (calculated as the amount of shoot dry weight 1
(DW) produced per unit of sulphur accumulated in shoots) of 38-dayold canola genotypes 2
(shoots with 5 unfolded leaves) grown at low (0 mg) sulphur supply in Lancelin soil. 3
Efficiency intervals (E, efficient; M, medium; I, inefficient) were constructed as explained 4
in Figure 3.1. 5
6
In our study sulphur starvation enhanced the sulphur uptake efficiency of canola 7
genotypes. Similar findings were reported by Ahmad et al. (2005) in study of sulphur 8
uptake efficiency in mustard genotypes, suggesting that differences in genotypes were due 9
to differences in sulphate uptake kinetics at the root cell membrane. The authors suggested 10
the existence of biphasic transporter systems in genotypes, i.e. combination of high- and 11
low-affinity transporters. There is a possibility that the sulphur-efficient genotype Castle 12
from our study has both high- and low-affinity transporter systems active at the root cell 13
membrane, whereas sulphur-inefficient genotype BLN 287 only has a low-affinity one. This 14
hypothesis will be tested in a future study. 15
It is well documented (Hawkesford and De Kok 2006) that sulphate is the most 16
common sulphur form for uptake and transport in the plant. The regulation of sulphur 17
transport can be dependent on cysteine (sulphate is reduced to sulphide before incorporation 18
into cysteine). On the other hand, glutathione and homoglutathione play an important role in 19
transporting sulphur in soybean and wheat (Anderson and Fitzgerald 2001). In our study, 20
canola genotypes with high sulphur utilisation efficiency may genetically differ in the 21
biosynthesis or transport efficiency of some or all three major sulphur-containing 22
compounds (cysteine, glutathione and homoglutathione). Study of xylem and phloem 23
transport of sulphur in genotypes differing in sulphur efficiency will be undertaken to 24
elucidate possible differences in the amounts and dynamics of these compounds. 25
In the present study sulphur starvation improved the sulphur utilisation efficiency. 26
Indeed, canola plants are considered to have relatively poor sulphur efficiency when 27
supplied with adequate sulphur (MacGrath and Zhao 1996). 28
Interestingly, it does not appear that significant improvement in the efficiency of 29
sulphur uptake and utilisation has occurred with modern breeding. More recent genotypes 30
such as Surpass 402 CL and Charlton released in the last decade had high sulphur utilisation 31
efficiency but out of 84 canola genotypes the older genotypes such as Wesway or Wesbell 32
were among the best performing ones. 33
57
No genotype was identified as having both nitrogen and sulphur efficiency based on all 1
three efficiency criteria. However, in terms of relative yield, two current commercial canola 2
genotypes (Surpass 600 and 46C74) were rated as efficient based on the growth at low vs 3
adequate nitrogen (Figure 3.3) or sulphur nutrient supply (Figure 3.4), and therefore could 4
be used as a reference for future breeding programs. 5
6
3.5 Conclusions 7
8
Nitrogen and sulphur efficiency of canola genotypes can be differentiated in the vegetative 9
growth stage. Identification of nitrogen- and sulphur-efficient genotypes in early growth 10
stages for selection purposes would lead to breeding genotypes that take up more nitrogen 11
and sulphur and utilise these nutrients better to produce higher yield and quality under 12
conditions of restricted availability of these nutrients. 13
14
3.6 Acknowledgements 15
16
The canola seed was contributed from Department of Agriculture and Food Western 17
Australia (South Perth and Northam), Canola Breeders Western Australia (Shenton Park), 18
and Australian Temperate Field Crops Collection (Horsham). The financial support from 19
the Australian Research Council is gratefully acknowledged. 20
21
Figure 3.A First screening experiment 22
58
3.7 References 1
2
Ahmad A, Abdin NZ (2000) Photosynthesis and its related physiological variable in 3
the leaves of Brassica genotypes as influenced by sulphur fertilization. 4
Physiologia Plantarum 110, 144-149. 5
Ahmad A, Ishrat K, Abdin NZ (2000) Effect of sulfur fertilisation on oil accumulation, 6
acetyl-CoA concentration, and acetyl-CoA carboxylase activity in the developing 7
seeds of rapeseed. Australian Journal of Agricultural Research 51, 1023-1029. 8
Ahmad A, Khan I, Nasar AA, Yash PA, Iqbal M (2005) Role of sulphate transporter 9
system in sulphur efficiency of mustard genotypes. Plant Science 169, 842-846. 10
Anderson JW, Fitzgerald MA (2001) Physiological and metabolic origin of sulphur 11
for the synthesis of seed storage proteins. Journal of Plant Physiology 158, 447-12
456. 13
Anon. (1979) Technicon Industrial Method 334-74W/B+, Technicon Industrial Systems, 14
Tarryown, N.Y. USA. 15
Brennan RH, Gartrell JW, Robson AD (1980) Reactions of copper with soil affecting its 16
availability to plants. I. Effect of soil type and time. Australian Journal of Soil 17
Research 18, 447-89. 18
Carmody P, Walton G (2003) A decade of development for the canola industry in Western 19
Australia. In Proceedings of the 11th
International Rapeseed Congress, Copenhagen, 20
Denmark pp. EO1: economics. 21
Cowling AW (2007) Genetic diversity in Australian canola and implications for crop 22
breeding for changing future environments. Field Crops Research 104, 103-111. 23
Fageria NK, Baligar VC (1997) Phosphorus-use efficiency by corn genotypes. Journal 24
of Plant Nutrition 20, 1267-1277. 25
Hawkesford MJ, De Kok LJ (2006) Managing sulphur metabolism in plants. Plant, 26
Cell &Environment 29, 382-395. 27
Hocking PJ, Pinkerton A, Good A (1996) Recovery of field-grown canola from sulfur 28
deficiency. Australian Journal of Experimental Agriculture 36, 79-85. 29
Holmes MRJ 1980 Nutrition of the Oilseed Rape Crop. Applied Science Publisher, 30
London, UK. 31
59
Kessel B, Becker HC (1999) Genetic variation of nitrogen-efficiency in field 1
experiments with oilseed rape (Brassica napus L.). In ‘Plant Nutrition – 2
Molecular Biology and Genetics’, Gissel-Nielsen G and Jensen A (Eds). pp. 3
391-395. Kluwer Academic Publishers, Dordrecht, The Netherlands. 4
Lancashire PD, Bleiholder H, Vandenboom T, Langeluddeke P, Stauss R, Weber E, 5
Witzenberger A (1991) A uniform decimal code for growth stages of crops and 6
weeds. Annals of Applied Biology 119, 561-601. 7
Maman N, Mason SC, Galusha T, Clegg MD (1999) Hybrid and nitrogen influence on 8
pearl millet production in Nebraska: yield, growth, and nitrogen uptake, and nitrogen 9
use efficiency. Agronomy Journal 91, 737-743. 10
Masson MG, Brennan RF (1998) Comparison of growth response and nitrogen uptake by 11
canola and wheat following application of nitrogen fertilizer. Journal of Plan 12
Nutrition 21, 1483-1499. 13
McGrath SP, Zhao FJ (1996) Sulphur uptake, yield responses and the interaction between 14
nitrogen and sulphur in winter oilseed rape (Brassica napus). Journal of 15
Agricultural Science 126, 53-62. 16
McQuaker NR, Brown DF, Klucker PD (1979) Digestion of environmental materials for 17
analysis by inductively coupled plasma-atomic emission spectrometry. Analytical 18
Chemistry 51, 1082-1084. 19
Mollers C, Bolune T, Wallbraun M, Lohaus G (1996) Protein content and amino acid 20
composition of the seeds and the phloem sap of one Brassica carinata and different 21
B. napus L. genotypes. In Proceedings of the Symposium Breeding for Oil and 22
Protein Crops, Zaporozhye, Ukraina. pp.148-154. 23
Osborne LD, Rengel Z (2002a) Genotypic differences in wheat for uptake and utilization of 24
P from iron phosphate. Australian Journal of Agricultural Research 53, 837-844. 25
Osborne LD, Rengel Z (2002b) Growth and P uptake by wheat genotypes supplied with 26
phytate as the only P source. Australian Journal of Agricultural Research 53, 845-27
850. 28
Osborne LD, Rengel Z (2002c) Screening cereals for genetic variation in efficiency of 29
phosphorus uptake and utilization. Australian Journal of Agricultural Research 53, 30
295-303. 31
Pluske WM, Osborne LD (2001) ‘Canola Symptoms of Nutrient Deficiency’ (Westfarmers 32
CSBP Ltd, Kwinana Western Australia) 33
60
Rengel Z, Graham R D (1995) Wheat genotypes differ in Zn efficiency when grown in 1
chelate-buffered nutrient solution. Plant Soil 176, 307-316. 2
Rengel Z, Hawkesford MJ (1997) Biosynthesis of a 34-kDa polypeptide in the root-cell 3
plasma membrane of a Zn-efficient wheat genotype increases upon Zn deficiency. 4
Australian Journal of Plant Physiology 24, 307-315. 5
Robson AD, Osborne LD, Snowball K, Simmons WJ (1995) Assessing sulphur status in 6
lupins and wheat. Australian Journal of Agricultural Research 35, 79-86. 7
Rossato L, Laine P, Ourry A (2001) Nitrogen storage and remobilization in Brassica napus 8
L. during the growth cycle: nitrogen fluxes within the plant and changes in soluble 9
protein patterns. Journal of Experimental Botany 52, 1655-1663. 10
Schjoerring JK, Bock JGH, Gammelvind L, Jensen CR, Mogensen VO (1995) Nitrogen 11
incorporation and remobilization in different shoot components of field-grown 12
winter oilseed rape (Brassica napus L.) as affected by rate of nitrogen application 13
and irrigation. Plant and Soil 177, 255-264. 14
Seiffert B, Ecke W, Lohaus G, Wallbraun M, Zhou Z, Mollers C (1999) Strategies 15
for the investigation of N-efficiency in oilseed rape. In Plant Nutrition – 16
Molecular Biology and Genetics, Gissel-Nielsen G and Jensen A (Eds.). pp 425-17
432. Kluwer Academic Publishers, Dordrecht, Netherlands. 18
Svecnjak Z, Rengel Z (2006a) Canola cultivars differ in nitrogen utilization efficiency at 19
vegetative stage. Field Crops Research 97, 221-226. 20
Svecnjak Z, Rengel Z (2006b) Nitrogen utilization efficiency in canola cultivars at grain 21
harvest. Plant and Soil 283, 299-307. 22
Yau SK, Thurling N (1987) Variation in nitrogen response among spring rape (Brassica 23
napus) cultivars and its relationship to nitrogen uptake and utilization. Field Crops 24
Research 16, 139-155. 25
Yuen SH, Pollard AG (1954) Determination of nitrogen in agricultural materials by the 26
Nessler reagent. II. Micro-determination in plant tissue and in soil extracts. 27
Preston Press Ltd, London, UK. 28
Zhao FJ, Evans EJ, Bilsborrow PE, Syers JK (1993) Influence of sulphur and nitrogen on 29
seed yield and quality of low glucosinolate oilseed rape (Brassica napus L). Journal 30
of Science Food and Agriculture 63, 29-37. 31
61
Chapter 4 Nitrogen efficiency of canola genotypes varies 1
between vegetative stage and grain maturity 2
3
4.1 Abstract 4
5
There is no information on whether N efficiency in canola at maturity can be reliably 6
determined by screening germplasm in the vegetative stage. Twelve canola genotypes 7
identified in preliminary screening study as having either high or low N efficiency indices 8
were tested for consistency in nitrogen efficiency between the vegetative stage and 9
maturity. Plants were grown in a glasshouse under low or adequate N supply and N 10
efficiency was assessed using the following criteria: dry weight at deficient N supply, 11
relative yield at low vs. adequate N supply, and nitrogen utilisation efficiency. None of the 12
12 tested genotypes was classified as efficient or inefficient under all three criteria. One 13
genotype (46C74) was classified as efficient under two criteria, and one genotype (Surpass 14
300 TT) was inefficient under two criteria. At maturity, three additional efficiency criteria 15
were used: harvest index, nitrogen harvest index, and oil and protein concentration in seed. 16
Two genotypes (Wesway and 46C74) (ranked as efficient at vegetative stage) remained 17
efficient at maturity under most of the efficiency criteria used. On the other hand, genotype 18
Surpass 603 CL ranked inefficient during the vegetative stage was ranked as efficient at 19
maturity under two criteria. Overall, there was little consistency in the N efficiency ranking 20
between vegetative stage and maturity in 12 tested genotypes. Screening canola germplasm 21
for N efficiency for breeding purposes would therefore require an assessment at maturity. 22
23
4.2 Introduction 24
25
Canola (Brassica napus), as the predominant oilseed crop in Australia, is grown on 1.4 26
million hectares annually. It is also the main oilseed crop in Western Australia, with 27
750,000 hectares grown annually (Seberry et al. 2010). Canola production in Australia is 28
constrained by poor soil fertility. In Western Australia, soils are among the most ancient and 29
therefore heavily leached in the world, and are particularly deficient in P and N (Moore 30
1998). 31
62
Selection and breeding of various crops has resulted in genotypes that respond to high 1
fertilizer applications. In contrast, traits necessary for growth under limited nutrient supply 2
have been suppressed or eliminated (Lynch 1998). Canola has a high demand for N 3
(Holmes 1980); therefore, high rates of N fertilizer are usually applied to canola in order to 4
obtain maximum seed yields, even though the contribution of fertilizer-derived N to total N 5
content is higher in vegetative than in reproductive shoot parts (Schjoerring et al. 1995). 6
Canola has high N uptake efficiency from soil, but relatively low N utilisation efficiency 7
(Rossato et al. 2001). Nitrogen uptake efficiency presents a quantitative measure of the 8
actual uptake of N by the plant in relation to the amount of the N added to the soil (Zapata, 9
1990). Nitrogen utilisation efficiency is calculated as total plant dry weight divided by N 10
content (Siddiqi and Glass 1981). Nitrogen efficiency decreases with an increase in N 11
supply (Maman et al. 1999). Thus, fine-tuned N nutrition together with identification of 12
more efficient genotypes could result in better use of N fertilizers (Mason and Brennan 13
1998) and may lead to an increase in crop productivity (Lynch 1998). 14
Genotypic differences in nutrient efficiency in various crops have been documented by 15
many authors. Differences in P efficiency were demonstrated in wheat (Triticum aestivum) 16
(Osborne and Rengel 2002) and maize (Zea mays) (Fageria and Baligar 1997). Differences 17
in S efficiency in mustard (Brassica juncea) (Ahmad 2005) and N efficiency in canola 18
(Brassica napus) (Yau and Thurling 1987, Kessel and Becker 1999, Svecnjak and Rengel 19
2006, Balint et al. 2008) have also been found. Most of these authors reported that 20
genotypic variations in utilization efficiency were more distinguishable under limited than 21
under adequate nutrient supply. 22
In a study of 70 German canola genotypes from four different types (breeding lines, 23
hybrids, and two resyntheses groups), significant differences were recorded in N efficiency, 24
with the N harvest index as the most important assessment criterion (Kessel and Becker 25
1999). However, genetic variation in N efficiency could be multi-factorial and may vary 26
with developmental stage (Seiffert et al. 1999). Factors such as total dry weight produced 27
per unit of N content at flowering, N uptake after flowering, total dry weight produced per 28
unit of N content of dropped leaves and N content of the seed have been used to assess N 29
efficiency. Differential N efficiency among canola genotypes is not well understood, but it 30
is likely that more than one mechanism is responsible for a given level of efficiency in a 31
particular genotype (cf. Rengel 1999, 2001). 32
63
Genotypic differences in N efficiency (based on dry weight and N accumulation in the 1
above-ground plant parts) during vegetative stage and grain maturity of 40 Australian 2
genotypes were recorded (Yau and Thurling 1987). More recently, the focus was on modern 3
Australian germplasm, including 84 canola genotypes (Svecnjak and Rengel 2006, Balint et 4
al. 2008); however, all these studies concentrated on N efficiency during the vegetative 5
stage. While it is advantageous for breeders to assess genetic differences in N efficiency 6
during vegetative growth (the earlier, the better), there is no information on whether such an 7
approach is likely to be relevant to N efficiency of canola genotypes at maturity. Hence, the 8
aim of the present study was to evaluate whether there is consistency in N efficiency from 9
the vegetative stage to physiological maturity in 12 canola genotypes shown in a 10
preliminary study to differ in N efficiency at vegetative stage. 11
12
4.3 Materials and methods 13
14
Six efficient and six inefficient canola genotypes were tested for N efficiency during 15
vegetative stage and at grain maturity. These chosen genotypes had either a high or low N 16
efficiency indices at vegetative stage in preliminary N screening study of 84 canola 17
genotypes (Balint et al. 2008). 18
A brown sandy soil (Uc4.22; Northcote 1971) was collected from a bushland site 15 19
km south-east of Lancelin (3156’S, 11520’E). Soil chemical characteristics of pH (H2O) 20
5.9, 2 % clay and 8 g/kg organic carbon with low availability of essential plant nutrients (N, 21
K, P, Mg, S, Zn and Cu) made this soil suitable for the present study. 22
The soil was air dried and sieved to 2 mm. Basal nutrient and treatment salt 23
solutions were added to soil, followed by air drying, thorough mixing and filling into plastic 24
pots lined with plastic bags (1,000 g of dry soil/pot). Basal nutrients were added at the 25
following rates (mg/kg soil): 65.8 N; 87.6 K; 20.9 P; 54.2 S; 92.7 Na; 40.9 Ca; 179.4 Cl; 26
7.10 Mg; 3.25 Mn; 2.85 Zn; 0.62 Cu; 0.12 B; 0.09 Co; and 0.08 Mo. Nitrogen was supplied 27
as NH4NO3. 28
The experiment was set up in a completely randomised block design with two 29
treatments [adequate N (control) and deficient N] in six replicates. The initial rate of N of 30
65.8 mg N/kg soil for the control and 16 mg N/kg soil for the deficient N treatment was 31
applied prior to sowing. Two subsequent N applications at 22 and 32 days after sowing (of 32
64
37.5 mg to control and 17.5 mg N/kg soil to deficient N treatment) brought the total N 1
applied to 140 mg N/kg soil for the adequate N treatment and 51 mg N/kg soil for the 2
deficient N treatment by the time of vegetative harvest. Total amount of N applied to plants 3
grown to grain maturity was 400 mg N/kg soil for the adequate and 180 mg N/kg soil for 4
the deficient N treatment. Most N was applied during the rosette and the flowering and/or 5
early grain filling stages. Potassium, S, P and micronutrients were reapplied at the initial 6
amounts at the rosette and the flowering stages. 7
The experiment was conducted in an evaporatively-cooled glasshouse in Perth as 8
described elsewhere (Balint et al. 2008) in early spring with average day/night temperatures 9
of 25/14 C. Canola genotypes were sown at ten seeds per pot in March and thinned to two 10
plants 8 days after sowing. Pots were watered with deionised water daily and weighed to 11
field capacity (10 % w/w) every second day. Insect pests were controlled using standard 12
chemical treatments as required. 13
One canola shoot in each replicate was harvested by cutting the plant just above the 14
soil surface 42 days after sowing when growth reduction and other N deficiency symptoms 15
were evident in most genotypes in the deficient N treatment. Shoot dry weight was 16
determined after drying in an oven at 65 C for 7 days. Nitrogen content was determined on 17
a 4-channel Autoanalyzer system, using modifications of manufacturer’s procedures (Anon 18
1979). After digestion with sulphuric acid and hydrogen peroxide, N was determined 19
colourimetrically by the indophenol blue method (Yuen and Pollards 1954). 20
The remaining plant in each replicate was harvested at grain maturity (early 21
maturing genotypes were Surpass 300 TT, Surpass 603 CL and Bugle; late maturing were 22
CBWA 005, Taparoo, 46C74 and Wesway). Dropped leaves were collected throughout the 23
growth period. Dry weights of stem, siliques and dropped leaves were determined after 24
drying in an oven at 65 C for 3 days. The N content in dropped leaves, stems and siliques 25
was measured as mentioned above. Oil and protein concentration in seed was determined 26
using near infrared spectrometry (NIR Systems 6500). 27
The N efficiency in vegetative stage and grain maturity was evaluated using the 28
following three criteria: 1) growth (assessed as dry weight) at deficient N supply; 2) dry 29
weight at deficient relative to adequate N supply; 3) N utilisation efficiency, expressed as 30
shoot (for vegetative stage) and/or seed (for maturity stage) dry weight per unit of N content 31
in shoots and/or seeds. In addition, the harvest index (HI) and N harvest index (NHI) 32
65
parameters were used for N efficiency evaluation at the grain maturity stage. Harvest index 1
was defined as the ratio of grain biomass to the total above-ground biomass at harvest. NHI 2
was defined as the ratio of N in grain to N in total above-ground biomass at harvest. 3
The N efficiency ranking (I, inefficient; M, medium efficient; E, efficient) was 4
calculated according to Rengel and Graham (1995); boundaries for the medium efficiency 5
interval were formulated by subtracting or adding the value of one standard error from the 6
median point of a particular efficiency criterion. The data were analysed by ANOVA using 7
GENSTAT 6.1 statistical program. 8
9
4.4 Results and discussion 10
11
Genotypic differences in N efficiency in canola have been documented for two Canadian 12
(Grami and La Croix 1977) and 40 Australian genotypes (Yau and Thurling 1987). In the 13
study of 70 German canola genotypes, N harvest index was chosen as the most important 14
assessment criterion (Kessel and Becker 1999). More recent work on differential N 15
efficiency in canola assessed four commercial cultivars (Svecnjak and Rengel 2006) and 84 16
genotypes (Balint et al. 2008). The reasons for the genotypic differences were related to 17
uptake, transport and/or utilisation of nutrients within the plant, but the underlying 18
mechanisms were not elucidated. 19
Plant growth is one of the most commonly assessed indicators of severity of stress 20
related to nutrient deficiency (Rengel 1999, 2001). In the present study, the first indicators 21
of N deficiency problems in canola shoots were visually detectable 20 days after sowing, 22
manifested by reduced growth and change in the leaf colour from dark to pale green 23
(chlorosis) only in plants grown under deficient nitrogen supply (51 mg N/kg of soil during 24
the vegetative stage). As the plants grew older, the deficiency symptoms became more 25
severe, with purple colouration developing on the upper surface of the leaves. Similar 26
deficiency symptoms in canola plants were reported by Pluske and Osborne (2001). The 27
extent of purple pigmentation under N deficiency varied among genotypes. 28
29
4.5 Vegetative stage 30
31
4.5.1 Effect of N supply on dry matter production 32
66
1
Canola genotypes differed considerably in growth and N uptake parameters. Shoot dry 2
weight was influenced by N treatment, but also by differences among genotypes, whereas 3
the N treatment x genotype interaction was not significant (Table 4.1). Dry matter 4
production under adequate N supply was approximately 3.5 times higher than under 5
deficient N supply (data not shown). In comparison, the amount of N applied in the 6
adequate N treatment was 2.8 times higher than in the deficient N treatment, indicating 7
higher N utilisation efficiency at deficient compared to adequate supply (Table 4.1). Similar 8
findings were reported by Yau and Thurling (1987) and Svecnjak and Rengel (2006). 9
Dry matter production under deficient N supply ranged between 550 and 900 10
mg/shoot (Figure 4.1). Three canola genotypes (Wesway, BLN 331 and Chikuzen) had dry 11
matter production above 750 mg/shoot in the deficient N treatment and were identified as 12
efficient. These genotypes had approximately 2-fold higher dry matter production than 13
genotypes CBWA 005, Eyre and Surpass 300 TT that were considered inefficient. A similar 14
range of growth differences in canola genotypes under limited N supply were reported by 15
Svecnjak and Rengel (2006). 16
200 350 500 650 800
Surpass 300 TT
Eyre
CBWA 005
Bugle
Surpass 603 CL
Westar
Hua You 1
Taparoo
46C74
Chikuzen
BLN 331
WeswayI M E
(mg/shoot)
17
Figure 4.1 Shoot dry weight of 12 canola genotypes grown at deficient nitrogen supply (51 18
mg N/kg soil) for 42 days. Genotypes are ranked in the order of decreasing nitrogen 19
efficiency (E, efficient; M, medium; I, inefficient). The boundaries for the medium 20
efficiency interval were created by subtracting or adding the value of one standard error to 21
the median point of the efficiency criterion. Black bars represent the most (46C74) and least 22
67
efficient genotypes (Surpass 300 TT) based on the combination of three N efficiency 1
criteria in the vegetative stage. 2
3
4
Table 4.1 Analyses of variance for dry weight, N concentration and content in shoot, and N 5
utilisation efficiency of canola genotypes in the vegetative stage. 6
7
Genotype
(G)
Nitrogen
treatment (N)
G x N
Shoot dry weight (mg/shoot) ** *** NS
N concentration in shoots (g N/kg shoot DW) *** *** **
N content in shoots (mg N/shoot) *** *** *
N-utilisation efficiency (g shoot DW/g plant N) *** *** *
NS: not significant; *, **, *** Significant at P=0.05, P= 0.01 and P=0.001, respectively. 8
9
10
4.5.2 Concentration of N in canola shoots 11
12
Nitrogen concentrations in shoots were significantly affected by the N treatment x genotype 13
interaction (Table 4.1). Nitrogen concentration in shoots of canola genotypes grown under 14
deficient N supply ranged from 16.5 (genotype 46C74) to 23.6 g N/kg shoot DW (genotype 15
CBWA 005) compared with plants grown under adequate N supply, with N concentration in 16
shoots ranging from 20.8 (genotype Chikuzen) to 32.2 g N/kg shoot DW (genotype Bugle). 17
Canola genotypes under adequate N supply (140 mg N/kg soil supplied during the 18
vegetative stage) accumulated 19 to 32 mg N/shoot, and only 20 % less (16 to 22 mg 19
N/shoot) under deficient N supply (51 mg N/kg soil during the vegetative stage). It is worth 20
noting again that the amount of N applied in the adequate N treatment was 2.8 times higher 21
than that applied in the deficient N treatment. Canola genotypes under adequate N supply 22
accumulated in shoots approximately 23 % of supplied N compared with the deficient N 23
treatment where plants utilised 50 % of the supplied amount. Genotypes Bugle and CBWA 24
005 (ranked as inefficient under all other efficiency criteria) accumulated significantly 25
higher amounts of N per shoot than genotypes 46C74, Wesway and Chikuzen (ranked as 26
efficient under most of the efficiency criteria) in the deficient N treatment (data not shown). 27
68
1
4.5.3 Dry matter production at deficient relative to adequate N supply 2
3
Dry matter production at deficient vs. adequate N supply (relative yield) was approximately 4
35 %. Genotypes with the relative yield higher than 47 % were rated efficient (Figure 4.2). 5
The efficient genotype 46C74 had small differences in dry matter production between 6
adequate and deficient N supply compared with inefficient genotype Surpass 300 TT for 7
which 3-fold differences in growth between the two N treatments were recorded. 8
20 40 60
Surpass 300 TT
Surpass 603 CL
Westar
Chikuzen
Eyre
CBWA 005
Taparoo
Hua You 1
Bugle
46C74
Wesway
M EI
(%)
9
Figure 4.2 Ranking of 12 canola genotypes based on the ratio between shoot dry weight at 10
deficient (51 mg N/kg soil) and adequate N supply (140 mg N/kg soil) after 42 days of 11
growth. For efficiency intervals (E, efficient; M, medium; I, inefficient) and genotypes 12
singled out with black bars see Figure 4.1. 13
14
4.5.4 Nitrogen utilisation efficiency 15
16
This index reflects how efficient canola genotypes were in converting N taken up into 17
organic matter. Significant differences were recorded in genotype x N treatment interaction 18
(Table 4.1), suggesting that genotypes that utilised N more efficiently under limited supply 19
were not necessarily the more efficient ones under adequate supply. For example, genotype 20
Wesway was superior to genotype Westar under N deficiency but not under N sufficient 21
69
supply (Figure 4.3), making these two genotypes valuable for a further study on 1
mechanisms of N efficiency. 2
The N utilisation efficiency ranged between 42 and 62 g shoot DW/g plant N (Figure 3
4.3). Two canola genotypes (46C74 and Chikuzen) were rated as efficient, with N 4
utilisation efficiency index above 60 g shoot DW/g plant N (Figure 4.3). 5
20 40 60
CBWA 005
Surpass 300TT
Bugle
Surpass 603 CL
Eyre
Wesway
Taparoo
Westar
Hua You 1
BLN 331
Chikuzen
46C74
M EI
(g shoot DW/
mg N in shoot)
6
Figure 4.3 Nitrogen utilisation efficiency [calculated as the amount of shoot dry weight 7
(DW) produced per unit of nitrogen in shoots) of 42-day-old canola genotypes grown at 8
deficient nitrogen supply (51 mg N/kg soil). For efficiency intervals (E, efficient; M, 9
medium; I, inefficient) and genotypes singled out with black bars see Figure 4.1. 10
11
Considerable genetic variation in N efficiency was identified in canola genotypes by 12
Yau and Thurling (1987) and Svecnjak and Rengel (2006). Increased efficiency of nutrient 13
use in plants may be due to an increasing rate of nutrient transport within the plant or 14
specific compartmentation in cells (Rengel and Graham 1995; Rengel and Hawkesford 15
1997); variations in these mechanisms may contribute to genotypic differences in canola 16
genotypes recorded in the present study. 17
18
4.5.5 Nitrogen efficiency based on all three criteria combined 19
20
70
None of the 12 tested genotypes was classified as efficient or inefficient under all three 1
criteria. One genotype was classified as efficient under two criteria (46C74) and one 2
genotype as inefficient under two criteria (Surpass 300 TT). 3
4
4.5.6 Conclusion 5
6
Canola genotypes significantly differed in N efficiency during vegetative stage. Differential 7
N efficiency of canola genotypes from the preliminary screening study was confirmed, with 8
six efficient and six inefficient genotypes registering such status based on at least one 9
efficiency criterion. If the efficiency ranking among 12 canola genotypes would remain the 10
same at grain maturity stage, screening a large number of canola genotypes during 11
vegetative stage would be sufficient for selection purposes in developing improved 12
genotypes for low-input canola production. 13
14
4.6 Grain maturity 15
16
4.6.1 Effect of N supply on growth 17
18
Symptoms of N deficiency were apparent during the period of rapid growth, during stem 19
elongation, and before and after flowering. Affected plants had pale green leaves. Flowering 20
in the deficient N treatment started approximately 10 days later than in the adequate N 21
treatment. Flowers in the deficient N treatment were pale creamy yellow, compared with 22
bright yellow flowers in the adequate N treatment. In the deficient N treatment there was 23
reduced silique set as well as some silique abortion. Also, leaves, stems and in some cases 24
siliques had purple colouration and the leaves were thick and small in size. The extent of 25
purple pigmentation varied among genotypes. 26
Canola genotypes differed considerably in growth and N uptake at grain maturity. 27
Dry matter of stems, leaves, siliques and seeds was strongly influenced by genotypic 28
differences. The N treatment effect was significant for dry weight of stems and seeds only. 29
Significant genotype x N treatment interaction for dry weight was recorded only for the 30
stems (Tables 4.2 & 4.3). Dry matter production of whole plants under adequate N supply 31
(400 mg N/kg soil for the whole growth cycle) was on average 20% higher than that under 32
71
deficient N supply (data not shown). In contrast, the amount of N applied in the deficient N 1
treatment was more than 2-fold lower (180 mg N/kg soil) in comparison with adequate N 2
supply. 3
4
Table 4.2 Analyses of variance for dry weight, N concentration and N uptake of stems, 5
siliques and dropped leaves of canola genotypes at maturity 6
7
Genotype
(G)
Nitrogen
treatment (N)
G x N
Stem
Stem dry weight (mg/plant) *** ** **
N concentration in stems (g N/kg stems DW) *** *** ***
N content in stems (mg N/stems) *** *** ***
Siliques
Siliques dry weight (mg/plant) * NS NS
N concentration in siliques (g N/kg siliques
DW)
*** *** ***
N content in siliques (mg N/siliques) *** *** ***
Dropped leaves
Dry weight of dropped leaves (mg/plant) NS NS NS
N concentration in dropped leaves (g N/kg DW) NS ** NS
N content in dropped leaves (mg N/leaves) NS *** NS
NS: not significant; *, **, *** Significant at P=0.05, P= 0.01 and P=0.001, respectively. 8
9
Seed yield at deficient N supply was approximately 35 % of that at adequate N 10
supply. Similar variations in seed production under deficient and sufficient N supplies were 11
reported by Svecnjak and Rengel (2006) for four canola genotypes. Seed production under 12
limited N supply ranged between 50 and 250 mg/plant (Figure 4.4A). Four canola 13
genotypes (Surpass 300 TT, Surpass 603 CL, Bugle and Wesway) produced more than 180 14
mg seeds/plant and were ranked efficient. These genotypes had approximately 2-fold higher 15
seed production than inefficient genotypes Chikuzen, Taparoo and Hua You 1 that produced 16
less than 74 mg seed/plant. 17
72
A)0 50 100 150 200 250
Chikuzen
Hua You 1
Taparoo
BLN 331
CBWA 005
Westar
Eyre
46C74
Wesway
Suprass 603 CL
Bugle
Surpass 300 TT
(mg/plant)
I M E
B)0 50 100 150 200
Westar
Eyre
Bugle
Hua You 1
Suprass 603 CL
Taparoo
46C74
Surpass 300 TT
BLN 331
CBWA 005
Chikuzen
Wesway
I M E
(%)
1
Figure 4.4 Seed dry weight (A) and ranking of 12 canola genotypes based on the ratio (B) 2
between total seed dry weigh at deficient (180 mg N/kg soil) and adequate nitrogen supply 3
(400 mg N/kg soil) at grain maturity. The boundaries for the medium efficiency interval 4
were formulated by subtracting or adding the value of one standard error to the median 5
point of the efficiency criterion. Black bars represent the most (Wesway) and least efficient 6
genotypes (Westar) based on the combination of N efficiency criteria at maturity. 7
8
9
Dry matter production of stems under deficient N supply ranged from 300 to 900 10
mg/plant. It was significantly influenced by genotype (P<0.001); Surpass 603 CL or CBWA 11
005 had considerably higher dry matter production than Surpass 300 TT or 46C74. Nitrogen 12
treatments also had a significant effect on dry matter production of stems (P<0.01). In the 13
present study, the significant interaction genotype x N treatment for dry matter production 14
was only recorded for stems. 15
Dry weight of dropped leaves under deficient N supply ranged from 10 to 40 16
mg/plant. No main effects (P=0.06 to 0.36) nor interaction (P=0.14) was significant 17
Dry matter production of siliques ranged from 98 mg/plant (Chikuzen) to 220 18
mg/plant (Surpass 300 TT) under deficient N supply. A significant effect on dry matter 19
production in siliques was recorded for the genotype treatment only. 20
21
73
4.6.2 Nitrogen concentration in dropped leaves, stems, siliques and seeds 1
2
Nitrogen concentration in siliques ranged from 4.5 to 10 g N/kg DW (BLN 331 < CBWA 3
005) at deficient N supply; and from 12.4 to 23.8 g N/kg DW (46C74 < Surpass 300 TT) at 4
adequate N supply. Concentration of N in stems was significantly affected by the N 5
treatment. It was also affected by the N treatment x genotype interaction, indicating 6
differential genotypic response at the two levels of N nutrition (Table 4.2). Concentration of 7
N in stems ranged from 13.7 to 26.7 under adequate and from 3.9 to 7.9 g N/kg DW under 8
deficient N supply. These concentrations of N under deficient N supply were lower than the 9
concentrations in shoots during the rosette stage (16 to 23 g N/kg). The majority of N 10
content from shoots was remobilized to seeds as the largest sink (around 70% of shoot N, 11
data not shown). These findings are consistent with Rossato et al. (2001) who suggested that 12
most of the N used for grain filling was derived from mobilization of N stored in vegetative 13
tissues (ie. shoots). 14
Canola genotypes grown under adequate N supply (400 mg N/kg soil during the whole 15
growth cycle) accumulated 19 to 32 mg N/stem, and only 20 % less (16 to 22 mg/stem) 16
under deficient N supply (180 mg N applied/kg soil) (data not shown, see Table 4.2 for 17
statistics). In contrast, N application in the adequate N treatment was 2.1 times higher than 18
in the deficient N treatment, suggesting that plants under adequate N supply were less 19
efficient users of N than plants supplied deficient amounts of N. 20
Genotype CBWA 005 accumulated significantly higher amount of N in stem than 21
genotypes 46C74, Wesway and Chikuzen under deficient N supply. Hence, genotype 22
CBWA 005 may not be efficient in remobilizing N towards the sink organs (seed). The two 23
groups of genotypes differing in the N content in stems might differ in the type and/or 24
amounts of amino acids involved in transporting N-containing compounds. The 25
predominant amino acid in canola phloem is glutamine (Mollers et al. 1996). However, 26
genotypes more efficient in remobilizing N might contain greater amounts or proportions of 27
amino acid asparagine, the more efficient N transporter with the N:C ratio of 0.5 compared 28
to 0.4 for glutamine (Seiffert et al. 1999). 29
Nitrogen concentration in dropped leaves of plants grown under deficient supply 30
ranged from 5.2 to 5.9 g N/kg DW; these values are similar to the ones obtained by 31
Svecnjak and Rengel (2006) and Hocking et al. (1997). Genotype Surpass 300 TT (ranked 32
74
as inefficient at vegetative stage) had the highest N concentration in dropped leaves 1
(making this genotype relatively inefficient due to N losses via leaf drop). In contrast, N 2
concentration in leaves at adequate N supply ranged from 9.8 to 14.2 g N/kg DW; these 3
values are higher than mentioned in the earlier reports (Svecnjak and Rengel 2006; Hocking 4
et al. 1997). This discrepancy might be due to higher amounts of N applied in our study 5
compared with the earlier reports, suggesting that canola could take up N in high amounts 6
when supplied luxuriously, but the remobilization of N from leaves remained low. 7
Under deficient N supply, canola seeds had N concentrations from 37.4 (Wesway) to 8
42.7 g N/kg DW (Westar), whereas seeds at sufficient supply had N concentration from 45 9
(CBWA 005) to 50.5 g N/kg DW (Surpass 300 TT). These differences in seed N 10
concentrations were proportionally smaller than differences in N applications, making 11
genotypes under deficient N supply more efficient in using available N compared with 12
genotypes in the adequate N treatment. Nitrogen content in seed under deficient N supply 13
ranged from 33 (genotypes BLN 331 and Hua You 1) to 100 mg N/seeds (Surpass 300 TT). 14
Nitrogen concentration in siliques under the adequate N supply ranged from 45 (BLN 15
331) to 93 g N/kg DW (genotype CBWA 005). Nitrogen content ranged from 5.3 (BLN 16
331) to 16.5 mg N/silique (Surpass 300 TT) (data not shown). 17
Nitrogen accumulation per whole above-ground plant under deficient N supply 18
ranged from 105 (Chikuzen) to 165 mg N/plant (Surpass 603 CL) (Figure 4.5). There was 19
no similarity among genotypes in amounts of N accumulated per plant parts. Genotypes 20
Hua you 1 and CBWA 005 lost more than Surpass 603 CL and Chikuzen by dropping off 21
N-rich leaves, suggesting poor N remobilization rate from leaves of Hua you 1 and CBWA 22
005 (Figure 4.5). Genotype Surpass 603 CL took up more N from the soil than any other 23
genotype under deficient N supply; interestingly, this genotype was ranked as inefficient at 24
the vegetative stage. Even though the largest N accumulation in canola shoots was reported 25
to occur during the flowering stage (Chamoro et al. 2002) or before anthesis (Hocking et al. 26
1997), the largest N accumulation in Surpass 603 CL occurred at the end of flowering (data 27
not shown). 28
75
0 20 40 60 80 100 120 140 160 180
Chickuzen
Hua You 1
Taparoo
BLN 331
Surpass 300 TT
Eyre
Westar
46C74
CBWA 005
Bugle
Wesway
Surpass 603 CL
Stem
Leaves
Siliques
Seeds
(mg N/plant)
1
Figure 4.5 Nitrogen content of stems, leaves, siliques and seed of 12 canola genotypes 2
grown at deficient nitrogen supply (180 mg N/kg soil) at grain maturity. 3
4
5
4.6.3 Growth at deficient relative to sufficient N supply 6
7
Relative seed yield at low N supply was 47 to 200 % of that at sufficient N supply. 8
Genotypes with relative seed yield higher than 112 % were rated efficient, whereas 9
genotypes with relative seed yield lower than 49% were recognized as inefficient (Figure 10
4.4B). Genotype Wesway (ranked as efficient under this assessment criterion) had smaller 11
differences in seed production between adequate and low N supply compared with 12
inefficient genotype Westar for which 3-fold differences in growth were observed between 13
the two N treatments. Genotypes Wesway and CBWA 005 yielded more seed under 14
deficient than under adequate N supply, even though seed yield of these two genotypes at 15
adequate N supply was lower than in other tested genotypes. 16
17
4.6.4 Nitrogen utilisation efficiency 18
19
Nitrogen efficiency in stem dry matter production was influenced by the main effects of 20
genotype (P<0.005) and N treatment (P<0.001). There was the significant genotype x N 21
treatment interaction (P<0.005) (Table 4.2). Nitrogen utilisation efficiency in stems under 22
76
deficient N supply ranged from 116 to 210 g DW/mg N. Genotypes with an efficiency index 1
above 189 g DW/mg N were ranked as efficient (ie. genotype Wesway). Three canola 2
genotypes (Westar, Taparoo and Chikuzen) had efficiency indices below 145 g DW/mg N 3
and were ranked as inefficient (data not shown). 4
Nitrogen efficiency in seed (seed dry weight per unit of N accumulated in the seed) was 5
influenced by the main effects of genotype (P<0.001), N treatment (P<0.001) and the 6
genotype x N treatment interaction (P<0.013) (Table 4.3). Nitrogen efficiency index in 7
seeds under sufficient supply ranged from 19.8 to 22 g DW/mg N. There was only a 17 % 8
difference between the two N treatments (indices under deficient supply ranged from 23.5 9
to 26.8 g DW/mg N). A similar decrease in N efficiency by increasing N nutrition rates 10
were reported elsewhere (Hocking et al.1997). Two canola genotypes (BLN 331 and 11
Wesway) were rated as efficient, with the N efficiency index above 25.7 g DW/mg N 12
(Figure 4.6). Other canola genotypes were ranked as medium efficient (Figure 4.6). 13
20 22 24 26 28
Westar
Hua You 1
Surpass 300 TT
Bugle
CBWA 005
Chickuzen
Surpass 603 CL
Taparoo
46C74
Eyre
Wesway
BLN 331
I M E
(g DW/mg N)
14
Figure 4.6 Nitrogen utilisation efficiency of seeds [calculated as the amount of seed dry 15
weight (DW) produced per unit of nitrogen in seeds] of canola plants grown at deficient 16
nitrogen supply (180 mg/kg soil). For efficiency intervals (E, efficient; M, medium; I, 17
inefficient) and genotypes singled out with black bars see Figure 4.4. 18
19
20
77
Table 4.3 Analyses of variance for seed dry weight, N concentration and content in seed and 1
N utilisation efficiency of canola genotypes at maturity. 2 3
Genotype
(G)
Nitrogen
treatment (N)
G x N
Seed dry weight (mg/plant) *** * NS
N concentration in seeds (g N/kg seeds DW) *** *** NS
N content in seeds (mg N/seed) *** *** NS
N-utilisation efficiency in seeds
(g DW of seeds/N in seeds)
*** *** *
Oil concentration in seed (g/kg) *** *** NS
Protein concentration in seed (g/kg) *** *** NS
NS: not significant; *, *** Significant at P=0.05 and P=0.001, respectively. 4
5
4.6.5 Harvest index and N harvest index 6
7
Harvest index under deficient N supply ranged from 2.9 to 18.5 %. Genotypes with a 8
harvest index above 15.4 % (Surpass 300 TT and Wesway) were ranked as efficient, 9
whereas genotypes with a harvest index below 8.1 % (Chikuzen and Taparoo) were ranked 10
as inefficient (Figure 4.7A). Two different N rates in this study did affect the harvest index 11
(data not shown). However, harvest index values in our study were lower than those 12
obtained by Svecnjak and Rengel 2006 (average 22 %) or Hocking et al. 1997 (27 to 34 %) 13
due to poor performance (low seed yield) of some genotypes (Chikuzen, Hua You 1 and 14
Taparoo). 15
Nitrogen harvest index ranged from 17.5 % (Chikuzen and Taparoo) to 73.4 % 16
(Surpass 300 TT and Bugle) for genotypes grown under deficient N supply (Figure 4.7B). 17
Nitrogen harvest index in canola is usually low because of dropping off N-rich leaves 18
(Rossato et al. 2001). In our study, genotypes Surpass 300 TT and Bugle had relatively high 19
seed yield and low dry matter production of leaves under deficient N supply that contributed 20
to higher nitrogen harvest index (compared to genotypes Taparoo and BLN 331 that had 21
poorer seed yield with a higher proportion of dry matter in dropped leaves). 22
78
A)0 5 10 15 20
Chikuzen
Taparoo
CBWA 005
BLN 331
Hua You 1
Westar
Eyre
Suprass 603 CL
46C74
Bugle
Wesway
Surpass 300 TT
I M E
(%) B)
0 20 40 60 80
Chickuzen
Taparoo
BLN 331
CBWA 005
Hua You 1
Westar
46C74
Eyre
Wesway
Surpass 603 CL
Bugle
Surpass 300 TT
I M E
(%)
1
2
Figure 4.7 Harvest index (A) [(seed DW/above-ground DW) x 100%] and nitrogen harvest 3
index (B) [(seed N content/above-ground N content) x 100%] of 12 canola genotypes 4
grown at deficient nitrogen supply. For efficiency intervals (E, efficient; M, medium; I, 5
inefficient) and genotypes singled out with black bars see Figure 4.4. 6
7
4.6.6 Oil and protein concentration in seed 8
9
10
Oil concentration in seed was influenced by the main effects of genotype (P<0.01) and N 11
treatment (P<0.01), but the genotype x N treatment interaction was non-significant (P=0.35) 12
(Table 4.3). In seed under deficient N supply oil concentration ranged from 435 to 475 g/kg 13
for different genotypes. Genotypes with oil concentration above 465 g/kg (Surpass 300 TT, 14
Surpass 603 CL, BLN 331 and Wesway) were ranked as efficient, whereas genotypes with 15
oil concentration below 448 g/kg (Westar and Hua You 1) were considered inefficient 16
(Figure 4.8A). On the other hand, oil concentration in seed under adequate N supply ranged 17
from 414 to 440 g/kg. 18
Protein concentration in seed was influenced by the main effects of genotype 19
(P<0.01) and N treatment (P<0.01), but the genotype x N treatment interaction was non-20
significant (P=0.18) (Table 4.3). Protein concentration in seed for the deficient N treatment 21
ranged from 234 to 264 g/kg. Genotypes with protein concentration above 262 g/kg (Westar 22
79
and Hua You 1) were ranked efficient, whereas genotypes with protein concentration less 1
than 247 g/kg (Wesway, Eyre, 46C74 and BLN 331) were ranked as inefficient (Figure 2
4.8B). Protein concentration for the adequate N treatment ranged from 282 to 315 g/kg. 3
A)400 420 440 460 480
Hua You 1
Westar
Taparoo
Chickuzen
46C74
Bugle
CBWA 005
Wesway
Eyre
BLN 331
Surpass 603 CL
Surpass 300 TT
I M E
(g/kg)
B)200 220 240 260 280
Wesway
BLN 331
46C74
Eyre
Taparoo
Surpass 603 CL
Chickuzen
CBWA 005
Surpass 300 TT
Bugle
Hua You 1
Westar
MI E
(g/kg)
4
Figure 4.8 Oil (A) and protein (B) concentration in seeds of 12 canola genotypes grown at 5
deficient nitrogen supply (180 mg N/kg soil). For efficiency intervals (E, efficient; M, 6
medium; I, inefficient) and genotypes singled out with black bars see Figure 4.4. 7
8
The association between oil and protein concentrations in seed is generally negative, 9
ie. if concentration of oil in seed increases, protein concentration decreases and vice versa 10
(Grami et al. 1997, Pritchard et al. 2000). In our study, two different N applications 11
produced the same negative correlation of protein and oil concentration in seed as explained 12
above (eg. high N application increased protein and decreased oil in seed of Wesway). 13
Under deficient N supply, this genotype produced seed with low protein, but high oil 14
concentration (data not shown). Similar findings were reported for different N applications 15
in the field (Brennan et al. 2000). The sum of oil and protein concentration in seed was 620 16
g/kg of field-grown canola in Victoria (Pritchard et al. 2000) and Western Australia 17
(Brennan et al. 2000). In our glasshouse study this sum was approximately 690 g/kg; this 18
value might be higher than that obtained by Pritchard et al. (2000) and Brennan et al. (2000) 19
due to an impact of favourable glasshouse conditions during the seed filling stage in 20
contrast to field conditions. 21
22
80
4.7 Conclusions 1
2
Canola genotypes differed significantly in N efficiency at grain maturity. Two genotypes 3
(Wesway and 46C74) (ranked as efficient at vegetative stage) remained efficient at maturity 4
under most of the efficiency criteria used. On the other hand, genotype Surpass 603 CL 5
(inefficient during vegetative stage) was ranked as efficient genotype at maturity under two 6
criteria. 7
Overall, there was little consistency in the N efficiency ranking from vegetative 8
stage to physiological maturity in 12 tested genotypes. Thus, vegetative-stage assessments 9
have poor predictive ability with respect to N efficiency at maturity and can not be used as 10
an efficient selection tool for N efficiency of canola cultivars. 11
12
4.8 Acknowledgements 13
14
We would like to thank Dr Zlatko Svecnjak for stimulating discussion and advice. The 15
canola seed was contributed by Department of Agriculture and Food Western Australia 16
(South Perth and Northam), Canola Breeders Western Australia (Shenton Park) and 17
Australian Temperate Field Crops Collection (Horsham). The financial support from the 18
Australian Research Council is gratefully acknowledged. 19
20
21
22
23
24
25
26
27
28
29
30
31
81
1 Figure 4.9 Nitrogen deficiency symptoms in canola shoots at vegetative stage (right pot). 2
3
4
5
6
7
8
9
10
11
12
13
14
82
4.9 References 1
2
3
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Balint T, Rengel Z, Allen D (2008) Australian canola germplasm differs in nitrogen an 8
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Brennan RF, Mason MG, Walton GH (2000) Effects of nitrogen fertilizer on the 10
concentration of oil and protein in canola (Brassica napus) seed. Journal of Plant 11
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Chamoro AM, Tamagno LN, Bezus R, Sarandon J (2002) Nitrogen accumulation, 13
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Maman N, Mason SC, Galusha T, Clegg MD (1999) Hybrid and nitrogen influence on 1
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Mollers C, Bolune T, Wallbraun M, Lohaus G (1996) Protein content and amino acid 7
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B. napus L. genotypes. In: Proceedings of the Symposium Breeding for Oil and 9
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Moore G (1998) Soil Guide: A Handbook for Understanding and Managing Agricultural 11
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Osborne LD, Rengel Z (2002) Screening cereals for genetic variation in efficiency of 16
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Pluske WM, Osborne LD (2001) ‘Canola Symptoms of Nutrient Deficiency’ (Westfarmers 19
CSBP Ltd Kwinana WA). 20
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Experimental Agriculture 40, 679-685. 23
Rengel Z (1999) Physiological mechanisms underlying differential nutrient efficiency of 24
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Implications. Z Rengel (ed) Binghamton, New York: The Haworth Press, pp 227-26
265. 27
Rengel Z (2001) Genotypic differences in micronutrient use efficiency in crops. 28
Communications in Soil Science and Plant Analyses 32, 1163-1186. 29
Rengel Z, Graham RD (1995) Wheat genotypes differ in Zn efficiency when grown in 30
chelate-buffered nutrient solution. Plant and Soil 176, 307-316. 31
Rengel Z, Hawkesford MJ (1997) Biosynthesis of a 34-kDa polypeptide in the root-cell 32
plasma membrane of a Zn-efficient wheat genotype increases upon Zn deficiency. 33
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Rossato L, Laine P, Ourry A (2001) Nitrogen storage and remobilization in Brassica napus 2
L. during the growth cycle: nitrogen fluxes within the plant and changes in soluble 3
protein patterns. Journal of Experimental Botany 52, 1655-1663. 4
Schjoerring JK, Bock JGH, Gammelvind L, Jensen CR, Mogensen VO (1995) Nitrogen 5
incorporation and remobilization in different shoot components of field-grown 6
winter oilseed rape (Brassica napus L.) as affected by rate of nitrogen application 7
and irrigation. Plant and Soil 177, 255-264. 8
Seberry DE, Mailer RJ, Parker PA (2010) The quality of Australian canola 2010. 9
Australian oilseed federation, Department of Primary Industries NSW. Volume 10
No. 16. 11
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investigation of N-efficiency in oilseed rape. In: Plant Nutrition – Molecular Biology 13
and Genetics, Gissel-Nielsen G and Jensen A (eds) Kluwer Academic Publishers, 14
Dordrecht, Netherlands, pp 425-432. 15
Siddiqi, KY, Glass ADM, Ruth TJ, Fernando M (1989) Studies of the regulation of the 16
nitrate influx by barley seedlings using 13
NO3-. Plant Physiology 90, 806-811. 17
Svecnjak Z, Rengel Z (2006) Canola cultivars differ in nitrogen utilization efficiency at 18
vegetative stage. Field Crops Research 97, 221-226. 19
Yau SK, Thurling N (1987) Variation in nitrogen response among spring rape (Brassica 20
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Research 16, 139-155. 22
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Nessler reagent. II. Micro-determination in plant tissue and in soil extracts. Journal 24
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Zapata F (1990) Isotope techniques in soil fertility and plant nutrition studies. In: Use of 26
nuclear techniques in studies of soil-plants relationships, International Atomic 27
Energy Agency, Vienna, pp 61-129. 28
29
30
31
32
33
85
Chapter 5 Differential sulphur efficiency in canola 1
genotypes at vegetative and grain maturity stage 2
3
5.1 Abstract 4
5
The breeding process can be facilitated if sulphur efficiency in canola at maturity can be 6
reliably determined by screening germplasm in the vegetative stage. Twelve canola 7
genotypes chosen from a preliminary screening study with either high or low S efficiency 8
indices were tested for consistency in S efficiency between vegetative stage and maturity. 9
Plants were grown under glasshouse conditions using low or adequate S supply. The criteria 10
for efficiency assessment at the vegetative stage were: shoot biomass at deficient S supply, 11
relative biomass production, and S utilisation efficiency. Considering these three criteria of 12
S efficiency only one canola genotype was classified as efficient (Surpass 402 CL). Three 13
genotypes were classified as inefficient under two criteria; genotypes IB 1337 and Surpass 14
300 TT for relative biomass production at low S supply and S utilisation efficiency and IB 15
1368 for biomass production and relative biomass production. 16
In addition to seed yield, relative seed yield and S utilisation, four additional 17
efficiency criteria were used for the maturity stage: harvest index, S harvest index, and oil 18
and protein concentrations in the seed. For all efficiency criteria used, highly significant 19
genotypic differences existed. IB 1368 was ranked efficient under all criteria. Genotypes 20
Surpass 402 CL and 46C74 were ranked inefficient at maturity but efficient at the 21
vegetative stage. In conclusion, screening canola germplasm for S efficiency requires an 22
assessment of the efficiency at maturity rather than during vegetative growth. 23
24
5.2 Introduction 25
26
Canola (Brassica napus) is the most widely grown oilseed crop in Australia. One of the 27
main constrains for a further increase in canola production in Australia is poor soil fertility, 28
particularly in Western Australia, where soils are predominantly sandy in texture, 29
moderately acidic, deficient in nitrogen, phosphorus and sulphur (Moore 1998) and prone to 30
leaching losses of S (Robson et al. 1995). 31
86
Sulphur is an essential nutrient needed for plant development; its concentration in 1
canola plants varies between 1 and 15 g/kg of dry weight depending on external supply. A 2
requirement for S in canola depends on the developmental stage. Shortage of S suppresses 3
the development of reproductive organs and may lead to silique abortion, resulting in 4
decreased seed yield (McGrath and Zhao 1996). Hence, S is one of the major nutrients 5
affecting canola growth and yield (Zhao et al. 1993, McGrath and Zhao 1996). 6
The S requirement depends on the plant species. A canola crop grown under UK 7
conditions has high S requirements (16 kg of S for 1 ton grain) compared with cereals (3 kg 8
of S for 1 ton of grain) (McGrath et al. 1996). Being susceptible to S deficiency, canola 9
responds well to S fertilization on low-S soils by increasing seed yield, as well as oil and 10
protein content (Hocking et al. 1996, McGrath and Zhao 1996, Ahmad and Abdin 2000). 11
Correcting S deficiency in plants only via fertilizers is not an economically and 12
environmentally favourable solution. Instead, identification and breeding of genotypes that 13
could acquire and use native soil S as well as fertilizer S more efficiently than standard 14
genotypes would be a sustainable long-term approach to dealing with (i) high S 15
requirements in canola and (ii) low content of S in Western Australian soils. Initial work on 16
screening of Australian canola germplasm for S efficiency (Balint et al. 2008) included 17
current commercial and some older genotypes and new breeding lines. In that study, 18
significant differences in S efficiency were recorded among canola genotypes during the 19
vegetative stage, but no reports could be found regarding S efficiency of Australian or any 20
other germplasm at the maturity stage. 21
The aim of the present study was to evaluate whether there is consistency in S efficiency 22
from the vegetative stage to grain maturity in 12 canola genotypes shown in a preliminary 23
study (Balint et al. 2008) to differ in S efficiency at the vegetative stage. The present study 24
also tried to elucidate some of the mechanisms responsible for transport and/or 25
remobilisation of S that may underlie differential S efficiency in canola. This information 26
will help in developing more efficient genotypes for low–input canola production on the 27
low-S Australian soils. 28
29
5.3 Materials and methods 30
31
87
Sulphur efficiency was tested in six efficient (Weswa, Argentina, IB 1368, Surpass 600, 1
46C74 and Surpass 402 CL) and six inefficient (IB 1363, CBWA 003, Karoo, Surpass 603 2
CL, Surpass 300 TT and IB 1337) canola genotypes ranked as such at the vegetative stage 3
in preliminary S screening experiment of 84 canola genotypes (Balint et al. 2008). These 4
genotypes comprised current commercial Australian genotypes (including some older ones), 5
new breeding lines and few yellow-seeded genotypes to increase genetic variability. The 6
yellow-seeded genotypes IB 1363, IB 1368 and IB 1337 are Brassica juncea, Indian 7
mustard genotypes with oil quality similar to that of standard canola varieties (eg. see 8
Norton et al. 2004). 9
A brown sandy soil (Uc4.22; Northcote 1971) was collected from a bushland site 15 10
km south-east of Lancelin (3156’S, 11520’E). Soil chemical characteristics of pH (H2O) 11
5.9, 2 % clay, 8 g/kg organic carbon and low levels of essential plant nutrients (N, K, P, 12
Mg, S, Zn, Cu) (Brennan et al. 1980) made this soil the standard one used for nutritional 13
studies at the University of Western Australia for more than three decades. 14
The experiment was conducted in Perth, Western Australia (3158’S, 11549’E) in 15
an evaporatively cooled glasshouse, as described elsewhere (Balint et al. 2008) in early 16
spring with average day/night temperatures of 25/14 C. In brief, the experiment was set 17
up in a randomized block design with two treatments [adequate S (control) and low S] in 18
five replicates. Basal mineral nutrients were added at the following rates (mg/kg soil): 65.8 19
N, 87.6 K and 20.9 P. Nitrogen was supplied as NH4NO3. The amounts of S added for 20
vegetative and grain maturity stages are presented in Table 5.1. 21
22
Table 5.1 Sulphur supply at vegetative and grain maturity stage 23
S supply, mg/kg soil (Na2SO4)
Treatments Vegetative stage
Additional for maturity stage Total
Control 54 mg 33 mg 87 mg
Low S 3 mg 3 mg 6 mg
24
25
The initial rate of S in both S treatments was applied pre-sowing and no further 26
application was made until vegetative harvest. The additional amount of S for plants grown 27
to grain maturity was applied during the flowering stage. Potassium, N, P and 28
88
micronutrients were reapplied at the initial rate (mg/kg soil): 87.6 K; 65.8 N; 20.9 P; 92.7 1
Na; 40.9 Ca; 179.4 Cl; 7.10 Mg; 3.25 Mn; 2.85 Zn; 0.62 Cu; 0.12 B; 0.09 Co; and 0.08 Mo 2
at the flowering and the initial silique filling stage. 3
Twenty seeds were sown per pot and thinned to two plants 10 days after sowing. 4
Pots were watered with deionised water daily and weighted to field capacity (10 % w/w) 5
every second day. Insect pests were controlled using standard methods as required 6
The vegetative harvest was done by cutting one canola shoot in each pot (roots were 7
not sampled) when S deficiency and growth reduction symptoms in the low-S treatment 8
were manifested in the majority of genotypes (day 48). Shoot dry weight was determined 9
after drying in an oven at 65 C for 7 days. Sulphur was determined by inductively coupled 10
plasma atomic emission spectroscopy (ICP-AES) after digestion with a mixture of nitric and 11
perchloric acids (McQuaker et al. 1979). Oil and protein concentration in seed was 12
determined in intact seeds using near infrared spectrometry (NIR Systems 6500). 13
Harvest at the grain maturity stage was performed on the remaining plant in each pot 14
[ for early maturing genotypes IB 1368, IB 1363, IB 1337 and Surpass 300 TT at day 115, 15
and for late maturing Surpass 402 CL, Surpass 603 CL, 46C74 and Argentina at day145]. 16
Dry weight of stems and siliques was determined after drying in an oven at 65 C for 3 17
days. Sulphur content in stems and siliques was measured as mentioned above. 18
The S efficiency ranking (I = inefficient; M = medium efficient; E = efficient) was 19
calculated according to Rengel and Graham (1995); boundaries for the medium efficiency 20
interval were formulated by subtracting or adding the value of one standard error from the 21
median point of a particular efficiency criterion. The data were analysed by ANOVA using 22
GENSTAT 10.1 statistical program. 23
24
5.4 Results 25
26
5.4.1 Vegetative stage 27
28
The earliest symptoms of S deficiency in canola genotypes under deficient S supply (3 mg S 29
kg/soil) occurred approximately 21 days after sowing and included reduced plant growth 30
and leaf thickening. Affected plants had pale green leaves and showed pale yellowish 31
interveinal mottling and cupping of younger leaves. The purple pigmentation developed on 32
89
the underside of leaves as plants grew older, with the leaf margins rolled inwards, creating 1
tubular appearance. Pluske and Osborne (2001) described similar S deficiency symptoms in 2
canola. 3
Shoot dry weight was influenced by the main effects of genotype (P<0.01) and S 4
treatment (P<0.001), but the interaction genotype x S treatment was non-significant 5
(P=0.38) (Table 5.2). 6
7
8
Table 5.2 Analysis of variance for dry weight, S concentration in shoots, shoot S content, 9
and S utilisation efficiency of 12 canola genotypes at the vegetative stage 10
11
Genotype (G) Sulphur treatment (S) G x S
Shoot dry weight [g/plant] ** *** NS
S concentration in shoots *** *** *
S content in shoots NS *** NS
S utilisation efficiency *** *** ***
NS: not significant; *, **, *** Significant at P=0.05, P= 0.01 and P=0.001, respectively. 12
13
14
Dry matter production in genotypes supplied with the adequate amount of S ranged 15
from 1.8 (IB 1363 and CBWA 003) to 2.5 g/shoot (Surpass 600 and IB 1337), ~ 60-70 % 16
higher than under limited S supply (data not shown). The interaction S treatment x genotype 17
for dry matter production was significant, suggesting the genotypes differed in their 18
responses to the two levels of S supply (Table 5.2). Three genotypes (Wesway, Surpass 402 19
CL and Surpass 603 CL) were classified as S-efficient, whereas IB 1363 and IB 1368 were 20
classified as inefficient (Figure 5.1A). 21
90
A) B)
20 40 60 80 100
Surpass 300 TT
Wesw ay
IB 1368
IB 1337
IB 1363
Surpass 600
CBWA 003
Argentina
Surpass 603 CL
Karoo
Surpass 402 CL
46C74
DW at deficient/DW at
adequate S supply (%)
EMI
1
Figure 5.1 Shoot dry weight (A), relative shoot dry weight (adequate S supply = 100 %, B), 2
and S utilisation efficiency (C) of 12 canola genotypes grown at deficient S supply (3 mg 3
S/kg soil) for 48 days. Genotypes are ranked in the order of decreasing S efficiency (E, 4
efficient; M, medium; I, inefficient). The boundaries for the medium efficiency interval 5
were formulated by subtracting or adding the value of one standard error from the median 6
point of the efficiency criterion. Black bars represent the most (Surpass 402 CL) and the 7
least efficient genotypes (IB 1368) based on the combination of the three efficiency criteria 8
in the vegetative stage. 9
C) 10
91
1
2
Sulphur concentration in shoots under adequate S supply ranged from 6.3 (genotypes 3
Wesway and 46C74) to 7.7 g S/kg shoot DW (Surpass 300 TT and CBWA 003), whereas 4
plants grown under limited S supply had S concentration in shoots ranging from 1.0 5
(genotype Wesway) to 1.7 g/S kg shoot DW (genotype Surpass 300 TT) (data not shown). 6
Shoot S contents differed significantly only between the S treatments, whereas the 7
genotype effect and the interaction were non-significant (Table 5.2). Genotypes supplied 8
with adequate S accumulated 10-17 mg S/shoot, utilising equivalent of 25 % of fertilizer 9
added (data not shown). On the other hand, genotypes grown at the deficient supply 10
accumulated only 0.7-2 mg S/shoot utilising approximately 50 % of the added S. 11
Shoot biomass production under low relative to adequate S supply ranged between 12
46 and 92 % for various genotypes (Figure 5.1B). Three genotypes (46C74, Surpass 402 CL 13
and Karoo) were ranked as efficient whereas five (Surpass 300 TT, IB 1363, IB 1368, IB 14
1337 and Wesway) were ranked as inefficient (less than 53 % relative growth) (Figure 15
5.1B). 16
Sulphur-utilisation efficiency reflects the efficiency of canola genotypes in using S 17
taken up for biomass production (Figure 5.1C). Two canola genotypes were ranked as 18
efficient, with S utilisation efficiency index above 847 g shoot DW/mg shoot S. On the 19
other hand, three canola genotypes had S utilisation efficiency indices below 681 g shoot 20
DW/mg shoot S and were recognised as inefficient (Surpass 300 TT, IB 1337) (Figure 21
5.1C). 22
Considering all three criteria of S efficiency, one genotype (Surpass 402 CL) was 23
classified as efficient. Three genotypes were classified as inefficient under two criteria: 24
genotypes IB 1337 and Surpass 300 TT for relative biomass production and S utilisation 25
efficiency and genotype IB 1368 for biomass production and relative yield. 26
27
5.4.2 Grain maturity stage 28
29
In the low-S treatment, symptoms of S deficiency were visible during the period of 30
rapid growth, during stem elongation, and before and after flowering. Affected plants had 31
pale green leaves. Flowering started 7 days later than the flowering in the control (adequate) 32
S treatment. The flowers had creamy yellow colour vs bright yellow at adequate S, and 33
92
there was impaired setting of siliques as well as some siliques abortion. In addition, many 1
siliques that did set seeds had fewer seeds in them than plants of the control treatment. The 2
leaves of S-deficient plants were thicker and smaller in size; the leaves, stems and in some 3
cases siliques had purple colouration with the leaf margins rolled inwards, creating tubular 4
appearance. The extent of purple pigmentation varied among genotypes. 5
Dry matter production of stems, siliques and seeds was greatly different among the 6
genotypes (P<0.001) and between S treatments (P<0.001). The interaction genotype x S 7
treatment in dry matter production was significant in siliques (P<0.01) and seed (P<0.001), 8
but not in stems (P=0.14) (Table 5.3). 9
Table 5.3. Analysis of variance for dry weight, S concentration and S content, and S 10
utilisation efficiency of stems, siliques and seeds of 12 canola genotypes at maturity. 11
Analysis of variance for oil and protein concentration in seeds was for 10 canola genotypes 12
(remaining two genotypes did not yield any seed) 13
14
Genotype (G) Sulphur treatment
(S)
G x S
Stem
Stem dry weight [g/plant] *** *** NS
S concentration in stems [g S/kg DW] * *** NS
S content in stems [mg S/stem] *** *** NS
Siliques
Silique dry weight [g/plant] *** *** ***
S concentration in siliques [g S/kg DW] *** *** ***
S content in siliques [mg S/plant] *** *** ***
Seed
Seed dry weight [g/plant] *** *** **
S concentration in seeds [g S/kg DW] *** *** ***
S content in seeds [mg S/plant] *** *** **
S utilisation efficiency of seeds
[g DW of seed/mg S in seed]
*** *** ***
Oil concentration in seed [g/kg] *** *** ***
Protein concentration in seed [g/kg] *** *** NS
NS: not significant; *, **, *** Significant at P=0.05, P= 0.01 and P=0.001, respectively. 15
16
93
Dry matter production of stems at low S supply ranged from 2.3 (IB 1368) to 8.9 1
g/plant (Surpass 600) among the genotypes (data not shown). The ranking of canola 2
genotypes by dry matter production at the grain maturity stage remained similar to the 3
ranking for shoot biomass at the vegetative stage. The S applications in our study had a 4
significant impact on dry matter production of stems (Table 5.3). 5
Dry matter production of siliques was significantly different among genotypes 6
(P<0.001) and between S treatments (P<0.001), and the interaction genotype x S was 7
significant (P<0.001) (Table 5.3). The plants grown at low S supply produced 0.18-2.17 g 8
siliques/plant, whereas the control plants produced 1.24-3.93 g siliques/plant (data not 9
shown). 10
Seed yields at adequate-S supply ranged from 0.78 (Argentina) to 2.54 g/plant (IB 11
1337) and at limited-S supply from 0.05 to 1.62 g/plant (Figure 5.2A); two genotypes (IB 12
1368 and IB 1363) produced more than 1.06 g seeds/plant under limited S supply and were 13
ranked as efficient. It is worth noting that these two genotypes (IB1368 and IB 1363) had 14
yellow-coated seeds and were ranked as inefficient during the vegetative growth stage 15
(Figure 5.2A). 16
A)
Seed DW at deficient S supply
(g/plant)
0 0.4 0.8 1.2 1.6
Surpass 603 CL
Karoo
Surpass 402 CL
46C74
Wesw ay
CBWA 003
Surpass 300 TT
IB 1337
IB 1363
IB 1368
I M E
B) 17
94
Figure 5.2 Seed dry weight (A) and relative seed yield (adequate S supply = 100 %, B) of 1
12 canola genotypes grown at deficient S supply (6 mg S/kg soil) and sufficient S supply 2
(87 mg S/kg soil) till maturity. The boundaries for the medium efficiency interval were 3
formulated by subtracting or adding the value of one standard error from the median point 4
of the efficiency criterion. Black bars represent the most (IB 1368) and the least efficient 5
genotypes (Karoo) based on the combination of three efficiency criteria used at the maturity 6
stage. 7
8
The impact of S deficiency was less severe on stem biomass than on siliques and 9
seed biomass, suggesting an essential role of S in reproductive growth and/or processes. 10
The proportion of the reproductive tissues (siliques with seeds) in total plant dry matter was 11
significantly increased by S application (data not presented). Plants at low S supply had 12
smaller and pale yellow siliques with high tendency of abortion, leaving canola plants with 13
only a few fertile siliques at maturity. In contrast, plants supplied with adequate amount of 14
S developed healthy, seed-rich siliques. 15
Among 12 genotypes tested, two (Argentina and Surpass 600) did not yield any seed 16
due to an extreme impact of S deficiency. Genotypes with relative seed yield higher than 17
55.4 % were rated as efficient (Figure 5.2B). These genotypes had smaller differences in the 18
seed dry weight between low and adequate S supplies compared to inefficient genotypes. 19
The most efficient genotype IB 1368 had almost 9 times higher relative seed yield than the 20
inefficient genotype Karoo. Yellow-coated genotypes had a higher relative yield than the 21
other tested genotypes (Figure 5.2B). 22
Sulphur concentration in stems was significantly affected by S treatment (P<0.001) and 23
genotype (P<0.05) (Table 5.3). No significant genotype x S interaction was recorded 24
(P=0.08). Sulphur concentration in stems ranged from 1.6 (Surpass 600) to 3.2 g S/kg DW 25
(Argentina) under adequate S supply, and from 0.6 (eg. Surpass 603 CL, Surpass 600) to 0.7 26
g S/kg DW (eg. Wesway, Argentina) under low S supply (data not presented). Sulphur 27
concentration in stems at maturity was approximately 50 % of S concentration in shoots at 28
anthesis under low S supply. Similarly, S concentration in stems at maturity under adequate 29
S supply was ~ 65 % of that at anthesis, suggesting poor S remobilisation from stems to 30
seed regardless of S supply. 31
Canola genotypes with adequate S supply (87 mg S/kg soil) accumulated 7-15 mg 32
S/stem, and only 2.5 times less (1.7-6 mg/stem) under limited S supply (6 mg S/kg soil) at 33
grain maturity (Figure 5.3). 34
95
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0
Argentina
Surpass 300 TT
Karoo
CBWA 003
Surpass 603 CL
46C74
Surpass 402 CL
Surpass 600
IB 1363
Wesw ay
IB 1337
IB 1368
S content in stems, siliques and seeds at deficient S supply
(mg S/plant)
Stem
Silique
Seed
1
Figure 5.3 Sulphur content of stems, siliques and seed of 12 canola genotypes grown at 2
deficient S supply (6 mg S/kg soil) till maturity. 3
4
Genotype Surpass 603 CL (ranked as inefficient under all other efficiency criteria) 5
accumulated a significantly higher amount of S in stems (5.2 mg S/stem) than genotype IB 6
1337 (1.9 mg S/stem) at limited as well as adequate S supply (Figure 5.3) suggesting that 7
Surpass 603 CL had less efficient mechanisms controlling S remobilisation from the stem in 8
comparison with IB 1337. 9
Sulphur concentration in seeds was significantly affected by S treatment (P<0.001) 10
and genotype (P<0.001). The interaction genotype x S treatment was also significant 11
(P<0.001) (Table 5.3). Seeds of 10 canola genotypes grown at low S supply (Argentina & 12
Surpass 600 did not yield seed due to severe S deficiency) had S concentration of 1.9-4.0 g 13
S/kg DW (Wesway the lowest and IB 1337 the highest), whereas S concentration in seeds at 14
adequate supply was 4.9-12.4 g S/kg DW (Surpass 600 the lowest and IB 1337 the highest) 15
(data not shown). 16
Sulphur content in seeds under deficient supply ranged from 0.015 (Surpass 603 CL) 17
to 0.50 mg S/seed (IB 1363) (data not shown). It appears that seed does not represent a large 18
sink for S remobilisation in the canola genotypes with dark in contrast to those with yellow 19
seed coats (IB 1368, IB 1363 and IB 1337) (Figure 5.3). In five out of 10 genotypes, seed S 20
accounted for only 20 % of total S content in canola shoots (Figure 5.3). 21
96
Sulphur concentration in siliques in the low S treatment ranged from 0.5 (Wesway) 1
to 1.1 g S/kg siliques (46C74) and in the adequate S treatment from 1.8 (IB 1337) to 6.3 g 2
S/kg siliques (Surpass 300 TT) (data not presented). Sulphur accumulation in siliques at low 3
S ranged from 0.00062 (Wesway) to 0.0021 mg S/siliques (CBWA 003), whereas control 4
plants accumulated from 0.002 (IB 1368) to 0.015 mg S/siliques (CBWA 003) (data not 5
shown). 6
Sulphur efficiency in seed at low S supply ranged from 8 to180 g of dry weight/mg 7
shoot S (Figure 5.4) and was higher at low S compared with adequate supply (data not 8
shown) (Figure 5.4). 9
0 50 100 150 200
Surpass 603 CL
46C74
Surpass 402 CL
Karoo
Wesway
CBWA 003
Surpass 300 TT
IB 1337
IB 1363
IB 1368
S efficiency in seed production at deficient S
supply (g seed DW/mg S in shoot)
I M E
10
Figure 5.4 Sulphur utilisation efficiency of seeds (seed dry weight produced per unit of S 11
accumulated in shoots) of 12 canola genotypes grown at deficient S supply (6 mg S/kg soil). 12
For efficiency intervals (E, efficient; M, medium; I, inefficient) and genotypes singled out 13
with black bars see Figure 5.2. 14
15
Harvest index for genotypes at low S supply ranged from 0.26 (Surpass 603 CL) to 16
33 % (IB 1363) (Figure 5.5A) and sulphur harvest index from 1.9 % (Surpass 603 CL) to 17
63.7 % (IB 1363) (Figure 5.5B). These extreme intervals in ranges of harvest indices and S 18
harvest indices in genotypes at low S supply were due to the relatively large differences in 19
seed yield among genotypes (Figures 5.5A&5B). 20
97
A) B)
0 20 40 60 80
Surpass 603 CL
Surpass 402 CL
Karoo
46C74
Wesw ay
Surpass 300 TT
CBWA 003
IB 1368
IB 1337
IB 1363
S harvest index at deficient S supply
(% )
EMI
8
1
Figure 5.5 Harvest index (A) [(seed DW/shoot DW) x 100%] and S harvest index (B) [(seed 2
S content/shoot S content)] of 12 canola genotypes grown under deficient S supply (6 mg 3
S/kg soil). For efficiency intervals (E, efficient; M, medium; I, inefficient) and genotypes 4
singled out with black bars see Figure 5.2. 5
6
7
Three genotypes (IB 1363 IB 1368 and IB 1337) were ranked as efficient, with 8
indices higher than 20 % (harvest index) and higher than 52 % (S harvest index) (Figures 9
5.5A&5B). Genotypes with indices lower than 5 % (harvest index) and lower than 15 % (S 10
harvest index) were ranked inefficient (Figures 5.5A&5B). 11
Oil concentration in seed was significantly affected by genotype (P<0.001) and S 12
treatment (P<0.001), and the interaction genotype x S treatment was significant (P<0.001) 13
(Table 5.3). The oil concentration at low S supply ranged from 400 (Karoo) to 440 g/kg (IB 14
1368). Genotypes with oil concentration above 435 g/kg were ranked as efficient (IB 1368 15
and Surpass 300 TT) and those below 414 g/kg as inefficient (Karoo, Surpass 603 CL and 16
Wesway) (Figure 5.6A). Concentration of oil under adequate S supply ranged from 430 (IB 17
1363 and 46C74) to 460 g/kg (Surpass 402 CL and IB 1337) (data not shown). 18
19
98
A)
390 410 430 450
Karoo
Surpass 603 CL
Wesway
46C74
CBWA 003
Surpass 402 CL
IB 1337
IB 1363
Surpass 300 TT
IB 1368
Oil concentration in seed at
deficient S supply (g/kg)
I M E
B)
200 220 240 260 280 300
Surpass 402 CL
46C74
Surpass 603 CL
Wesway
Surpass 300 TT
Karoo
CBWA 003
IB 1368
IB 1363
IB 1337
Protein concentration in seed at
deficient S supply (g/kg)
I M E
1
Figure 5.6 Oil (A) and protein (B) concentrations in seeds of 10 canola genotypes grown at 2
deficient S supply (6 mg S/kg soil). For efficiency intervals (E, efficient; M, medium; I, 3
inefficient) and genotypes singled out with black bars see Figure 5.2. 4
5
6
Protein concentration in seed was significantly different among genotypes (P<0.001) 7
and between S treatments (P<0.001), but the interaction genotype x S treatment was non-8
significant (P=0.27) (Table 5.3). Protein concentration in seed under low S supply ranged 9
from 229 (Surpass 402 CL) to 279 (IB 1337) g/kg (Figure 5.6B). 10
The sum of oil and protein content in seed in genotypes under adequate S supply 11
ranged from 680 (46C74) to 750 g/kg (Surpass 603 CL and CBWA 003), whereas the sum 12
under deficient S supply ranged from 650 (Karoo) to 710 g/kg (IB 1368) (data not shown). 13
These differences caused by differential S supply were significant. 14
Considering all criteria of S efficiency at maturity stage (Figures 5.2A-5.6B), one 15
canola genotype (IB 1368) was classified as efficient. Genotype Karoo was classified as 16
inefficient under all efficiency criteria except one (protein concentration in seed), for which 17
it was medium efficient. 18
99
1
5.5 Discussion 2
3
Differences in S efficiency in mustard genotypes (Ahmad et al. 2005) or canola genotypes 4
(Balint et al. 2008) have been reported in the past. Such differential S efficiency among 5
canola genotypes is not well understood, but it is likely that more than one mechanism is 6
responsible for a particular level of efficiency in a particular genotype (cf. Rengel 1999). 7
Sulphur starvation enhanced the S uptake efficiency of canola genotypes in our 8
study (83% of soil S was taken up by plants in the low-S treatment compared with only 9
34% in the adequate-S treatment) as well as in previous published ones (eg. Massonneau et 10
al. 1997). Similar relationship between S supply and S uptake efficiency as was also 11
observed in a study with mustard genotypes (Ahmad et al. 2005) where differences among 12
genotypes were hypothesized to be due to differential S uptake kinetics at the root-cell 13
plasma membrane. These authors suggested the existence of biphasic transporter systems in 14
mustard, i.e. combination of high- and low-affinity transporters. Recent research on 15
Arabidopsis indeed showed that at least some high-affinity sulphate transporters are up-16
regulated by S starvation (Rouached et al. 2008). Even though no information as yet exists 17
about potential genotypic differences in the sulphate transporter expression and function, we 18
hypothesise that canola genotypes differing in S efficiency in our study may have 19
differential activity of sulphate transporters. 20
It is well documented (Hawkesford and De Kok 2006) that sulphate is the most 21
common S form for uptake and transport in the plant. Cysteine is involved in the regulation 22
of the sulphate transport from roots to shoots. The main source of S for the grain protein 23
synthesis was glutathione (a protein-S metabolite) derived from the flag leaf in wheat 24
(Anderson and Fitzgerald 2001). In the soybean, homoglutathione from the pod wall (the 25
sulphate metabolite) was the main source of S for developing grain (Anderson and 26
Fitzgerald 2001). In our study, canola genotypes with high S utilisation efficiency may 27
genetically differ in the biosynthesis or transport efficiency of S-containing compounds (eg. 28
cysteine, glutathione and homoglutathione) towards or within the shoot. Study of xylem and 29
phloem transport of S in canola genotypes differing in S efficiency is in progress to 30
elucidate possible differences in the amounts and dynamics of these compounds (see 31
chapter 6). 32
100
Canola growth at deficient S supply in studies by McGrath and Zhao (1996) and 1
Hocking et al. (1996) resulted in differences in seed yield of 20 % between deficient S10 and 2
adequate S40 supply (10 and 40 kg/ha S supply, respectively). In the study presented here, 3
genotypes Argentina and Surpass 600 did not produce any yield in the low S treatment, with 4
genotype Argentina being also the lowest seed-producing genotype under adequate S supply 5
(0.78 g/plant seed yield). For the remaining 10 genotypes seed yield at deficient S supply 6
was approximately 50 % of that at sufficient supply. The smaller differences between 7
deficient and adequate S supplies in the two published studies (Hocking et al. 1996, 8
McGrath and Zhao 1996) conducted in the field compared with our glasshouse study were 9
likely due: either to an increase in availability of organic S as a result of rising soil 10
temperature in the field experiments during the season; or due to uptake of sulphate-S (that 11
was leached to depth; (Janzen and Bettany 1984b) via a canola root system that can 12
penetrate more than 1 m down the soil profile by late flowering (Kirkegaard JA and 13
Hocking PJ, unpublished data). This increased S availability contributed to yield recovery at 14
deficient S supply. 15
Genotypes IB 1337, IB 1363 and IB 1368 in our study accumulated higher amounts 16
of S in seed than other genotypes (Figure 5.3). These three genotypes have yellow coated 17
seed. Further work is required to elucidate links between yellow seed coats and S 18
accumulation in the seed of canola genotypes. 19
Considerable genetic variation of S efficiency during vegetative stage was identified 20
in canola genotypes (Balint et al. 2008). Increased nutrient efficiency may be due to an 21
increased rate at which the nutrient is transported within the plant or compartmented in cells 22
(Rengel and Graham 1995, Rengel and Hawkesford 1997). It was hypothesized by 23
Lappartient and Touraine (1996) that glutathione is responsible for mediating responses to S 24
availability through demand-driven processes that involve the translocation to roots of 25
phloem-transported message that provides information about the nutritional status of canola 26
leaves. The S-efficient genotypes in our study might contain larger amounts of glutathione 27
and/or phloem transported messages compared to inefficient S genotypes. The 28
concentration or amounts of these compounds are most likely variable during the plant 29
development, contributing to differential S efficiency in given genotypes. Differential S 30
efficiency may also be due to differential remobilisation of sulphate reliant on differential 31
efficiency of transporters involved in remobilising vacuolar sulphate (cf. Hawkesford 2000). 32
101
Genetic modification of these transporters would be the answer to future improvement in S 1
efficiency in plants. 2
The association between oil and protein concentrations in seed is generally negative, 3
ie. if concentration of oil in seed increases, protein concentration decreases and vice versa 4
(Grami et al. 1997, Prichard et al. 2000). This association in our study under both S supplies 5
was not regular (in four cases, genotypes with high oil concentration also had the high 6
protein concentration). Similar results with increased concentrations of both components in 7
seeds were reported by Zhang et al. (2006) for yellow coated rapeseed. High protein and 8
high oil content in canola genotypes represents a desirable combination and should be 9
explored further in breeding. 10
11
5.6 Conclusion 12
13
Canola genotypes differed in S efficiency in the vegetative compared with maturity 14
stage. Genotype IB 1368 that ranked inefficient under two efficiency criteria during the 15
vegetative stage was ranked efficient under all criteria at maturity. On the other hand, two 16
canola genotypes (Surpass 402 CL and 46C74) that ranked as efficient under the two 17
criteria at the vegetative stage did not remain efficient at maturity under most efficiency 18
criteria. Overall, there was little consistency in the S efficiency ranking from the vegetative 19
stage to maturity in 12 tested genotypes. Hence, screening canola germplasm for S 20
efficiency for breeding purposes would therefore require an assessment of efficiency at 21
maturity. 22
23
5.7 Acknowledgements 24
25
The canola seed was obtained from Department of Agriculture and Food Western Australia 26
(South Perth and Northam), Canola Breeders Western Australia (Shenton Park), and 27
Australian Temperate Field Crops Collection (Horsham). The financial support from the 28
Australian Research Council is gratefully acknowledged. 29
30
31
102
1
Figure 5.7 Sulphur deficiency symptoms in canola plants at vegetative stage (1st pot from 2
the left- canola plant grown at adequate S supply) 3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
103
5.8 References 1
2
Ahmad A, Abdin NZ (2000) Photosynthesis and its related physiological variables in the 3
leaves of Brassica genotypes as influenced by sulphur fertilization. Physiologia 4
Plantarum 110, 144-149. 5
Ahmad A, Khan I, Nasar AA, Abrol YP, Iqbal M (2005): Role of sulfate transporter system 6
in sulfur efficiency of mustard genotypes. Plant Science 169, 842-846. 7
Anderson JW, Fitzgerald MA (2001) Physiological and metabolic origin of sulphur for the 8
synthesis of seed storage proteins. Journal of Plant Physiology 158, 447-456. 9
Balint T, Rengel Z, Allen D (2008) Australian canola germplasm differs in nitrogen and 10
sulphur efficiency. Australian Journal of Agricultural Research 59, 167-174. 11
Brennan RH, Gartrell JW, Robson AD (1980) Reactions of copper with soil affecting its 12
availability to plants. I. Effect of soil type and time. Australian Journal of Soil 13
Research 18, 447-489. 14
Grami B, LaCroix LJ (1977) Cultivar variation in total nitrogen uptake in rape. Canadian 15
Journal of Plant Science 57, 619-624. 16
Hawkesford MJ (2000) Plant responses to sulphur deficiency and the genetic manipulation 17
of sulphate transporters to improve S-utilization efficiency. Journal of experimental 18
botany 51, 131-138. 19
Hawkesford MJ, DeKok LJ (2006) Managing sulphur metabolism in plants. Plant Cell and 20
Environment 29, 382-395. 21
Hocking PJ, Pinkerton A, Good A (1996) Recovery of field-grown canola from sulphur 22
deficiency. Australian Journal of Experimental Agriculture 36, 79-85. 23
Lappartient AG, Touraine B (1996) Demand-driven control of root ATP sulfurylase and 24
SO42-uptake in intact canola: the role of phloem-translocated glutathione. Plant 25
Physiology 111, 147-157. 26
Massonneau AN, Cathala N, Grignon C, Davidian JC (1997) Effect of sulphate deficiency 27
on the plasma membrane polypeptide composition of Brassica napus. Journal of 28
Experimental Botany 48, 93-100. 29
McGrath S.P, Zhao FJ, Withers PJA (1996) Development of sulphur deficiency in crops and 30
its treatment. Proceedings of the Fertilizer Society No. 379. The Fertilizer Society, 31
Peterborough, UK. 32
104
McQuaker NR, Brown DF, Klucker PD (1979) Digestion of environmental materials for 1
analysis by inductively coupled plasma-atomic emission spectrometry. Analitical 2
Chemistry 51, 1082-1084. 3
Moore G (1998) Soil Guide: A Handbook for Understanding and Managing Agricultural 4
Soils. Bull. 4243. Agriculture Western Australia, Perth, Western Australia. 5
Pluske WM, Osborne LD (2001) Canola Symptoms of Nutrient Deficiency. Wesfarmers 6
CSBP Ltd, Kwinana, Western Australia. 7
Pritchard FM, Eagles HA, Norton RM, Salisbury PA, Nicolas M (2000) Environmental 8
effects on seed composition of Victorian canola. Australian Journal of Experimental 9
Agriculture 40, 679-685. 10
Rengel Z, Graham RD (1995) Wheat genotypes differ in Zn efficiency when grown in 11
chelate-buffered nutrient solution. Plant Soil 176, 307-316. 12
Rengel Z, Hawkesford MJ (1997) Biosynthesis of a 34-kDa polypeptide in the root-cell 13
plasma membrane of a Zn-efficient wheat genotype increases upon Zn deficiency. 14
Australian Journal of Plant Physiology 24, 307-315. 15
Rengel Z (1999) Physiological mechanisms underlying differential nutrient efficiency of 16
crop genotypes. In Mineral Nutrition of Crops: Fundamentals Mechanisms and 17
Implications. Z Rengel (ed.). Binghamton, New York: The Haworth Press, pp. 227- 18
265. 19
Robson AD, Osborne LD, Snowball K, Simmons WJ (1995) Assessing sulfur status in 20
lupins and wheat. Australian Journal of Agricultural Research 35, 79-86. 21
Rouached H, Wirtz M, Alary R, Hell R, Bulak Arpat A, Davidian JC, Fourcroy P, 22
Berthomieu P (2008) Differential regulation of the expression of two high-affinity 23
sulfate transporters, SULTR1.1 and SULTR1.2, in Arabidopsis. Plant Physiology 24
147, 897-911. 25
Zhang XK, Yang GT, Chen L, Yin JM, Tang ZL, Li JN (2006) Physiological differences 26
between yellow-seeded and black-seeded rapeseed (Brassica napus L.) with different 27
testa characteristics during artificial ageing. Seed Science and Technology 34, 373-28
381. 29
Zhao FJ, Evans EJ, Bilsborrow PE, Syers JK (1993) Influence of sulphur and nitrogen on 30
seed yield and quality of low glucosinolate oilseed rape (Brassica napus L). Journal 31
of Science Food and Agriculture 63, 29-37. 32
33
105
Chapter 6 Nitrogen and sulphur uptake and remobilisation 1
in canola genotypes with varied N- and S-use efficiency differ at 2
vegetative and maturity stages 3
4
5
6.1 Abstract 6
7
Eight canola genotypes chosen from a preliminary screening study with either high 8
or low N and/or S efficiency indices were tested for consistency in S and/or N efficiency 9
between vegetative stage and maturity. Soil labelling was used to assess N/15
N or S/34
S 10
uptake, whereas leaf-feeding/labelling technique was used to study transport and 11
remobilisation of N and S. Plants were grown under glasshouse conditions using deficient 12
or adequate N and/or S supply. Nitrogen and S uptake in plants was assessed using the 13
following criteria: growth, N and/or S concentration and 15
N and/or 34
S atom excess in 14
above-ground plant parts. Transport and remobilisation of N and S were assessed via the 15
same assessment criteria in plants 2 days (1st harvest) and 6 days after the commencement 16
of leaf-labelling (2nd
harvest). 17
Genotype Wesway was more efficient than Westar in taking up N during the 18
vegetative stage as well as in remobilizing N from leaves, stems and siliques to seeds at 19
maturity. Genotype Surpass 402 CL appeared to be more efficient than Karoo in taking up S 20
during the vegetative and particularly during the maturity stage, but was less efficient than 21
Karoo in remobilizing S from leaves and stems to siliques and seeds at maturity. Soil and 22
leaf labelling techniques using 15
N and 34
S appeared to be a useful tool for studying uptake, 23
transport and remobilization of N and S during the vegetative and maturity stages in canola 24
genotypes differing in N- or S-use efficiency. 25
26
6.2 Introduction 27
28
Canola (Brassica napus) is the most widely grown oilseed crop in Australia. One of the 29
main constrains for a further increase in canola production in Australia is poor soil fertility. 30
Soils in Australia and particularly in Western Australia are predominantly sandy in texture, 31
106
moderately acidic, deficient in nitrogen, phosphorus and sulphur (Moore 1998) and prone to 1
leaching losses of S (Robson et al. 1995). The selection and breeding of canola crops 2
resulted in genotypes that are responsive to high fertilizer applications. In contrast, the traits 3
necessary for growth under limited nutrient supply are likely to have been suppressed or 4
eliminated, resulting in genotypes with relatively poor N- and S-use efficiency. Hence, one 5
of the foci of the future development of Australian canola cultivars should become 6
improvement of N-use efficiency as suggested for European canola cultivars (Mollers et al. 7
1996; Mollers and Schierholt 2002). In addition, identification and breeding of genotypes 8
that could use native soil S as well as added fertilizer S more efficiently than standard 9
genotypes would be an environmentally-friendly long-term approach to dealing with both 10
the low S availability in Western Australian soils and the high S requirements of canola. 11
Considerable genetic variation in N efficiency of Australian canola genotypes was 12
identified by Yau and Thurling (1987). The more recent work on differential N efficiency in 13
four Australian commercial canola cultivars (Svecnjak and Rengel 2006) and a large 14
screening of 84 genotypes (Balint et al. 2008) suggested that the genotypic differences in 15
efficiency were related to uptake and/or transport and utilisation of N within the plant, but 16
the underlying mechanisms were not elucidated. Remobilisation of N was not studied in the 17
above-mentioned reports, but differences in N remobilisation could contribute to genotypic 18
differences in N-use efficiency (eg. in barley, Mickelson et al. 2003), particularly in canola 19
because most of N used for seed filling in canola was derived from mobilization of N stored 20
in vegetative tissues (Rossato et al. 2001, Gombert et al. 2010) particularly senescing leaves 21
(Malagoli et al. 2005; Diaz et al. 2008). 22
Genotypic differences in S efficiency during vegetative stage in mustard (Ahmad et al. 23
2005) and canola (Balint and Rengel 2009) have been reported. In the study of S uptake 24
efficiency in mustard (Ahmad et al. 2005), differences among genotypes were hypothesized 25
to be in differential S uptake kinetics at the root-cell plasma membrane. The authors 26
suggested the existence of biphasic transporter systems in mustard, i.e. a combination of 27
high-and low-affinity transporters. In canola, differential S efficiency among various 28
genotypes is not well understood (Balint and Rengel 2009). It is likely that not only uptake 29
and transport mechanisms, but also remobilization mechanisms play an important role in 30
genotypic differences in S-use efficiency. The S remobilization from vegetative tissues was 31
particularly prominent in low-N canola plants (Sunarpi and Anderson 1997). Significant S 32
107
remobilization from leaves to roots in low-S plants was also reported (Abdallah et al. 2010), 1
but that physiological adaptation was observed only during the vegetative growth. 2
The present study was aimed at elucidating differences in N and S uptake and 3
remobilization in canola genotypes differing in N- and S-use efficiency using stable 4
isotopes 34
S and 15
N at vegetative and maturity stages via soil and leaf labelling. 5
6
7
6.3 Materials and Methods 8
9
All experiments were performed using the brown sandy soil (Uc4.22; Northcote 10
1971) collected from a bushland site 15 km south-east of Lancelin (3108’S, 11529’E), air 11
dried and sieved to 2 mm. Soil chemical characteristics of pH (H2O) 5.9, 2 % clay and 8 12
g/kg organic carbon, with low levels of essential plant nutrients (N, K, P, Mg, S, Zn, Cu) 13
(Balint et al. 2008), made this soil suitable for the present study. 14
The seed of four canola genotypes for each N and S experiments were sown at ten 15
seeds per pot and thinned to four plants 8 days after sowing. Pots were watered with 16
deionised water daily and weighed to field capacity (10 % w/w) every second day. Insect 17
pests were controlled using standard chemical treatments as required. 18
All experiments were conducted in an evaporatively cooled glasshouse as described 19
elsewhere (Balint et al. 2008). The average day/night temperatures were 25/14 C for the 20
uptake experiments and 26/15 C for the remobilisation ones. 21
22
6.3.1 Uptake experiments (soil labelling) 23
24
6.3.1 A) Nitrogen uptake 25
26
Vegetative stage 27
Soil labelling technique was used to assess 15
N/N uptake in four canola genotypes 28
(Wesway, 46C74, Westar and Surpass 300 TT). They had either high or low N efficiency 29
indices at the vegetative stage in two preliminary N screening studies (Balint et al. 2008; 30
and Balint and Rengel 2008). 31
32
108
1
The basal nutrients were supplied to the soil at the following rates (mg/kg soil): 87.6 2
K; 20.9 P; 54.2 S; 92.7 Na; 40.9 Ca; 179.4 Cl; 7.10 Mg; 3.25 Mn; 2.85 Zn; 0.62 Cu; 0.12 B; 3
0.09 Co; and 0.08 Mo. The soil was labelled with 15
N-Urea (~10 atom % enriched, SerCon 4
Australia Pty Ltd, Fulham Gardens, South Australia). After addition of basal and treatment 5
salt solutions, soils were air dried, thoroughly mixed and filled into plastic pots lined with 6
plastic bags (5,000 g of air-dry soil/pot). 7
The amount of 15
N-urea applied was calculated to keep an average 15
N content of 2A 8
% 15
N excess in plant N during the growing period. The quantity of 15
N required (15
NR) to 9
achieve the particular target enrichment was estimated by using relevant data from 10
preliminary studies of canola genotypes (Balint et al. 2008; Balint and Rengel 2008) for the 11
percentage of N in dry matter and the total plant dry weight. The 15
N natural abundance in 12
plants was calculated using the formula by Unkovich et al. (1994b). 13
14
15NR = [(N % x DW/100) (2A %
15N)] -
15NNA 15
A % 15
NS 16
17
15NR –the quantity of
15N required to achieve the particular target enrichment 18
N % - percentage of N in dry matter 19
DW – total plant dry weigh 20
15NNA-
15N natural abundance in plants 21
A % 15
NS (enrichment used/supplied) 22
23
Nitrogen at the initial rate was supplied pre-sowing as 15
N-urea [CO(NH2)2] solution 24
at 60 mg N plus 6 mg 15
N/kg soil for the control treatment and 14.4 mg N plus 1.6 mg 25
15N/kg soil for the low N treatment. Two subsequent enriched
15N-urea applications (at 21 26
and 37 days after sowing) brought the total N applied to 126 mg N plus 14 mg 15
N/kg soil 27
for the control and 45.9 mg N plus 5.1 mg 15
N/kg soil for the low N treatment by the time of 28
vegetative harvest (46 days after sowing) (see Table 6.1A). 29
30
31
32
33
109
Table 6.1 Supplies of N as 15
N-urea (~10 atom % enrichment) (A) and supplies of S as 1
K234
SO4 (~20 atom % enrichment) (B) to canola plants in the experiments (the amounts for 2
mature stages include those applied in the vegetative stages) 3
4
(A)
Treatments
mg N supply/kg soil
Plants harvested in the vegetative stage
mg
mg N supply/kg soil
Plants harvested at maturity (grain)
Adequate N 66 360
Deficient N 16 120
(B) mg S supply/kg soil
Plants harvested in the vegetative stage
mg
mg S supply/kg soil
Plants harvested at maturity (grain)
Adequate S 50 87
Deficient S 4 8
5
6
Plants were harvested 46 days after sowing. Shoot dry weights were determined 7
after drying in an oven at 65C for 7 days. 15
N analyses of dry canola shoots employed 8
Dumas principles and a continuous flow system by a 20/20 Isotope Ratio Mass 9
Spectrometer (IRMS) and an ANCA-SL preparation system, manufactured by Europa 10
Scientific Ltd., Crewe, UK. Briefly, a sample in a tin capsule was combusted at temperature 11
1,700-1,800°C, forming NOx gases that were reduced at 650°C to produce N2 (Skrzypek 12
and Paul 2006; Paul et al. 2007). These yielded gases were carried through water, and CO2 13
was trapped in a stream of He, while nitrogen was introduced into the IRMS in transient 14
peaks. Isotopic compositions were reported in the standard δ-notation (e.g. Paul et al. 2007) 15
after normalization of raw isotopic data to the isotope reference scale. 16
17
Maturity stage 18
The total amount of N applied to plants grown to maturity was 324 mg N plus 36 mg 19
15N/kg soil for the control and 108 mg N plus 12 mg
15N/kg soil for the low N treatment 20
(see Table 1A). Most of the enriched 15
N was applied during the rosette, flowering and early 21
grain filling stages (56, 77 and 102 days after sowing). Potassium, S, P and micronutrients 22
were reapplied at the initial amounts at the rosette and the flowering stages (56 and 77 days 23
after sowing). 24
110
Genotypes Westar and Wesway were harvested at maturity. Dry weights as well as 1
the 15
N content of stem, siliques, seeds and dropped leaves were determined after drying in 2
an oven at 65C for 3 days. The methodology for 15
N content analyses in plant parts at 3
maturity was the same as described above. 4
5
6.3.1 B) Sulphur uptake 6
7
Vegetative stage 8
Soil labelling was used to assess S/34
S uptake in four canola genotypes (Karoo, 9
Surpass 402 CL, IB 1368 and Surpass 300 TT) with either high or low S efficiency indices 10
at the vegetative stage in two preliminary S screening studies (Balint et al. 2008, and Balint 11
and Rengel 2009). 12
The basal nutrients were applied to the soil at the following rates (mg/kg soil): 66 N; 13
87.6 K; 20.9 P; 92.7 Na; 40.9 Ca; 179.4 Cl; 7.10 Mg; 3.25 Mn; 2.85 Zn; 0.62 Cu; 0.12 B; 14
0.09 Co; and 0.08 Mo. The initial rate of 50 mg 34
S/kg soil for the adequate and 4 mg 34
S/kg 15
soil for the deficient S treatment (see Table 1B) was applied pre sowing, and no additional 16
applications of S were made by the time of vegetative harvest (46 days after sowing). 17
The soil was labelled with 34
S (~ 99 atom % enriched, SerCon Australia Pty Ltd) 18
which was converted quantitatively to sulphate using digestion procedure by HNO3 as 19
described by Zhao et al. (2001). One hundred milligrams of the 34
S label was weighed into a 20
Pyrex digestion tube, to which 2.96 mL of 2 M KNO3 was added to give sufficient (K+) to 21
accompany SO42-
produced. Five mL of fuming HNO3 was then added. Digestion was 22
carried out in a programmable heating block with the temperature rising slowly to 160o
C, 23
and was then kept at this level for 2 h. The tube was then cooled, and 3 mL of H2O2 (30 % 24
v/v) was added to remove any excess HNO3. The temperature was raised and maintained at 25
120o
C until the content was dry. After cooling, the contents were dissolved in deionized 26
water and transferred to a 250-ml volumetric flask (Zhao et al. 2001). 27
The amount of 34
S applied was calculated to keep an average 34
S content of 2 A % 28
34S excess in plant S during the growing period. The quantity of
34S required (
34SR) to 29
achieve the particular target enrichment was estimated by using relevant data from 30
preliminary studies of canola genotypes (Balint et al. 2008; Balint and Rengel 2009) for the 31
percentage of S in dry matter (S %) and the total plant dry weight (DW). The 34
S natural 32
111
abundance in plants (34
SNA) was calculated using the formula by Unkovich et al. (1994) (see 1
above for the 15
NNA). 2
The
34S analyses of dry canola shoots were undertaken by an Elemental Analyzer - 3
Isotopic Ratio Mass Spectrometer (EA-IRMS) (Sercon Ltd., Cheshire, UK). Tin capsules 4
containing reference or sample material plus vanadium pentoxide catalyser were loaded into 5
an automatic sampler. Samples were dropped into a furnace held at 1,080o C and combusted 6
in the presence of oxygen. Tin capsules flash-combust, rising the temperature in the region 7
of the sample to ~1,700o C. The combusted gases were then swept in a helium stream over 8
combusting catalyst (tungstic oxide/zirconium oxide) and through a reduction stage of high 9
purity copper wires to produce SO2, N2, CO2 and water. Water was removed using a 10
NafionTM
membrane. Sulphur dioxide was resolved from N2 and CO2 on a packed GC 11
column at the temperature of 45o C. The resultant SO2 peak entered the ion source of the 12
IRMS where it was ionized and accelerated. Gas species of different mass were separated in 13
a magnetic field then simultaneously measured on a Faraday cup universal collector array. 14
Analysis was based on monitoring of m/z 48, 49 and 50 of SO+ produced from SO2 in the 15
ion source. 16
17
Maturity stage 18
The total amount of 34
S applied to plants grown to maturity was 87 mg 34
S/kg soil 19
for the adequate and 8 mg 34
S/kg soil for the deficient S treatment (see Table 1B). Most of 20
the enriched 34
S was applied during the flowering stage (65 days after sowing). Nitrogen, K, 21
P and micronutrients were reapplied at the initial amounts at the rosette, flowering and 22
initial pod filling stages (51, 65 and 95 days after sowing). 23
Genotypes Karoo and Surpass 402 CL were harvested at maturity. Dry weights as 24
well as the 34
S content of stem, siliques, seeds and dropped leaves were determined after 25
drying in an oven at 65C for 3 days. The methodology for 34
S content analyses in plant 26
parts at maturity was the same as described above for the soil labelling at the vegetative 27
stage. 28
29
6.3.2 Remobilisation experiments (leaf labelling) 30
A) Nitrogen remobilisation 31
32
112
1
Vegetative stage 2
A leaf-feeding technique was used to study transport and remobilisation of N during 3
the vegetative stage in the same four canola genotypes (Westar, Wesway, Surpass 300 TT 4
and 46C74) as used in the soil labelling. Non-labelled urea was used as soil N fertilizer. The 5
rest of the basal nutrients and treatment salts solutions were added to the soil (7 kg of air-6
dry soil/pot) as described in soil labelling experiment. 7
Two canola shoots of each genotype in each treatment were labelled at the 8
vegetative stage (50 days after sowing) when growth reductions as well as N deficiency 9
symptoms were evident in the low N treatment. The amount of 15
N KNO3 (~60 % 10
enrichment) solution used was calculated to keep an average 15
N content of 2% 15
N excess 11
of plant N. After selection of plants with similar number and size of leaves, the tip of a fully 12
expanded leaf was immersed in a small plastic bag containing 2.0 mL of 1.75 % (w/v) 13
KNO3 solution 15
N ~60 % enriched. Similar technique by using plastic vials for leaf 14
labelling with 2.5% of ~99 % enriched 15
N-urea in wheat was described by Palta (1991). 15
Concentration of 1.75 % (w/v) KNO3 was estimated to be safe for leaf labelling to avoid the 16
leaf burning (Krogmeier et al. 1989; Bremner 1990). Each plastic bag was attached to the 17
leaf, and the micropore™
3M surgical tape (Smith and Nephew Pty Limited, West Perth, 18
Western Australia) was used around the edges to avoid evaporative losses. 19
Leaf tips were immersed into the labelled solution by 9:00 am and allowed to absorb 20
it for 2 days. At the end of time allocated for labelling (2 days), the first sampling was done 21
immediately after the bags and KNO3 solution were removed and the second sampling 4 22
days afterwards. Shoot dry weights were determined after drying at 65 C for 7 days. The 23
15N analyses of dry canola shoots (excluding the leaf used for labelling) employed the same 24
principles as described for 15
N analyses of canola shoots in the soil labelling experiment. 25
The N remobilization was estimated from the difference in the N content between the two 26
harvests. 27
28
29
Maturity stage 30
Leaf labelling at maturity was done using two genotypes, Westar and Wesway. The 31
initial and two subsequent N applications at the vegetative stage, amounts and intervals of N 32
113
applications to plants grown to maturity as well as K, S, P and micronutrients applications, 1
sowing and plant maintenance were as described in the soil labelling experiment. 2
Plants had four to five well-developed siliques with seeds at the time of labelling 3
(105 days after sowing). The amount of labelling solution per plastic bag was adjusted for a 4
smaller size of leaves at maturity (1 mL/plastic bag). Concentration of KNO3, length of 5
exposure as well as the harvest times were the same as described for leaf labelling at the 6
vegetative stage. The 15
N analyses of siliques, stems, seeds and leaves (excluding the leaf 7
used for labelling) were done as described for 15
N analyses in the soil labelling experiment. 8
9
6.3.2 B) Sulphur remobilisation 10
11
Vegetative stage 12
Leaf labelling was done in two canola genotypes: Karoo with low and Surpass 402 13
CL with high S efficiency indices at maturity in the preliminary S screening study (Balint 14
and Rengel 2009). Two shoots of each genotype in each treatment were labelled at the 15
vegetative stage (50 days after sowing) when growth reductions as well as S deficiency 16
symptoms were evident in the low S treatment. Initial and subsequent S applications were 17
done as explained in the soil labelling experiment. The amount of 34
S as K2SO4 (~99 % 18
enrichment) solution used was calculated to keep an average 34
S content of 1A% 34
S excess 19
of plant S. After selection of plants with a similar number and size of leaves, the tip of a 20
fully expanded leaf was immersed in a small plastic bag containing 2.0 mL of 1.50 % (v/v) 21
34S ~99 % enriched K2SO4 solution. Dilution used was safe for leaf labelling to avoid leaf 22
burning. The 34
S analyses of dry canola shoots (excluding the leaf used for labelling) were 23
done as described in the soil labelling experiment. 24
25
Maturity stage 26
The second leaf labelling was done at maturity using the same genotypes, Karoo and 27
Surpass 402 CL. Non-labelled K2SO4 was used as S fertilizer. The rest of the basal nutrients 28
and treatment salts solutions were added to the soil (7 kg of air-dry soil/pot) as described for 29
the soil labelling experiment. 30
Remaining plants of each treatment had four to five well-developed siliques with 31
seeds at the time of labelling (105 days after sowing). The methodology for 34
S leaf labeling 32
114
at maturity was the same as described above for 34
S leaf labelling at the vegetative stage, 1
except that only 1 mL of the labelling solution was used per bag. The 34
S analyses of 2
siliques, stems, seeds and leaves (excluding the leaf used for labelling) were the same as 3
described for the soil labelling experiment. 4
5
6.3.3 Statistics 6
7
Each experiment was set up in a completely randomised block design with two 8
treatments [adequate N or S (control) and low N or S] and two or four genotypes in three 9
replicates. The data were analysed by 2-way ANOVA using GENSTAT 10.1. Least 10
significant difference (LSD 0.05) was used to determine significant differences between 11
treatment means. For each ANOVA, standard error of the mean, significance and LSD 0.05 12
were reported. 13
14
6.4 Results 15
6.4.1 Uptake experiments (soil labelling) 16
A) Nitrogen uptake 17
18
Growth, N concentration and 15
N atom excess in above-ground plant parts 19
20
Vegetative stage 21
Dry matter production in shoots was influenced by N treatment only (P<0.01) (Table 22
6.2A), ranging from 0.53 (genotype 46C74) to 1.00 g/plant (Surpass 300 TT) at deficient N 23
supply. Dry matter production under adequate N supply was approximately 2 times higher 24
than under deficient N supply (data not shown). 25
Nitrogen concentration in shoots was influenced by the main effects of genotype and 26
N treatment and the interaction, indicating differential genotypic response at the two levels 27
of N nutrition (P<0.01) (Table 6.2A). Nitrogen concentration in shoots ranged from 13 to 28
14.5 g N/kg DW (Wesway<46C74) at deficient and 2-3 times that at adequate N supply 29
(31.9 to 39.2 g N/kg DW) (46C74 < Wesway) (Figure 6.1A). 30
31
115
A)
0
5
10
15
20
25
30
35
40
Wesw ay Westar Surpass 300
TT
46C74 Wesw ay Westar Surpass 300
TT
46C74
Genotypes and N treatments
N c
on
cen
trati
on
(g
/kg
DW
)
Shoots
(stems+ leaves)
Adequate N Deficient N
1
B)
0
10
20
30
40
50
Wesway Westar Wesway Westar Wesway Westar Wesway Westar
Genotypes and plant tissues
N c
on
cen
trat
ion
(g
/kg
DW
pla
nt
tiss
ue)
Adequate N
Deficient N
seeds leavesstemssiliques
2
3
Figure 6.1 Nitrogen concentration in shoots (stems + leaves) in the vegetative stage (A) and 4
different plant tissues at maturity (B) in canola genotypes grown at adequate and deficient N 5
supply (soil labelling). The error bars represent ±SE. 6
Note: Y-axis labels: Figure A): N concentration (g/kg DW). Figure B): N concentration 7
(g/kg DW plant tissue). 8
Nitrogen treatment had the major effect on 15
N atom excess in shoots (P<0.001) (Table 9
6.2A). 15
N atom excess in shoots at adequate N supply ranged from 3.18 (Westar) to 3.58 10
mg 15
N/shoot (Surpass 300 TT) and at deficient N from 0.44 (Surpass 300 TT) to 0.91 mg 11
15N/shoot (Westar) (data not shown). 12
13
116
Maturity stage 1
Dry matter production of stems, siliques and seeds was influenced by N treatment 2
only (Table 6.2B), whereas that of leaves by both, N treatment and genotype (Table 6.2B). 3
Compared to Westar, genotype Wesway had the higher dry matter production of all above 4
ground plant parts at adequate N supply. Wasway also had higher production of leaves and 5
stems at deficient N supply, where as Westar had higher dry matter production of seeds and 6
siliques at deficient N supply (data not shown). 7
Table 6.2 Analyses of variance for shoot dry weight and N parameters in the soil-labelling 8 experiments for plants in the vegetative stage (A) and at maturity (B) 9 10 (A) Genotype
(G)
Nitrogen
treatment (N)
G x N
Shoot dry weight [mg/shoot] ns *** ns
N concentration in shoots [g N/kg shoot DW] * *** ** 15N atom excess in shoots [mg 15N/shoot] ns *** ns
(B) Genotype
(G)
Nitrogen
treatment (N)
G x N
Leaves Dry matter production in leaves [g/leaves] * ** ns
N concentration in leaves [g N/kg leaves DW] * ** * 15N atom excess in leaves [mg 15N/leaves] ns * *
Stems
Dry matter production in stems [g/stems] ns *** ns
N concentration in stems [g N/kg stems DW] * ns ns 15N atom excess in stems [g 15N/stem] * ns ns
Siliques
Dry matter production in siliques [g/stems] ns *** ns
N concentration in siliques [g N/kg siliques DW] ns ns ns 15N atom excess in siliques [mg 15N/siliques] ns * ns
Seeds
Seed dry weight [g/plant] ns ** ns
N concentration in seeds [g N/kg seeds DW] ns ** ns 15N atom excess in seeds [mg 15N/plant] * * ns
ns: not significant; *, **, *** significant at P≤0.05, P<0.01 and P<0.001, respectively. 11
12
Nitrogen concentration in leaves was influenced by differences in genotypes, N 13
treatment and their interaction (Table 6.2B). Compared to the other genotype, Wesway had 14
higher N concentration in leaves at deficient N, and Westar at adequate N supply (Figure 15
6.1B). Nitrogen concentration in stems was influenced by genotypic differences only; 16
117
genotype Wesway had higher N concentration in stems than Westar at both N supplies 1
(Table 6.2B, Figure 6.1B). No significant differences were recorded for silique N 2
concentrations (Table 6.2B, Figure 6.1B). Nitrogen treatment had a significant effect on N 3
concentration in seeds, from around 33 g N/kg DW (Westar = Wesway) at deficient to 38 - 4
44 g N/kg DW (Westar < Wesway) at adequate N supply (Table 6.2B, Figure 6.1B). 5
Differences in seed N concentrations were considerably smaller than differences in N 6
supply, suggesting that genotypes were more efficient users of available N under deficient 7
compared to adequate N supply. 8
15
N atom excess in leaves was influenced by the main effects of N treatment and the 9
interaction (Table 6.2B). Compared to the other genotype, Wesway had higher 15
N atom 10
excess in leaves at deficient N and Westar at adequate N supply (Figure 6.2). Two canola 11
genotypes differed in 15
N atom excess in stems (P≤0.05) (Table 6.2B). Genotype Wesway 12
had the higher 15
N atom excess in stems compared to Westar at deficient N supply (Figure 13
6.2), suggesting greater 15
N remobilization from leaves to stems (Figure 6.2). 14
0
5
10
15
20
25
30
Wesway Westar Wesway Westar
Genotypes and N treatments
15N
ato
m e
xc
es
s (
mg
/tis
su
e)
seeds
siliques
stems
leaves
Deficient N Adequate N
15
Figure 6.2 15
N atom excess in plant tissues of canola genotypes grown to maturity stage – 16
(soil labelling). The error bars represent ±SE. 17
Note, Y-axis label: 15
N atom excess (mg/tissue) 18
19
15N atom excess in siliques was influenced by N treatment and ranged from 3.5 to 20
4.5 mg 15
N/plant (Westar < Wesway) at deficient and 2.6 to 5.1 mg 15
N/plant at adequate N 21
supply (Westar < Wesway) (Table 6.2B; Figure 6.2). 15
N atom excess in seeds was 22
influenced by the main effects of genotype and N treatment (Table 6.2B). Wesway had 23
118
higher 15
N atom excess in seeds than Westar at deficient N, suggesting greater N 1
remobilization from siliques to seeds in Wesway (Figure 6.2). 2
3
B) Sulphur uptake 4
Growth, S concentrations and 34
S atom excess in shoots 5
6
Vegetative stage 7
Sulphur deficiency symptoms (reduced plant growth, leaf thickening and purple 8
colouration on the underside of leaves) were in accordance to those reported for the same 9
genotypes in two earlier S screening studies of canola genotypes (Balint et al. 2008; Balint 10
and Rengel 2009). In the present study, dry matter production in four tested genotypes was 11
influenced by differences in genotypes only (P≤0.05) and not by differential S supply 12
(Table 6.3A). 13
Sulphur concentration in shoots of four tested genotypes was influenced by the 14
interaction (P<0.001), indicating differential genotypic response at the two levels of S 15
nutrition (Table 6.3A). Sulphur concentration ranged from 0.13 to 0.28 g S/kg DW (Surpass 16
300 TT < IB 1368) at deficient S supply and three times that at adequate S supply (Surpass 17
402 CL < Surpass 300 TT) (Figure 6.3A). 18
Sulphur treatment had the major effect on 34
S atom excess in shoots (P<0.008) 19
(Table 6.3A). 34
S atom excess in shoots ranged from 0.61 (Surpass 300 TT) to 3.0 mg 20
34S/shoot (Surpass 402 CL) at adequate S supply and from 0.21 (Surpass 300 TT) to 0.43 21
mg 34
S/shoot (IB 1368) at deficient S (Figure 6.3B). 22
119
A) 1
B)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Surpass 402 CL Karoo Surpass 300 TT IB 1368
Genotypes and S treatments
34 S
ato
m e
xcess [
mg
/sh
oo
t D
W]
Adequate S
Deficient S
2
Figure 6.3 Sulphur concentration in shoots (A) and 34
S atom excess in shoots (B) of canola 3
genotypes grown under adequate and deficient S supply in the vegetative stage (soil 4
labelling). The error bars represent ±SE. 5
Note, Y-axis labels: Figure A): S concentration (g/kg shoot DW); Figure B): 34
S atom 6
excess (mg/shoot DW) 7
8
9
10
11
Table 6.3 Analyses of variance for dry weight and S nutrient parameters in soil labelling 12
experiments in the vegetative stage (A) and at maturity (B) 13
14
15
120
(A) Genotype
(G)
Sulphur
treatment (S)
G x S
Shoot dry weight [mg/shoot] * ns ns
S concentration in shoots [g S/kg shoot DW] *** *** *** 34
S atom excess in shoots [g 34
S kg/shoot] ns ** ns 1 (B) Genotype
(G)
Sulphur
treatment (S)
G x S
Leaves Dry matter production in leaves [g/leaves] ns *** ns
S concentration in leaves [g S/kg DW] *** *** ** 34
S atom excess in leaves [mg 34
S/leaves] ns *** ns
Stems
Dry matter production in stems [g/stems] ns ** ns
S concentration in stems [g S/kg stems DW] ns *** ns 34
S atom excess in stems [mg 34
S/stem] ns ** ns
Siliques
Dry matter production in siliques [g/siliques] ns ** ns
S concentration in siliques [g S/kg siliques DW] ns *** ns 34
S atom excess in siliques [mg 34
S/siliques] ns *** ns
Seeds Seed dry weight [mg/plant] ns * ns
S concentration in seeds [g S/kg seeds DW] *** *** *** 34
S atom excess in seeds [mg 34
S/seeds] ns ** ns
ns: not significant; *, **, *** Significant at P≤0.1, P<0.01 and P<0.001, respectively. 2
3
Maturity stage 4
Sulphur concentration in leaves and seeds was influenced by genotype, S treatment 5
and the interaction (Table 6.3B). Genotype Surpass 402 CL had higher S concentration in 6
seeds at adequate but lower at deficient S supply than Karoo (Figure 6.4A). Sulphur 7
concentration in leaves was higher in Karoo than Surpass 402 CL under both S supplies 8
(Figure 6.4A). 9
The S treatment had a major effect on S concentration in stems and siliques (Table 10
6.3B). Genotype Surpass 402 CL had higher S concentrations in seed than genotype Karoo 11
at adequate S supply, but the reverse was measured at deficient S supply (Figure 6.4A). 12
34
S atom excess in leaves, stems, siliques and seeds was influenced by the S 13
treatment only (Table 6.3B) and ranged from 8.1 (Karoo) to 10.1 (Surpass 402 CL) mg 14
34S/plant tissues
at adequate S (Figure 6.4B) and from 0.58 (Karoo) to 0.63 (Surpass 402 15
CL) mg 34
S/plant tissues at deficient S (Figure 6.4C). 16
121
1
0
0.1
0.2
0.3
0.4
0.5
0.6
Plant tissues and S treatments
S c
on
ce
ntr
ati
on
(g
/kg
DW
pla
nt
tis
su
e)
Surpass 402 CL
Karoo
leaves stems stemsleaves siliquessiliques seedsseeds
Adequate S Deficient S
2
A) 3
4
B)
0
2
4
6
8
10
Surpass 402 CL Karoo
Genotypes and S treatment
34S
ato
m e
xces
s (m
g/tis
sue)
seeds
siliques
stems
leaves
Adequate S
5
122
C)
0
0.2
0.4
0.6
0.8
Surpass 402 CL Karoo
Genotypes and S treatment
34S
ato
m e
xces
s (m
g/tis
sue)
seeds
siliques
stems
leaves
Deficient S
1
Figure 6.4 S concentration (A) and 34
S atom excess in plant tissues at adequate S (B) and 2
deficient S (C) supply of canola genotypes grown to maturity (soil labelling). The error bars 3
represent ±SE (A) or +SE (B and C). 4
Note, Y-axis labels: in Figure A): S concentration (g/kg DW plant tissue); in Figures B) and 5
C): 34
S atom excess (mg/tissue) 6
7
6.4.2 Remobilisation experiments (leaf labelling) 8
A) Nitrogen remobilisation 9
Growth, N concentration and 15
N atom excess in shoots (leaves and stems) 10
11
Vegetative stage 12
Growth, N concentration and 15
N atom excess in leaves and stems in plants 2 days 13
(1st harvest) and 6 days after commencement of labelling (2
nd harvest) was affected by 14
genotype, N treatment and the interaction (data not presented). Genotype Wesway had the 15
highest leaf dry matter production under deficient N supply 2 days (2.62 g/plant) and 6 days 16
after commencement of labelling (2nd
harvest) (3.62 g/plant), whereas Surpass 300 TT had 17
the lowest dry matter production at the 1st (0.93 g/plant) and the 2
nd harvest (1.45 g/plant) 18
(data not shown). Genotype 46C74 had the lowest stem dry matter production at deficient N 19
supply at the 1st (1.37 g/stems) and the 2
nd harvest (1.39 g/stems), whereas genotype Westar 20
had the highest stem production at both harvests (data not shown). 21
Nitrogen concentration and the 15
N atom excess in leaves 2 days (1st harvest) and 6 22
days after commencement of labelling (2nd
harvest) were both affected by genotype, N 23
123
treatment and the interaction (data not presented). Genotype Wesway had the highest 15
N 1
atom excess in leaves at the 1st (1.23 mg
15N/plant) but lowest at the 2
nd harvest (1.03 mg 2
15N/plant) (Figure 6.5A). Genotype Westar had the highest
15N atom excess in stems at the 3
1st harvest (4.01 mg
15N/plant) and Wesway at the 2
nd (7.03 mg
15N/plant) (Figure 6.5A). 4
5
Growth, N concentrations and 15
N atom excess in plants 2 days (1st harvest) 6
and 6 days after the commencement of labelling (2nd
harvest) 7
8
Maturity stage 9
Nitrogen concentrations in various plant tissues in two tested genotypes differed 10
significantly (Table 6.4). Genotype Wesway had higher N concentration (10.4 vs. 4.1 g 11
N/kg DW) and dry matter production in leaves (3.80 vs. 2.45 g/plant) than Westar under 12
deficient N supply at the 1st harvest (data not shown). Genotype Westar in contrast had 13
higher N concentrations in stems than Wesway under deficient N supply at both harvests 14
(data not shown). Compared with Westar, N concentrations in Wesway were higher in 15
siliques but lower in seeds at deficient N supply at both harvests (data not shown). 16
17
Table 6.4 Analyses of variance for N concentration and 15
N atom excess in plant tissues 2 18
days (1st harvest) and 6 days (2
nd harvest) after leaf labelling commenced (maturity stage) 19
20
(1st harvest) (2
nd harvest)
Genotype
(G)
(N)
trtratment
G x N
Genotype
(G)
(N)
treatment
x
G x N
Leaves N concentration in leaves [g N/kg leaves DW] ns *** *** *** ** ***
15N atom excess in leaves [mg
15N/leaves] ns *** *** ** *** ***
Stems N concentration in stems [g N/kg stems
DW] *** ** *** ** *** **
15N atom excess in stems [mg
15N/stems] * *** ** ** *** **
Siliques N concentration in siliques [g N/kg
silique
DW] *** *** *** *** *** ns
15N atom excess in siliques [mg
15N/siliques] ** * ** *** ns **
Seeds N concentration in seeds [g N/kg seeds DW] *** * *** *** ns ** 15
N atom excess in seeds [mg 15
N/seeds] ** ** ** *** ns ***
ns: not significant; *, ** , *** significant at P≤0.05, P<0.01 and P<0.001, respectively. 21
124
0
2
4
6
8
10
Wesw ay Westar 46C74 Surpass 300 TT
Genotypes and duration of the labelling
15 N
ato
m e
xc
es
s (
mg
/tis
su
e)
stems
leaves
6 days after labelling started- 2nd harvest
1
A) 2
0
2
4
6
8
10
12
14
16
18
20
Wesway
1st harvest
Wesway
2nd harvest
Westar
2nd harvest
Genotypes, duration of the labelling and N treatment
15 N
ato
m e
xcess (
mg
/tis
su
e)
seeds
siliques
stems
leaves
Deficient N
3
B) 4
Figure 6.5 15
N atom excess in plant tissues 6 days (2nd
harvest) after labelling started at 5
vegetative stage (A) and 2 days (1st harvest) and 6 days (2
nd harvest) after labelling started 6
at maturity (B) in canola genotypes grown under deficient N supply – (leaf labelling). The 7
error bars represent +SE. 8
Note, Y-axis labels in Figure A) and B): 15
N atom excess (mg/tissue). 9
10
125
A higher 15
N atom excess in leaves was found in Westar than Wesway at deficient N 1
supplies at both harvests (Figure 6.5A). Compared with Westar, genotype Wesway had a 2
significantly higher 15
N atom excess in stems at deficient N at the 1st harvest (Figure 6.5B). 3
The 15
N atom excess in siliques in Wesway under deficient N supply was 4
significantly lower at the 1st than the 2
nd harvest, suggesting intense N remobilisation from 5
vegetative to generative plant parts (Figure 6.5B). Under deficient N supply, genotype 6
Wesway had a higher 15
N atom excess in seeds than Westar at both harvests (Figure 6.5B). 7
8
B) Sulphur remobilsation 9
10
S concentrations and 34
S atom excess in shoots 2 days (1st harvest) and 6 days 11
after commencement of labelling (2nd
harvest) 12
13
Vegetative stage 14
Sulphur concentration and the 34
S atom excess in leaves at the 1st and 2
nd harvest 15
were affected by genotype, S treatment and the interaction (Table 6.5A). The only 16
exemption was the 34
S atom excess in leaves at the 2nd
harvest that was affected by the S 17
treatment only (P<0.018) (Table 6.5A). Genotype Surpass 402 CL had a higher 34
S atom 18
excess in leaves at the 2nd
harvest (0.27 mg 34
S/plant) than Karoo (0.19 mg 34
S/plant), 19
suggesting lower S remobilisation from leaves to stems in Surpass 402 CL (Figure 6.5A) 20
21
Table 6.5A. Analyses of variance for S concentration and 34
S atom excess in leaves and 22
stems 2 days (1st harvest) and 6 days (2
nd harvest) after leaf labelling commenced at 23
vegetative stage (A) 24
25
(1st harvest) (2
nd harvest)
(A) Genotype
(G)
(S)
treatment
G x S
Genotype
(G) (S)
treatment
G x S
Leaves S concentration in leaves[g S/kg leaves DW] *** *** *** *** ** *** 34
S atom excess in leaves [mg 34
S/leaves] * * ** ns * ns
Stems
S concentration in stems [g S/kg stems DW] ** ** ** ** ** ** 34
S atom excess in stems [mg 34
S/stems] * * ** *** *** ***
ns: not significant; *, ** , *** Significant at P≤0.05, P<0.01 and P<0.001, respect 26
126
A) 1
Figure 6.6A Figure 6.6 34
S atom excess in leaves 2 days (1st harvest) and 6 days (2
nd 2
harvest) after labelling started in the vegetative stage 3
4
5
S concentration and 34
S atom excess in plant tissues 2 days (1st harvest) and 6 6
days after commencement of labelling (2nd
harvest) 7
Maturity stage 8
9
Table 6.5B. Analyses of variance for S concentration and 34
S atom excess in leaves, stems 10
and siliques with seeds 2 days (1st harvest) and 6 days (2
nd harvest) after leaf labelling 11
commenced at maturity (B). 12 13
(1st harvest) (2
nd harvest)
(B)
Genotype
(G)
(S)
treatment
G x S
Genotyp
(G)
(S)
treatment
G x S
14 Leaves
S concentration in leaves [g S/kg leaves
DW]
*** *** *** *** *** ***
34S atom excess in leaves [mg
34S/plant] *** *** ns ns ns ns
Stems
S concentration in stems [g S/kg stems DW] *** ns *** ** *** ns 34
S atom excess in stems [mg 34
S/stems] ns ** ns *** *** ns
Siliques with seeds
S concentration in siliques with seeds
[g S/kg DW siliques with seeds]
*** ** ** * *** **
34S atom excess in siliques with seeds
[mg 34
S/plant]
* * ns ** ** **
ns: not significant; *, ** , *** Significant at P≤0.05, P<0.01 and P<0.001, respect 15
127
B) 1
Figure 6.6B 34
S atom excess in leaves 2 days (1st harvest) and 6 days (2
nd harvest) after 2
labelling started in the vegetative stage (A) and 34
S atom excess in plant tissues 6 days (2nd
3
harvest) after labelling started at maturity (B) in canola genotypes grown under deficient S 4
supply (leaf labeling). The error bars represent ±SE (A) or + SE (B). 5
6
Differences in S concentration in leaves were affected by S supply, genotypes and 7
their interaction (P<0.001) (Table 6.5B). Compared to genotype Karoo, S concentration in 8
leaves in genotype Surpass 402 CL increased from the 1st to the 2
nd harvest at deficient S 9
supply (0.065 < 0.09 g S/g leaves DW) (data not shown). In contrast, S concentration in 10
leaves decreased from the 1st to the 2
nd harvest at adequate S supply (0.18 > 0.16 S/g leaves 11
DW)(data not shown). Differences in S concentration in stems were influenced by genotype 12
and interaction after the 1st, and by genotype and S treatment after the 2
nd harvest (Table 13
6.5B). Sulphur concentration in siliques with seeds was influenced by genotype, S treatment 14
and the interaction (Table 6.5B), suggesting the genotypes differed in their responses to two 15
different levels of S supply. Genotype Surpass 402 CL had highest S concentration in 16
siliques with seeds after the 2nd
harvest at deficient S supply (0.14 g S/g siliques and seeds 17
DW), and genotype Karoo at adequate S supply (0.43 g S/g siliques and seeds DW) (data 18
not presented). 19
The 34
S atom excess in leaves at the 1st harvest was influenced by differences in 20
genotypes and the S treatment (Table 6.5B). There were no significant differences in 34
S 21
atom excess in leaves at the 2nd
harvest (Table 6.5B). The 34
S atom excess in stems was 22
128
influenced by the S treatment at the 1st harvest (Table 6.5B) and by the S treatment and 1
differences in genotypes at the 2nd
harvest (Table 6.5B). Genotype Surpass 402 CL had 2-2
fold higher (0.8 mg 34
S/stems) 34
S atom excess in stems than Karoo (0.38 mg 34
S/stems) at 3
the 2nd
harvest (Figure 6.6B). Differences in genotypes and the S treatment affected the 34
S 4
atom excess in siliques with seeds after both harvests, whereas the interaction was 5
significant at the 1st harvest only (Table 6.5B). 6
7
6.5 Discussion 8
6.5.1 Vegetative stage (uptake and remobilisation study) 9
A) Nitrogen 10
11
In the soil-labelled experiment, N-efficient and N-inefficient genotypes showed 12
similar recovery of the 15
N label (about 50% at adequate and 30% at deficient N supply, 13
except Westar with 70% recovery under deficient N). The relatively low 15
N recovery might 14
be caused by a large amount of 15
N that had been taken up remaining in roots (cf. Pessarakli 15
and Tucker 1985). Genotype Surpass 300 TT had the lowest 15
N excess in shoots at 16
deficient N supply (with the highest dry matter production of all genotypes at deficient N 17
supply), suggesting a 15
N dilution effect (see also Greenwood et al. 1991; Plenet and 18
Lemaire 2000). 19
In the leaf-labelling experiment under deficient N supply, among all genotypes 20
tested Wesway had the highest 15
N excess in leaves and stems (Figure 6.3A). Wesway had 21
the lowest 15
N excess in leaves and highest in stems 6 days after commencement of 22
labelling, suggesting more efficient N remobilization from leaves to stems in Wesway 23
compared with other genotypes (Figure 6.3A). Stems were the main sink for N remobilized 24
from leaves in the period from stem elongation to flowering in canola field experiment 25
(Gombert et al. 2010). Interestingly, Wesway had the lowest 15
N recovery in shoots at 26
deficient N supply in the soil-labelling experiment (data not shown), suggesting that this 27
genotype would assimilate greater proportions of 15
N via leaves than via roots under 28
deficient N supply. Similar findings were reported for the wild-type and nitrate reductase-29
deficient mutants of pea (Pisum sativum L. var. Juneau) (Lexa and Cheeseman 1997), 30
suggesting an existence of genetically-inherited mechanism contributing to efficiency of 31
15N/N remobilization in Wesway. 32
129
In the present study, only about 60 % of labelled 15
N supplied via leaves was 1
recovered in the leaves and stems. Relatively low 15
N recovery might have been caused by 2
the leaf labelling technique with urea-N potentially resulting in some gaseous loss of N as 3
ammonia during initial hydrolysis in the treated leaf as in the study by Palta et al. (1991). In 4
addition, relatively low 15
N recovery might have been due to 15
N retention in roots as 5
observed during 15
N leaf labelling in legumes (McNeill et al. 1997) and oilseed rape where 6
vegetative storage protein (VSP) in the taproot was linked to N being made available via 7
leaf senescence rather than via direct N uptake (Rossato et al. 2001). These authors 8
hypothesized that by increasing the N storage through VSP in taproots and/or stems, the leaf 9
N would be more efficiently mobilized to seed, with a result that low N efficiency of oilseed 10
rape could be improved. In our study with canola genotypes, Wesway (with the highest 15
N 11
atom excess in stems and lowest in leaves under deficient N supply 6 days after 12
commencement of labelling, and with greatest remobilization of N from leaves to stems), 13
could also have the large N storage capacity in VSP (potentially in stems). Further work is 14
recommended to characterize VSP (23 kDa protein) in stems and taproots in canola 15
genotypes differing in N efficiency. 16
17
B) Sulphur 18
19
Dry matter production in four tested genotypes (Surpass 402 CL, Karoo, IB 1368 20
and Surpass 300 TT) was influenced by genotypic differences only and not by the S 21
treatment, the latter being inconsistent with earlier S screening studies of canola genotypes 22
(Balint et al. 2008; Balint and Rengel 2009). This inconsistency might be due to a relatively 23
high S application in the deficient S treatment in the current study resulting in accumulation 24
of S in vegetative matter initially, followed by remobilization of endogenous S compounds 25
to maintain growth during subsequent exhaustion of external S supply (as reported by 26
Dubousset et al. 2009). 27
In the soil-labelled experiment, S-efficient and S-inefficient genotypes showed 28
similar recovery of the 34
S label in above-ground parts (approximately 20 % at adequate and 29
50 % at deficient S supply). Relatively low 34
S recovery under both S supplies (in particular 30
at adequate S supply) could be due to a large amount of 34
S that had been taken up 31
remaining in roots as reported for the 15
N label used for canola plants in the studies by 32
130
Pessarakli and Tucker (1985) and in this work (see above). Sulphur limitation changed the 1
34S partitioning within different plant tissues in the study by Abdallah et al. (2010), with 65 2
% of S taken up being found in roots, and only 23 % in leaves. Relatively higher recovery 3
of 34
S label at deficient compared to adequate S supply might have been related to 4
activation of high-affinity S transporters in roots at deficient S supply (for Arabidopsis see 5
Joshimoto et al. 2002; Rouached et al. 2008). The similarity of Brassica sulphate 6
transporters to the homologous Arabidopsis genes was reported to fall in the range of 84 to 7
89 % (Bruchner et al. 2004) because both belong to the Brassicaceae family. 8
Sulphur concentrations in shoots of canola genotypes tested here fluctuated 9
considerably (Figure 6.1B). In our study potassium sulphate (K234
SO4) was the major source 10
of S supply. It is well documented (Hawkesford and DeKok 2006) that sulphate is the most 11
common S form for uptake and transport in the plant. Compared to other tested genotypes at 12
deficient S supply, genotype IB 1368 had the highest S concentration in shoots (Figure 13
6.1B) and the xylem sap (Balint and Rengel 2011a), indicating higher capacity to take up S 14
from the soil compared to other tested genotypes (genotype IB 1368 was ranked as S-15
efficient at the vegetative stage in our earlier studies, Balint et al. 2008; Balint and Rengel 16
2009). It is worth noting that IB 1368 (B. juncea, Indian mustard) genotype had oil quality 17
similar to that of standard canola varieties (eg. see Norton et al. 2004). Some earlier studies 18
on sulphate uptake and xylem loading indicated close dependence on the species analysed 19
and the sulphur nutrition status (Herschbach et al. 1995a, 1995b, Westermann et al. 2000). 20
21
6.5.2 Maturity stage (uptake and remobilisation study) 22
A) Nitrogen 23
24
In canola, N uptake usually decreases after the flowering stage (Malagoli et al. 25
2005); therefore, N remobilization of N accumulated in plants during the vegetative stage 26
would be the main N source for pod/grain filling at maturity. Conversely, Gabrielle et al. 27
(1998), Rossato et al. (2001) and Malagoli et al. (2004) reported that N uptake continued 28
even after flowering, making about 11% of the total N taken up in the whole life cycle. 29
Two tested genotypes in our study had approximately the same N concentration in 30
leaves at deficient N supply (Figure 6.1A), with genotype Wesway having higher dry matter 31
production (data not presented) at similar tissue levels of N, suggesting better N utilisation 32
131
in Wesway compared to Westar. In general, N concentration was higher in leaves than 1
stems (Figure 6.1A), indicating poor N remobilization from leaves to stems as reported 2
earlier by Hocking et al. (1997), Dreccer et al. (2000), Rossato et al. (2001) and Svecnjak 3
and Rengel (2006). Relatively high N concentration in leaves might be a concern with 4
respect to N losses via leaf drop. However, N losses due to leaf drop near maturity might be 5
less important than losses via leaves in the vegetative stage (due to a smaller number and 6
size of leaves at maturity) (see Malagoli et al. 2005). 7
The amount of N loss via drop-off of N-rich leaves in our study was higher in 8
Westar (12.8 %) than in Wesway (9.2 %) due to poorer N remobilization from leaves in the 9
former genotype, and was in agreement with amounts of leaf N losses (12 %) reported by 10
Hocking et al. (1997) in their field study. Nevertheless, not only the N concentration and/or 11
N accumulation in leaves was contributing to differential N efficiency in canola, but also 12
the leaf size, as reported by Mickelson et al. (2003) who found negative correlation between 13
leaf N concentration at maturity and leaf size (i.e. genotypes with large leaves were more 14
efficient at N remobilization than small-leaved ones). Further work is recommended to 15
identify if this negative correlation existed in canola genotypes tested here, and whether it 16
contributed to differential N efficiency. 17
In our soil-labelling experiment, at deficient N supply, leaf 15
N atom excess was 18
higher in Wesway than Westar. However, the contribution of leaf 15
N in above-ground 15
N 19
recovery was 9 % in Wesway and 10.5 % in Westar, making the two genotypes equally 20
efficient in remobilizing N from leaves to stems (Figure 6.2). Further, the 15
N atom excess 21
in siliques and seeds was higher in Wesway than Westar, indicating greater 15
N 22
remobilization from stems to siliques and seeds in Wesway (Figure 6.2). Genotypic 23
differences in N remobilization in 10 winter wheat genotypes were reported by Barbottin et 24
al. (2010) and four canola cultivars by Svecnjak and Rengel (2006). 25
In spite of source limitation at deficient N supply, both canola genotypes were 26
unable to withdraw majority of N/15
N from leaves before they were dropped. Further, both 27
genotypes recovered similar amounts of 15
N: in stems 9-10 %; in siliques 18-19 %; in seeds 28
60-62.5 %. Similar results on 15
N recovery in plant parts were reported by Rossato et al. 29
(2001) in their study on N storage and remobilization in B. napus. 30
Seeds had the highest N concentration of all above-ground plant parts (Figure 6.2) 31
and represented the largest N sink (on average 69 % of the amount of shoot N). The seed in 32
our study were slightly smaller sinks than in studies by Malagoli et al. (2005) (74 % of 33
132
shoot N was incorporated into seeds) or Schjoerring et al. (1995) who reported that stems 1
and siliques each contained 10 % of total shoot N, and the remaining 80 % were 2
incorporated into seeds. It is worth bearing in mind that the differences in seed N 3
concentrations in Wesway and Westar at different N supplies in the present study were 4
proportionally smaller than differences in N applications, indicating that both genotypes 5
used available N more efficiently at deficient compared to adequate N supply. 6
7
B) Sulphur 8
9
In our study, genotype Surpass 402 CL had lower S concentration in leaves at deficient S 10
supply and higher dry matter production in leaves (data not shown) at lower tissue 11
concentrations of S than genotype Karoo (Figure 6.3A), suggesting more efficient use of 12
available S compared to Karoo. Further, S concentrations in leaves at maturity of both 13
genotypes were higher then those in stems, indicating relatively poor capacity of canola to 14
mobilize S from leaves at the same extent as from stems (Figure 6.4A). However, lower S 15
concentration in stems compared to leaves at maturity could also be associated with 16
differential structural components of their tissues. 17
Limited sulphate supply caused accumulation of Sultrl;3 (a sulphate transporter gene 18
essential in sulphur redistribution) in leaves and roots of 14-day-old Arabidopsis plants 19
(Yoshimoto et al. 2003). In oilseed rape, a limited S supply during the vegetative stage 20
caused a decline in sulphate concentration in maturing leaves via expression of BnSultr4;1 21
gene (coding for a vacuolar sulphate transporter) considered to be involved in sulphate 22
efflux from the vacuoles of old leaves towards young organs (Dubousset et al. 2009). 23
Expression of these two genes, Sultrl;3 and BnSultr4;1, important in the vegetative stage in 24
Arabidopsis and oilseed rape plants might also play an important role during the maturity 25
stage in canola. Further work is required to ascertain whether differential expression of 26
these two genes contributes to differential S efficiency of genotypes studied here under 27
limited S supply. 28
Sulphur concentration in stems at maturity was approximately 50 % of that in shoots 29
at anthesis under deficient S supply and approximately 70 % at adequate S supply, 30
suggesting poor S remobilisation from stems to seed regardless of S supply. In contrast, 31
silique walls were a relatively large sink for S in our tested genotypes (containing on 32
133
average 35 % of shoot S, Figure 6.4A), with the remainder of shoot S being distributed in 1
leaves (33 %), stems (10 %) and seeds (22 %). These findings are in accordance with our 2
previous study on S efficiency in 12 canola genotypes at maturity (Balint and Rengel 2009). 3
Silique walls were also a major S sink in other oilseed rape genotypes; in a study by Mason 4
and Hocking (1993) S redistribution from the silique walls to the seeds was negligible. A 5
study by Fismes et al. (1999) revealed that the silique walls retained from 39 to 61 % of 35
S 6
in different treatments, whereas seeds accumulated only 1 to 16 % of applied 35
S. 7
The lower S concentration in seeds of Surpass 402 CL compared to Karoo at 8
deficient S supply indicated lower remobilization of S from silique walls to the maturing 9
seeds (Figure 6.4A). In our previous study on S efficiency, dark-coated seeds of B. napus 10
genotypes (such are Surpass 402 CL and Karoo) did not represent as large S sink as yellow-11
coated seeds of B. juncea (Balint and Rengel 2009). 12
In our soil-labelling experiment, 34
S atom excess in leaves and stems of Surpass 402 13
CL was higher than in Karoo, suggesting lower 34
S remobilization from leaves via stems to 14
siliques and seeds in Surpass 402 CL. As a consequence, greater 34
S losses via dropped 15
leaves in the maturity stage in Surpass 402 CL might have contributed to this genotype 16
being less efficient than Karoo. Genotypic differences in uptake and remobilization of S 17
from leaves to stems, siliques and seeds were reported in four Brassica oilseed cultivars 18
(Malhi et al. 2007). In the study reported here, the 34
S atom excess in siliques and seeds at 19
deficient S supply was higher in Karoo than Surpass 402 CL, indicating existence of genetic 20
control supporting greater 34
S remobilization from stems to siliques and seeds in Karoo 21
(Figure 6.4C). However, in spite of source limitation at deficient S supply, neither canola 22
genotype was able to withdraw all S/34
S from leaves before they were dropped. Further, 23
both genotypes recovered similar amounts of 34
S: in leaves 30 %, stems 40 %, siliques 10 % 24
and seeds 20 %. A similar pattern of 35
S recovery in plant parts of field-grown oilseed rape 25
was reported by Fismes et al. (1999) (stems 27-46 %; silique walls 39-6 1%, and seeds 1-16 26
%). A substantially higher amount of 35
S in silique walls than other plant parts in the study 27
by Fismes et al. (1999) was related to the sampling regime (pod filling stage, when 28
remobilization of S from the pod walls to the seeds is usually still intense and far from 29
complete). 30
Compared with Karoo, genotype Surpass 402 CL had a higher 34
S atom excess in 31
stems 6 days after commencement of leaf labelling (2nd
harvest) in the vegetative as well as 32
maturity stages (Figures 6.6A and 6B). In the soil-labelling experiment, genotype Surpass 33
134
402 CL also had a higher 34
S atom excess in both stems and leaves (Figures 6.4B and 4C), 1
suggesting higher remobilization rate of 34
S from the leaves to stems in Surpass 402 CL 2
than Karoo. In addition, Surpass 402 CL had a higher capacity to accumulate 34
S than 3
Karoo, regardless of S applications, via leaves or roots (Figures 6.6A and 6B; 6.4B and 4C). 4
However, in both experiments, the 34
S atom excess in siliques and seeds (in soil labelling) 5
and silliques with seeds (in leaf labelling) as a proportion of that in stems was higher in 6
Karoo than Surpass 402 CL (Figures 6.6A and 6B, 6.4B and 4C), suggesting greater 7
remobilization of 34
S from stems in Karoo. 8
9
6.6 Conclusions 10
11
Genotype Wesway was more efficient than Westar in taking up N during the 12
vegetative stage as well as in remobilizing endogenous N from leaves, stems and siliques to 13
seeds at maturity. Genotype Surpass 402 CL appeared to be more efficient than Karoo in 14
taking up S during the vegetative and particularly during the maturity stage, but was less 15
efficient than Karoo in remobilizing endogenous S from leaves and stems to siliques and 16
seeds at maturity. 17
18
6.7 Acknowledgment 19
20
We would like to thank Ms Lydia Bednarek for mass spectrometry analyses, and Ms 21
Kristina Djekic and Mr Robert Balint for excellent technical assistance with harvests and 22
sample preparations. The canola seed was contributed by Department of Agriculture and 23
Food Western Australia, South Perth. The financial support from the Australian Research 24
Council is gratefully acknowledged. 25
26
27
28
29
30
31
32
135
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140
Chapter 7 Amino acid composition of xylem and phloem
sap varies in canola genotypes differing in N- and S-use
efficiency
7.1 Abstract
There is little information on amino acid composition of xylem and phloem sap in
canola genotypes differing in N- and/or S-use efficiency. Four canola genotypes with
either high or low N- and/or S-use efficiency indices were grown in a glasshouse using
deficient or adequate N and/or S supply. Xylem sap was obtained by vacuum extraction 46
days after sowing. Phloem sap was collected via bleeding from the peduncle of the
maturing siliques 120 days after sowing.
Among the genotypes, Wesway (N-use efficient) had the highest concentrations of
total N, glutamine (235 mmol/L) and asparagine (14 mmol/L) in the xylem sap in the
vegetative stage. In contrast, genotype Westar (classified previously as N-use inefficient)
had the highest concentrations of most amino acids in the phloem sap among genotypes
grown at deficient N supply.
Compared to the other tested genotypes, genotype Surpass 402 CL (S-use efficient)
had higher S concentration, and genotype Karoo (S-use inefficient) had higher
concentrations of glutamate and methionine, in the xylem sap in the vegetative stage.
Similarly, genotype Karoo had higher concentration of methionine under adequate S
supply and of glutamate under deficient S in the phloem sap at maturity.
In conclusion, in the canola phloem sap, the most important N-transporting amino
acid is glutamine for N transport and methionine and the glutathione-precursor glutamate
for S transport. It does not appear there is a relationship between differential N- or S-use
efficiency of canola genotypes and the concentration of N- or S-transporting amino acids
in the phloem sap.
141
7.2 Introduction
Canola (Brassica napus) is the main oilseed crop in Western Australia. More than
one million tones of canola were produced in 2008, worth over 300 million dollars to the
Western Australian economy.
Poor soil fertility is one of the main problems affecting canola production in
Western Australia because soils are sandy in texture, moderately acidic, deficient in
nitrogen, phosphorus and sulfur (Moore, 1998) and prone to leaching losses of S (Robson
et al. 1995). Existing genotypes with relatively poor S- and N-use efficiency tend to lack
the characteristics that are necessary for growth under limited nutrient supply.
Identification and breeding of genotypes that could use N and S from both native soil and
fertilizer more efficiently than standard genotypes would be an environmentally-friendly
long-term approach to dealing with the N and S deficiency in Western Australian soils and
the high N and S requirements of canola.
Genetic variation in N efficiency of Australian canola genotypes was identified by
Yau and Thurling (1987). More recent work on N- and S-use efficiency of 84 canola
genotypes (Balint et al. 2008) and N-use efficiency of four Australian commercial canola
cultivars (Svecnjak and Rengel 2006) suggested that the differences in efficiency were
related to uptake and/or transport and utilisation of nutrients within the plant. However, the
underlying mechanisms were not elucidated. In the study of N-use efficiency in 12 canola
genotypes (Balint and Rengel 2008) it was suggested that the differences in stem N content
among genotypes were due to differences in the type and/or amounts of amino acids
involved in transporting N-containing compounds from roots to shoots.
Amino acids, the major transport form of reduced N are translocated predominately in
the phloem (Reins et al. 1991). In the phloem exudate from pea shoots, the predominant
amino acid was asparagine (Urquhart and Joy 1981). The predominant amino acid in
canola phloem is glutamine (Mollers et al. 1996), but genotypes more efficient in
remobilizing N were proposed to contain greater amounts and/or proportions of asparagine,
the more efficient N transporter with an N:C ratio of 0.5 compared to 0.4 for glutamine
(Seiffert et al. 1999).
Genotypic differences in the S uptake efficiency of mustard (Ahmad et al. 2005)
were hypothesized to be due to differences in S uptake kinetics at the root-cell plasma
142
membrane. The authors suggested the existence of biphasic transporter systems in mustard,
i.e. combination of high- and low-affinity transporters. Differential S-use efficiency in 84
canola genotypes during the vegetative stage (Balint et al. 2008) and 12 canola genotypes
during the vegetative and maturity stages (Balint and Rengel 2009) have also been
reported. Differential S-use efficiency and differences in stem S content of canola
genotypes were suggested to be due to differences in the type and/or amounts of amino
acids involved in transporting S-containing compounds from roots to shoots (Balint and
Rengel 2009).
Even though differential S efficiency among canola genotypes is not well understood it
is likely that more than one mechanism is responsible for a particular level of efficiency in
a particular genotype (cf. Rengel 1999) It has been well documented (Hawkesford and
DeKok 2006; Davidian and Kopriva 2010) that sulphate is the most common S form for
uptake and transport in plants: its transport from roots to shoots can be dependent on
and/or regulated by amino acid cysteine (sulphate is reduced to sulphide before
incorporation into cysteine). On the other hand, the thiol tripeptides [glutathione (Glu-Cys-
Gly) and homoglutathione (Glu-Cys-Ala)] were reported to play an important role in
transporting S in soybean and wheat (Anderson and Fitzgerald 2001). In contrast, the role
of amino acids and other S transporters in partitioning of S into various organs in canola
has not been reported yet.
The present study was aimed at investigating the differences in amino acid content of
xylem and phloem sap at vegetative and maturity stages in canola genotypes with
differential N- and S-use efficiency.
7.3 Materials and Methods
The brown sandy soil (Uc4.22; Northcote 1971) was collected from a bushland site
15 km south-east of Lancelin (3108’S, 11529’E), Western Australia. Soil had pH (H2O)
5.9, 2 % clay, 8 g/kg organic carbon and low levels of essential plant nutrients (N, K, P,
Mg, S, Zn, Cu) (Balint et al. 2008). The soil was air dried, sieved to 2 mm, fertilized with
basal nutrient and treatment salt solutions, followed by air drying, thorough mixing and
filling into plastic pots lined with plastic bags (5,000 g of air-dry soil/pot).
143
Nitrogen in N experiment was supplied pre-sowing and 21 days after sowing as
urea CO(NH2)2 at 140 mg in the adequate and 51 mg N/kg soil in the deficient N treatment
by the time of xylem sap collection (46 days after sowing). Total amount of N applied to
plants grown to grain maturity was 360 mg in the adequate and 120 mg N/kg soil in the
deficient N treatment. Most N was applied during the rosette, flowering and early grain
filling stages (56, 77 and 112 days after sowing). Potassium, S, P and micronutrients were
re-applied at the initial amounts at the rosette and the flowering stages (56 and 77 days
after sowing).
Sulphur in S experiment was supplied as K2SO4 at 50 mg S/kg soil in the adequate
and 4 mg S/kg soil in the deficient S treatment pre-sowing, and no additional S was applied
by the time of xylem sap collection (46 days after sowing). Total amount of S applied to
plants grown to grain maturity was 87 mg/kg for adequate and 8 mg/kg soil in the deficient
S treatment. Most S was applied during the flowering stage (65 days after sowing).
Nitrogen, K, P and micronutrients were re-applied at the initial amounts at the rosette,
flowering and initial pod-filling stages (51, 65 and 95 days after sowing).
Seeds were prepared and sown (10 seeds per pot, thinned to five plants 8 days after
sowing) and pots watered and weighted daily to field capacity (10 % w/w) as described
earlier (Balint et al. 2008). Genotypes grown were chosen from the previous studies (Balint
et al. 2008; Balint and Rengel 2008; 2009). For N experiment, we used Wesway and
46C74 as N-use efficient, and Westar and Surpass 300 TT as N-use inefficient. For S
experiment, Surpass 402 CL and IB 1368 were S-use efficient, and Karoo and Surpass 300
TT as S-use inefficient. All canola genotypes used in the study were B. napus except IB
1368, the only B. juncea (Indian mustard) genotype. All genotypes were used for xylem
sap analyses. Wesway and Westar from N experiment and Surpass 402 CL and Karoo from
S experiment were used for phloem sap analysis at maturity stage (120 days after sowing).
7.3.1 Xylem sap sampling and analyses
Three canola shoots (stems with leaves attached) from each replicate pot were
harvested by cutting just above the soil surface 46 days after sowing. Sampling of xylem
exudates was done in laboratory within the first hour after stem cutting, i.e. between 10.00-
11.00 am. The outer/epidermis layer 2 cm from the stem base was peeled/removed to
144
prevent phloem contamination of xylem sap that was obtained by vacuum extraction. The
xylem sap was collected by a pipette into a 1-mL eppendorf tube and stored at -20 oC.
Shoot dry weights were determined after drying in an oven at 65 C for 7 days.
Each sample was placed in an aluminium tin capsule and freeze-dried to measure
the N content in the xylem sap [g N/kg xylem sap DW], 200 µL of; the remaining dry
matter in the tin capsules was analysed on a 4-channel Autoanalyzer system, using
modifications of manufacturer’s procedures (Anon 1979). Nitrogen was determined
colourimetrically by the indophenol blue method (Yuen and Pollards 1954) after digestion
with sulphuric acid and hydrogen peroxide.
For the measurement of S content in the xylem sap [g S/kg xylem sap DW], each
sample in eppendorf tubes was freeze-dried, then 200µL of the remaining dry matter was
analysed by inductively coupled plasma atomic emission spectroscopy (ICP-AES) after
digestion with a mixture of nitric and perchloric acids (McQuaker et al. 1979).
The Phenomenex EZ:faast® analysis kit (Lane Cove, NSW, Australia) was used for
free amino acid analyses by Gas Liquid Chromatography - Flame Ionisation Detection
(GC-FID). The EZ:faast® amino acid analysis procedure consist of five stages: pH
adjustment, washing, elution, derivatisation and liquid extraction. A Shimadzu® GC 17-A
gas chromatograph coupled with an AOC-20i auto sampler and flame ionisation detector
(Shimadzu, Kyoto, Japan) controlled by GC Solution software (version 2.30) was used.
The carrier gas was high purity helium (He) at a constant flow rate of 1.5 mL/min.
Hydrogen and air was used for the flame ionisation detection. The oven temperature
regime was as follows: initial temperature 110 °C with a 32 °C per minute increase to 320°
C and held at 320° C for 0.5 minutes. Injection port temperature was 250 °C, whereas a
detector was held at 320° C. Samples were injected at 2 µL in the split mode (1:15, v/v and
1:5, v/v). A Hewlett-Packard 5970 Mass selective (MS) detector (Wilmington, DE, USA)
was used for positive identification of the amino acid peaks. The column used was the
same as that for the GC-FID. Samples were injected at 1 and 2 µL in the split-less method.
MS detector data acquisition occurred from 45-550 mass units at 1.2 scans/s.
7.3.2 Phloem sap sampling and analyses
145
Two remaining shoots in each replicate pot containing genotypes Wesway and
Westar in N experiment and Karoo and Surpass 402 CL in S experiment were grown to
maturity. The maturing siliques with seeds were used for phloem sap collection (bleeding
from silique peduncle) as described by King and Zeevaart (1974). Five independent
replicates (siliques) were used for each measurement. At the time of harvest (120 days
after sowing, with the seeds in the late cotyledon developmental stage), cuts were made 6-8
hours after light onset. Immediately after cutting, the silique peduncles were re-cut under
distilled water and placed in 250 µL eppendorf tubes containing 200 µL of 20 mM
ethylenediaminetetraacetate (EDTA) solution (pH 6.5). Tightly-closed tubes had an
opening in the lid big enough to put silique peduncles through and immerse them in the
EDTA solution (Figure 7.A). During collection of phloem exudate (6 h), samples were
incubated in an air-tight chamber at room temperature (23o C) and relative humidity of ~95
%. Following exudation, samples with ‘diluted’ phloem sap were stored at -20 oC.
Methodology for the N and S content in the phloem sap [g S/kg phloem sap DW] at
maturity was the same as described for the N and S content in the xylem sap.
The two experiments (N and S) were each set up in a completely randomised block
design with factorial arrangement of genotypes x fertilization treatments with three
replicates. The data were analysed by 2-way ANOVA using GENSTAT 10.1 statistical
program.
7.4 Results
7.4.1 Vegetative stage
A) Growth, N and amino acid concentration in the xylem sap (N experiment)
Dry matter production in shoots was significantly affected by N treatment (P<0.01)
and under deficient N supply ranged from 0.53 (genotype 46C74) to 1.00 g/shoot (Surpass
300 TT). Dry matter production under deficient N supply was approximately 2 times lower
than under adequate N supply (data not shown).
Nitrogen concentration in the xylem sap was influenced by differences in
genotypes, N treatment and genotype x N treatment (all P<0.001) (Table 7.1). Nitrogen
concentration under adequate N supply ranged from 19 to 47 g N/kg xylem sap DW
146
(46C74<Surpass 300 TT) and was about 4 times lower at deficient N supply
(46C74<Wesway) (Figure 7.1). In contrast, the amount of N applied in the adequate N
treatment was only 2.8 times higher than that at deficient N supply.
Table 7.1 Analyses of variance for N and S concentration in xylem and phloem sap in
genotypes differing in N- or S-use efficiency
Genotype
(G)
Nitrogen
treatment (N)
G x N
N concentration in xylem sap (g N/kg xylem sap DW) *** *** ***
N concentration in phloem sap (g N/kg phloem sap DW) ns ns ns
Genotype
(G)
Sulphur
treatment (S)
G x S
S concentration in xylem sap (g S/kg xylem sap DW) *** *** ***
S concentration in phloem sap (g S/kg phloem sap DW) * * ns
ns: not significant; *, *** Significant at P≤0.05 and P<0.001, respectively
0
10
20
30
40
50
Wesway Westar Surpass 300 TT 46C74
Genotypes and N treatments
N c
on
ce
ntr
ati
on
(g
N/k
g x
yle
m s
ap
DW
)
Adequate N
Deficient N
Figure 7.1 Nitrogen concentration in the xylem sap in four canola genotypes grown at
adequate and deficient N supply at vegetative stage.
Note, Y-axis label: N concentration (g N/kg xylem sap DW)
147
Concentrations of glutamine, asparagine, aspartate, glutamate, leucine, methionine
and proline were significantly influenced by N treatment and genotype x N treatment
interaction (Table 7.2, Figure 7.2). Differences in concentration of alanine were influenced
by genotypes and genotype x N treatment interaction, whereas concentration of serine was
influenced by N treatment only (Table 7.2). The amino acids glycine, histidine and lysine
were influenced by the main effects of genotype and N treatment (Table 7.2).
Table 7.2 Analyses of variance for amino acid concentrations in the xylem sap (46 days
after sowing, flowering stage) in genotypes differing in N-use efficiency (N experiment).
Amino acids Genotype (G) Nitrogen treatment (N) G x N
Ala ** ns *
Gly ** * ns
Val ns *** **
Leu ** *** ***
Ser ns *** ns
Pro ** *** **
Asn * *** *
Tpr ns * ns
Asp ** *** *
Met *** *** ***
Glu * *** *
Phe ns ns ns
Aaa ns ns ns
Gln ** *** **
Lys ** *** ns
His * *** *
Tyr ns * ns
ns: not significant; *, **, *** significant at P≤0.05, P<0.01 and P<0.001, respectively.
148
Glutamine was the most abundant amino acid under both N supplies, followed by
alanine, serine, aspartate and methionine (Figure 7.2). All four canola genotypes had
higher aspartate compared to asparagine concentrations under both N supplies (Figure 7.2).
Some amino acids showed 2, 3 and up to 10 times higher concentrations at
adequate compared to deficient N supply (Figure 7.2).
0
40
80
120
160
200
240
ALA VAL LEU PRO ASN ASP MET GLU GLN HIS
Am
ino
ac
id c
on
ce
ntr
ati
on
(m
mo
l/L
)
Wesway
Westar
Surpass 300 TT
46C74
Adequate N
ASN (mmol/L)
0
3
6
9
12
15
0
20
40
60
80
100
ALA VAL LEU PRO ASN ASP MET GLU GLN HIS
Amino acids and N treatments
Am
ino
acid
co
ncen
trati
on
(m
mo
l/L
) Wesway
Westar
Surpass 300 TT
46C74
Deficient N
ASN (mmol/L)
0
1
2
3
4
Figure 7.2 Amino acid concentration (mmol/L) in the xylem sap in four canola genotypes
grown under adequate and deficient N supply at vegetative stage. Only amino acids for
which the interaction genotype x N treatment was significant were shown.
Note, Y-axis labels in both graphs: Amino acid concentration (mmol/L).
149
Of all the genotypes at adequate N supply, Wesway had the highest concentration of
alanine, valine, methionine and glutamate. Most importantly, this genotype also had the
highest concentration of glutamine (235 mmol/L) and asparagine (14 mmol/L) in the
xylem sap, which are the two amino acids relevant for N transport (Figure 7.2). On the
other hand, at deficient N supply, genotype Westar had the highest concentration of not
just these two amino acids (glutamine 60 and asparagine 3.68 mmol/L xylem sap), but also
of alanine, leucine, proline, methionine, glutamate and histidine (Figure 7.2).
B) Growth, S and amino acid concentration in the xylem sap (S experiment)
In the present study, dry matter production in four tested genotypes was influenced
by differences in genotypes only (P≤0.05) and not by differential S supplies (data not
shown). Sulphur concentration in the xylem sap was influenced by genotype, S treatment
and interaction (all P<0.001) (Table 7.1). At adequate S supply, S concentration ranged
from 0.21 to 0.89 g S/kg xylem sap DW (Karoo<Surpass 402 CL) and up to 5 times less
(see Surpass 300 TT) at deficient S supply (Karoo<IB 1368) (Figure 7.3).
0.0
0.2
0.4
0.6
0.8
1.0
Surpass 402 CL Karoo Surpass 300 TT IB 1368
Genotypes and S treatments
S c
on
cen
trati
on
(g
S/k
g x
yle
m s
ap
DW
)
Adequate S
Deficient S
Figure 7.3 Sulphur concentration in the xylem sap in four canola genotypes grown at
adequate and deficient S supply at vegetative stage.
Note, Y-axis label: S concentration (g S/kg xylem sap DW)
150
Amino acids alanine, glutamine, lysine, histidine, proline, asparagine,
phenylalanine, α-aminoadipic acid, glutamate and methionine were influenced by genotype
x S treatment interaction (Table 7.3). Sulphur treatment had a significant effect on amino
acids glycine, tryptophane, aspartic acid and tyrosine (Table 7.3). Amino acids valine and
leucine were influenced by both main effects (genotypes and S treatment) (Table 7.3).
Table 7.3 Analyses of variance for amino acid concentrations in the xylem sap (46 days
after sowing, flowering stage) in genotypes differing in sulphur-use efficiency (S
experiment)
Amino acids Genotype (G) Sulphur treatment (S) G x S
Ala * *** **
Gly ns * ns
Val *** * ns
Leu *** * ns
Ser ns ns ns
Pro *** ns *
Asn ns ns *
Trp ns * ns
Asp ns * ns
Met ns ns **
Glu ns * *
Phe ns ** *
Aaa ns ns *
Gln *** * ***
Lys * ** *
Hys *** ** *
Tyr ns * ns
Cys ns ns ns
Hyp ns ns ns
ns: not significant; *, **, *** Significant at P<0.1, P<0.01 and P<0.001, respectively
Several amino acids showed 20 to 30 % higher concentrations at adequate
compared to deficient S supply (Figure 7.4). Genotype Surpass 300 TT had significantly
151
higher concentrations of alanine, serine, asparagine, aspartate (data not presented),
methionine and glutamate (Figure 7.4) and aspartate (data not presented) compared to
Surpass 402 CL and Karoo and non-significantly higher than IB1368 (Figure 7.4). At
deficient S supply, Karoo had higher concentrations of glutamate and methionine than
Surpass 300 TT (and non-significantly higher than Surpass 402 CL). In contrast,
concentrations of glycine and cysteine (the building blocks of gluthathione, the S
transporter) were similarly low in all genotypes (Figure 7.4).
0
10
20
30
40
50
60
GLY MET GLU C-C
Adequate S
Am
ino
ac
id c
on
ce
ntr
ati
on
s (
mm
ol/L
)
Surpass 402 CL
Karoo
Surpass 300 TT
IB 1368
0
10
20
30
40
50
60
GLY MET GLU C-C
Amino acids and S treatments
Am
ino
acid
con
cent
ratio
n (m
mol
/L)
Surpass 402 CL
Karoo
Surpass 300 TT
IB 1368
Deficient S
Figure 7.4 Amino acid concentration (mmol/L) in the xylem sap in four canola genotypes
grown under adequate and deficient S supply at vegetative stage. Only amino acids
relevant for S transport, and those for which the interaction genotype x S treatment was
significant were shown (Gly, Met, Glu and Cys).
Note, Y-axis labes in both graphs: Amino acid concentration (mmol/L)
7.4.2 Maturity stage
152
A) Nitrogen and amino acid concentrations in the phloem sap
Two tested genotypes did not differ in N concentration of the phloem sap. In
contrast, there were significant differences in amino acid concentrations of the phloem sap
(Figure 7.5). The concentrations of α-aminoadipic acid, alanine and proline were
influenced by N treatment; the concentrations of serine, glutamine and alanine by the main
effect of genotype (P<0.01). In general, genotype Westar had higher concentration of most
amino acids (except glutamic acid and α-aminoadipic acid) compared with genotype
Wesway under deficient N supply, except glutamic acid and α-aminoadipic acid (Figure
7.5).
0
100
200
300
400
ALA VAL SER PRO ASN GLU AAA GLN
Amino acids and N treatment
Am
ino
acid
co
ncen
trati
on
s (
mm
ol/L
)
Wesway
Westar
Deficient N
0
100
200
300
400
ALA VAL SER PRO ASN GLU AAA GLN
Am
ino
acid
co
cn
cen
trati
on
s (
mm
ol/L
)
Wesway
Westar
Adequate N
Figure 7.5 Amino acid concentration (mmol/L) in the phloem sap in two canola genotypes
grown at adequate and deficient N supply at maturity.
153
Note, Y-axis labels in both graphs: Amino acid concentration (mmol/L).
B) Sulphur and amino acid concentration in the phloem sap
Sulphur concentration in the phloem sap was significantly affected by S treatment
(P<0.001) and genotype (P=0.038) and no significant genotype x S interaction was
recorded (P=0.26) (data not presented). Sulphur concentration in the phloem sap ranged
from 2.06 (Karoo) to 2.76 g S/kg phloem sap DW (Surpass 402 CL) under adequate S
supply, and from 0.93 (Karoo) to 1.51 g S/kg phloem sap DW (Surpass 402 CL) under
deficient S supply. Compared with S concentrations at deficient and adequate S supplies in
the xylem sap at anthesis (Figure 7.3), S concentrations in the phloem sap at maturity were
approximately 5 times greater under deficient and 2.5 times greater under adequate S
supply (data not presented).
Concentration of methionine and glutamate were influenced by genotype x S
treatment interaction (Figure 7.6). Genotypic differences in concentrations of glycine and
cysteine in the phloem sap were non-significant. Genotype Karoo had higher concentration
of methionine under adequate S supply and of glutamate under deficient S (Figure 7.6).
0
50
100
150
200
SER MET GLU SER MET GLU
Amino acids and S treatments
Am
ino
acid
s c
on
cen
trati
on
(m
mo
l/L
)
Surpass 402 CL
Karoo
Adequate S Deficient S
Figure 7.6 Amino acid concentrations (mmol/L) in the phloem sap in two canola genotypes
grown at adequate and deficient S supply at maturity. Only amino acids relevant for S
transport and those for which the interaction genotype x S treatment was significant were
shown (Ser, Met and Glu).
Note, Y-axis label: Amino acid concentration (mmol/L).
154
7.5 Discussion
7.5.1 Nitrogen and S in the xylem and phloem sap
Nitrogen concentrations in the xylem sap of various canola genotypes tested here differed
considerably with the N treatment (Figure 7.1). Indeed, the xylem N concentrations can be
affected by the amount and form of N in the soil (Andrews 1986; Lexa and Cheeseman
1997). In our study, urea fertilizer [which would have been converted to ammonium
(NH4+) in soil] was the major form of N supply. While working on expression analysis of
genes that encode starvation-induced high-affinity NO3- and NH4
+ transporters in
Arabidopsis, Gansel et al. (2001) proposed that the uptake system for NH4+ is less efficient
than the one for NO3-. In the study presented here, canola genotypes Surpass 300 TT and
Wesway, which differed in the xylem sap N concentration at deficient N supply might
have had differential regulation/expression of the homologues of NO3- and NH4
+
transporter genes (controlled by the root N status as well as the shoot N demand) (cf.
Gansel et al. 2001).
Compared to other tested genotypes at deficient N supply, Wesway had the highest
N concentration in the xylem sap (Figure 7.1), indicating a higher capacity to take up N
from the soil (genotype Wesway was ranked as efficient at the vegetative stage in our
earlier studies, Balint et al. 2008; Balint and Rengel 2008). Similar results for N
concentration in the xylem sap of N-efficient and -inefficient genotypes were reported for
maize (Niu et al. 2007). However, N concentration in the xylem sap is regulated via several
factors, which vary among species (eg. tomato and sunflower) (Jia and Davies 2007) with
changes in the soil such as soil flooding (Jackson et al. 2003); and NO3- availability (Lexa
and Cheeseman 1997) and above-ground environmental conditions such as air humidity
(Brewitz et al. 1996).
In our study, sulphate was the major form of S supply. Sulphate was also a major
from of S supply in the study by Hawkesford et al. (2003) whereby it was taken up by
roots and transported in the inorganic form to developing leaf tissues (Anderson 2005).
Sulphate in the leaf tissue can be either stored or reduced to sulphide and incorporated into
organic forms of S such as amino acid cysteine (Cys), the major precursor for amino acid
methionine (Met) and tripeptide glutathione (GSH) and its homologues, e.g.
homoglutathione (Schmidt and Jager 1992; Hell 1997; Hawkesford and Wray 2000;
155
Leustek et al. 2000; Kopriva and Rennenberg 2004). In our study, sulphate was most likely
reduced to sulphide and via cysteine was subsequently incorporated into methionine
because methionine was the most abundant S-containing amino acid in the xylem sap
under both S supplies (Figure 7.4).
Compared to other tested genotypes at deficient S supply, genotype IB 1368
(Brassica juncea) had the highest S concentrations in the shoots (Balint and Rengel 2011)
as well as in the xylem sap (Figure 7.3), indicating a higher capacity to take up S from the
soil compared to other genotypes (genotype IB 1368 was ranked as S-efficient at the
vegetative stage in our earlier studies, Balint et al. 2008; Balint and Rengel 2009). Given
that some earlier reports on sulphate uptake and xylem loading indicated close dependence
on the species analysed and S nutrition (Herschbach et al. 1995a, 1995b and Westermann
et al. 2000), further work is needed to elucidate potentially differential processes/regulation
of xylem loading in B. napus and B. juncea.
7.5.2 Amino acid composition of the xylem and phloem sap of different
genotypes as influenced by N or S supply
In the earlier study on differential N-use efficiency of canola genotypes (Balint and
Rengel 2008) we proposed that genotypes more efficient in remobilizing N might contain
greater amounts or proportions of the amino acid asparagine, the more effective N
transporter with an N:C ratio of 0.5 compared to 0.4 for glutamine (see also Seiffert et al.
1999). However, in the present study, glutamine (as one of the immediate products of
ammonia assimilation, Miflin and Lea 1977) was the most abundant N amino compound in
the xylem sap, with 8-fold higher concentrations at adequate compared to deficient N
supply (Figure 7.2); this is a greater difference than that reported by Finneman and
Schjoerring (1999) (only 4-fold) in their study of NH4+ translocation in oilseed plants.
Glutamine was also the predominant amino compound in barley fed NO3-, with glutamine
concentration increasing 3-fold with a change from nitrate to ammonium nutrition (Lewis
et al. 1982). On the other hand, maize plants fed NO3- did have higher glutamine
concentrations in the xylem, but when fed ammonium, the asparagine concentration
exceeded that of glutamine (Murphy and Lewis 1987). In our study, concentration of
asparagine in the xylem sap was significantly lower than that of glutamine under both N
156
supplies; hence, asparagine is unlikely to play a major role in differential N efficiency in
the four canola genotypes tested. Asparagine appears prefer, or has more important role in
long-range transport especially in legumes species (Lam et al. 2003) rather than Brassica
species.
Decreased N supply caused impaired N assimilation in soybean and a few non-N2-
fixing species (tomato, maize and sunflower), leading to specific changes in the amino acid
composition of the xylem sap (elevated aspartate/asparagine ratio and a decrease of
glutamine concentration compared to plants grown at adequate N supply) (Amarante et al.
2006). Canola genotypes in our study had higher concentrations of aspartate compared to
asparagine under both deficient and adequate N supplies (Figure 7.2), indicating that this
ratio might be a species-specific trait and may differ for legumes and non-N2-fixing
species. The elevated aspartate/asparagine ratio in tomato, maize and sunflower in the
study by Amarante et al. (2006) could be related to a shorter exposure of plants to the N-
free conditions (4 days only compared to 46 days of exposure to deficient N supply for
canola genotypes in our study). It was reported that some AMT genes (ammonium
transporters gene family) showed increased expression during early N deficiency, whereas
the expression of other transporters increased only after prolonged starvation (Loque and
von Wiren 2004).
In our earlier study on differential S-use efficiency of canola genotypes (Balint and
Rengel 2009) we proposed that genotypes more efficient in remobilizing S might
genetically differ in the biosynthesis or transport efficiency of S-containing compounds
(eg. cysteine, methionine, glutathione, homoglutathione). In the study presented here,
methionine was the most abundant S-containing amino acid in the xylem sap at deficient S
supply (Figure 7.4). The concentration of cysteine was relatively low (10 mmol/L, Figure
7.4), suggesting continuous cysteine use for methionine synthesis. The low concentration
of cysteine was also reported in the work on S assimilatory metabolism by Leustek et al.
(2000) and Saito (2000), whereby the turnover rate of cysteine was high, but the cellular
concentration of free cysteine was maintained low (approximately 20 mmol/L).
Genotype Karoo, recognized as S-inefficient in our previous studies (Balint et al.
2008, Balint and Rengel 2009) had the highest concentrations of methionine (49.7
mmol/L) and glutamate (36.5 mmol/L) in the xylem sap (Figure 7.4). In our study,
genotypes at deficient S supply did not accumulate arginine [(in contrast to a study
reported by Thomas et al. (2000) for sugar beet)] or asparagine [(as reported by Prosser et
157
al. (2001) for spinach) in the xylem sap when concentrations of methionine and cysteine
were low. The absence of asparagine and arginine accumulation in our study could be due
to poor shoot-to-root signaling to regulate sulphate uptake and expression of sulphate
transporters as reported for Brassica oleracea by Bruchner et al. (2004).
Several amino acids (alanine, valine, serine, proline, glutamate, α-aminoadipic acid
and glutamine) had significantly different concentrations in the phloem sap (collected
when seeds were in the late cotyledon developmental stage) of the two genotypes in the N
experiment (Figure 7.5). N-use inefficient genotype Westar had higher amino acid
concentrations than Wesway at deficient N supply, suggesting negative correlation
between the N-use efficiency (NUE) rating of the genotypes and the amino acid
concentrations. In the study of N uptake and remobilisation in field-grown oilseed rape by
Malagoli et al. (2005), it was established that the phloem-loading process was a key step in
efficient N remobilisation, but it remained unclear whether this process was limiting for
NUE. In our study, differences in N concentration as well as in the concentration of the
important N-transporter amino acids asparagine and glutamine in the phloem sap were not
related to differential N-use efficiency (NUE) in the canola genotypes tested.
Compared with S-use inefficient Karoo, the S-use efficient genotype Surpass 402
CL (cf. Balint et al. 2008, Balint and Rengel 2009) had higher S concentrations in the
xylem sap (Figure 7.3) at flowering and the phloem sap at maturity (data not shown).
Amino acid S-methylmethionine, a methionine derivate was a key contributor to phloem S
transport in (Arabidopsis thaliana) HMT2, loaded into the phloem for transport
to seeds
(Lee et al. 2008). In the study presented here, methionine was the most abundant S-
containing amino acid in the xylem sap (Figure 7.4) but not in the phloem sap (Figure 7.6).
In the earlier studies on S uptake in canola, the rate of sulphate uptake was correlated with
the glutathione concentration in the phloem and the roots (Lappartient and Touraine 1996;
Davidian et al. 2000), but whether glutathione biosynthesis and transport was limiting for
S-use efficiency (SUE) remained unclear. In the present study, the highest concentration of
glutamate (one of building blocks of gluthathione) in the phloem sap at deficient S supply
was recorded in the S-inefficient genotype Karoo (Figure 7.6). The phloem saps of canola
genotypes differing in SUE were collected from plants 120-day-old in our study vs 21-day-
old in the study by Lappartient and Touraine (1996), however, despite that difference, the
results from the two studies were consistent with the suggestion that glutathione and its
precursors are important in regulating S transport. In addition, the S-use inefficient
158
genotype Karoo had a higher glutamate concentration in the phloem sap than the S-use
efficient Surpass 402 CL at deficient S supply (Figure 7.6), suggesting the poor capacity of
the S-use inefficient genotype to convert glutamate to glutathione; this hypothesis remains
to be tested in a further study.
7.6 Conclusions
In conclusion, genotype Wesway was more efficient than Westar in taking up N, whereas
Surpass 402 CL was more efficient than Karoo in taking up S during the vegetative stage.
No clear relationship between amino acid composition of the xylem and the phloem sap
with the N- or S-use efficiency of canola genotypes was found. Glutamine may be the most
important N-transporting amino acid in the canola phloem sap. Regarding S
remobilization, S-containing methionine and the glutathione-precursor glutamate may be
the most important amino acids involved in S transport in the canola phloem sap.
7.7 Acknowledgment
We would like to thank Mr Greg Cawthray for Gas Liquid Chromatography (GC-FID)
analyses, and Mr Robert Balint and Ms Kristina Djekic for excellent technical support with
amino acid sample preparations. The canola seed was contributed by Department of
Agriculture and Food Western Australia, South Perth. The financial support from the
Australian Research Council is gratefully acknowledged.
Figure 7.A Phloem sap collection
[(silique peduncles immersed in 250µL
Eppendorf tubes containing 200 µL of
20 mM ethylenediaminetetraacetate
(EDTA) solution (pH 6.5)].
159
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164
Chapter 8 General discussion and conclusions
The objective of this thesis was to characterize N and S uptake, transport and
remobilization in canola genotypes differing in N and/or S efficiency. These objectives
were addressed by preliminary screening of Australian canola germplasm (84 genotypes)
for N and/or S efficiency at the vegetative stage. The screening at maturity was conducted
on a group of chosen genotypes identified in preliminary screening (as having either high
or low N and/or S efficiency indices). Advanced studies related to uptake, transport and
remobilization of N and/or S on the small group of chosen genotypes were conducted using
15N and/or
34S stable isotopes and xylem and phloem amino acids assays.
In a study of 70 German canola genotypes, significant differences in N efficiency
were recorded by using the N harvest index as the important assessment criterion (Kessel
and Becker 1999). In the study by Yau and Thurling (1987) using 40 Australian genotypes,
differences in N efficiency were based on dry weight and N accumulation in the above-
ground plant parts during the vegetative and grain maturity stages. In the study presented
here, the modern Australian canola germplasm was assessed for variations in N and S
efficiency in the vegetative growth stage (Balint et al. 2008) using the following three
evaluation criteria: i) growth at low N or S supply; ii) growth at low relative to adequate N
and S supply, and iii) N or S utilisation efficiency expressed as shoot dry weight per unit of
N or S content in shoots.
The results obtained from this study indicated existence of genotype-specific traits for
N and S uptake efficiency because significant treatment x genotype interactions for N and
S concentration and content in shoots were recorded (Balint et al. 2008). When considering
nutrient utilisation efficiency in canola genotypes, 16 out of 84 canola genotypes were
rated as N efficient and 25 genotypes as S efficient.
Based on all three efficiency criteria, there was no genotype identified as having both
N and S efficiency. Further, two current commercial canola genotypes (Surpass 600 and
46C74) were rated as N and S efficient in terms of relative yield. On the other hand, two
genotypes were ranked efficient (Chikuzen and 46C74) or inefficient (CBWA-005 and
Beacon) in uptake and utilisation of N under all three criteria. In terms of S efficiency,
165
genotype Argentina was ranked efficient and CBWA 003 and IB 1363 were classified
inefficient under all three criteria (Balint et al. 2008).
While it is advantageous for breeders to assess genetic differences in N and S
efficiency during vegetative growth (the earlier, the better), prior to this study there was no
information on whether N and S efficiency in canola at maturity can be reliable determined
by screening germplasm in the vegetative stage to shorten the process. In other words, if
the efficiency ranking among chosen canola genotypes from preliminary screening study
remained the same at the seed maturity stage, screening a large number of canola
genotypes during the vegetative stage would be sufficient for selection purposes.
Therefore, following the preliminary screening of 84 canola genotypes (Balint et al. 2008),
the second screening study on 12 chosen genotypes from each of the groups (differing in N
or S efficiency) was conducted for comparison of efficiency in the vegetative and the seed
maturity stage. The same three criteria used in preliminary study were applied to evaluate
N or S efficiency at the vegetative stage, with additional four efficiency criteria used at
seed maturity: harvest index, nitrogen harvest index, and oil and protein concentration in
seed.
Genotype Surpass 300 TT (ranked as inefficient at the vegetative stage) had the
highest N concentration in dropped leaves at maturity (making this genotype relatively
inefficient due to N losses via leaf drop). Genotype Wesway was superior to Westar (in
both stages, vegetative and grain maturity, under most of the efficiency criteria used) in N
utilisation efficiency under deficient N but not under adequate N supply (Figure 4.3 &
Figure 4.6) (Balint and Rengel 2008). Hence, out of 12 genotypes, only two genotypes
(Wesway and 46C74) (ranked as efficient at vegetative stage) were still ranked efficient at
maturity under most of the efficiency criteria used. On the other hand, genotype Surpass
603 CL (ranked inefficient at the vegetative stage) was ranked as efficient at maturity
under two criteria (Balint and Rengel 2008).
In terms of S screening study, the results revealed that dry matter production in
tested genotypes at the vegetative stage was influenced by genotypic differences only
(Balint and Rengel 2009), the observation inconsistent with the first S screening study of
canola genotypes (Balint et al. 2008) where S treatment x genotypes interaction existed.
This inconsistency was probably due to S application in the deficient-S treatment in the
second screening study at the vegetative stage (first S screening study had no added S to
deficient S treatment) that resulted in accumulation of S in the vegetative matter initially,
166
followed by remobilization of endogenous S compounds to maintain growth during the
subsequent exhaustion of external S supply (as also reported by Dubousset et al. 2009).
At maturity, the interaction genotype x S treatment in dry matter production was
significant for silique and seed biomass only, indicating an essential role of S in
reproductive growth and/or processes, whereas the impact of S deficiency was less severe
on stem biomass (Balint and Rengel 2009). Canola seed of most genotypes did not
represent a large sink for S remobilisation in the canola genotypes because seed S
accounted for only 20 % of the total S content in canola shoots (Balint and Rengel 2009).
In the study presented here, the ranking of canola genotypes for S utilisation
efficiency significantly differed in the vegetative compared with maturity stage. Genotype
IB 1368 that ranked inefficient under two efficiency criteria during the vegetative stage
was ranked efficient under all criteria at maturity. Conversely, two canola genotypes
(Surpass 402 CL and 46C74) that ranked as efficient under the two criteria at the
vegetative stage did not remain efficient at maturity under most efficiency criteria.
Generally, there was no or little consistency in the S efficiency ranking from the vegetative
stage to maturity in 12 tested genotypes. Therefore, screening canola germplasm for S as
well as for N efficiency for breeding purposes would require an assessment of efficiency at
maturity.
It should be noted that results obtained here, from studies by screening genotypes
for nutrient efficiency in controlled/glasshouse environment could differ (to some extent)
from results obtained via screening under field conditions (e.g. see K-efficiency screening
in barley by Damon and Rengel 2007). Plants grown under the field conditions may be
exposed to various abiotic stresses from which they are protected in the controlled
environment, including drought, high temperature, and frost.
Genotypes with yellow seed coat (IB 1368, IB 1363 and IB 1337) were B. juncea
(Indian mustard) rather than B. napus species. Indian mustard genotypes were ranked
inefficient for biomass production, S accumulation in shoots and SUE in shoots at the
vegetative stage (Balint and Rengel 2009). However, those genotypes at seed maturity
accumulated the highest amount of S in seed, had higher seed yields, higher harvest and S
harvest indices and higher relative seed yields compared to the standard B. napus
genotypes, suggesting better genotypic mobilization of S from vegetative tissues for seed
filling at maturity (Balint and Rengel 2009). These results are in agreement with findings
167
reported by Malhi et al. (2007), whereby S uptake in seed was highest in B. juncea mustard
cultivar compared to B. juncea or B. napus canola cultivars.
Following the second screening study, a group of genotypes with differential N and
S utilisation efficiency in the vegetative and maturity stages were used to investigate the
differences in amino acid concentration in the xylem and phloem sap at the vegetative and
maturity stages. Based on the literature related to N transport, the predominant amino acid
in canola phloem was reported to be glutamine (Mollers et al. 1996), but genotypes more
efficient in remobilizing N were proposed to contain greater amounts and/or proportions of
asparagine, the more efficient N transporter with an N:C ratio of 0.5 compared to 0.4 for
glutamine (Seiffert et al. 1999). The results obtained here revealed that glutamine was the
most abundant N-transporting amino acid in the xylem sap in N-efficient (Wesway and
4674C) and N-inefficient (Westar and Surpass 300 TT) genotypes under both N supplies
(Figure 7.2) (Balint and Rengel 2011a). Interestingly, the concentration of asparagine in
the xylem sap was significantly lower than that of glutamine under both N supplies (Figure
7.2) (Balint and Rengel 2011a). Therefore, asparagine is unlikely to play a major role in
differential N efficiency of the canola genotypes tested.
An interesting observation in the study presented here was that all canola genotypes
tested had higher concentration of aspartate compared to asparagine in xylem sap under
both N supplies (Figure 7.2) (Balint and Rengel 2011a). In other studies, decreased N
supply caused impaired N assimilation in several crop species, leading to specific changes
in the amino acid composition of the xylem sap (elevated aspartate/asparagine ratio and a
decrease in glutamine concentration) compared to plants grown at adequate N supply
(Amarante et al. 2006), indicating that the aspartate/asparagine ratio might be a species-
specific trait.
Genotype Wesway (ranked as N-use efficient) had the highest concentration of
glutamine (235 mmol/L) and asparagine (14 mmol/L) in the xylem sap at adequate N
supply (Figure 7.2) (Balint and Rengel 2011a). In contrast, genotype Westar (ranked as N-
use inefficient) had the highest concentration of these two amino acids (glutamine 60 and
asparagine 3.68 mmol/L xylem sap) at deficient N supply (Figure 7.2) (Balint and Rengel
2011a). The obtained results are indicating negative correlation between the N efficiency
rating of the genotypes and the amino acid concentrations in the xylem sap.
Based on the literature related to S transport, sulphate was the most common S form for
uptake and transport in plants, and its transport from roots to shoots is dependent
168
on/regulated via the amino acid cysteine (Hawkesford and DeKok 2006). On the other
hand, the thiol tripeptides [glutathione (Glu-Cys-Gly) and homoglutathione (Glu-Cys-Ala)]
were reported to play an important role in transporting S in soybean and wheat (Anderson
and Fitzgerald 2001). The results obtained here (Balint and Rengel 2011a) revealed that
concentration of S-containing amino acid cysteine was relatively low (10 mmol/L, Figure
7.4) suggesting continuous cysteine use for methionine synthesis. Therefore, the sulphate
was most likely reduced to sulphide and then via cysteine incorporated into methionine
because methionine was the most abundant S-containing amino acid in the xylem sap
under both S supplies (Figure 7.4) (Balint and Rengel 2011a). In other studies on S
assimilatory metabolism, similar finding was reported, i.e. that the turnover rate of cysteine
was high, but the cellular concentration of free cysteine was maintained low
(approximately 20 mmol/L) (Leustek et al. 2000; Saito 2000). Interestingly, the genotype
Karoo, recognized as S-use inefficient in our previous studies (Balint et al. 2008, Balint
and Rengel 2008), had the highest concentrations of methionine (49.7 mmol/L) as well as
glutamate (36.5 mmol/L) in the xylem sap at deficient S supply (Figure 7.4). Therefore, the
results indicate that the S efficiency rating of the genotypes is negatively correlated with
the concentration of specific amino acid in the xylem sap (genotype Karoo, rated as S
inefficient in two previous studies was ‘expected’ to have, if not lowest than lower
concentration of methionine and/or glutamate compared with S efficient Surpass 402 CL).
Methionine was the most abundant amino acid in the phloem sap of four tested
genotypes but only at the adequate S supply at maturity (Figure 7.6). On the other hand,
the glutamate (one of building blocks of gluthathione) was the most abundant amino acids
in the phloem sap at deficient S supply at maturity (Figure 7.6) (Balint and Rengel 2011a).
In the earlier studies on S uptake in canola, the rate of sulphate uptake was correlated with
the glutathione concentration in the phloem and the roots (Lappartient and Touraine 1996;
Davidian et al. 2000). Therefore, it could be speculated that the S-containing methionine
and the glutathione-precursor glutamate may be the most important amino acids involved
in S transport in the canola genotypes tested here.
8.1 N and S remobilization in canola genotypes
169
Based on the literature, differences in N remobilization could contribute to genotypic
differences in N efficiency (eg. in barley, Mickelson et al. 2003); the same may apply
particularly to canola because most of N used for seed filling in canola was derived from
mobilization of N stored in vegetative tissues (Rossato et al. 2001; Gombert et al. 2010).
The results obtained in the 15
N leaf-labelling study revealed that among all
genotypes tested at the vegetative stage at deficient N supply, Wesway had the lowest 15
N
excess in leaves, but highest in stems 6 days after commencement of labelling, suggesting
more efficient N remobilization from leaves to stems in Wesway compared with other
genotypes (Figure 6.5A) (Balint and Rengel 2011b). Interestingly, Wesway had the lowest
15N recovery in shoots at deficient N supply in the soil-labelling experiment (data not
shown), suggesting that this genotype would assimilate greater proportions of 15
N via
leaves (if given a chance) than via roots under deficient N supply. Similar findings were
reported for the wild-type and nitrate reductase-deficient mutants of pea (Pisum sativum L.
var. Juneau) (Lexa and Cheeseman 1997).
The results obtained at the maturity stage in the soil-labelling study revealed that
contribution of leaf 15
N in above-ground 15
N recovery was 9 % in Wesway and 10.5 % in
Westar, making the two genotypes equally efficient in remobilizing N from leaves to stems
(Figure 6.5B) (Balint and Rengel 2011b). However, the 15
N atom excess in siliques in
Wesway under deficient N supply was significantly lower at the 1st than the 2
nd harvest in
the leaf-labelling study, suggesting intense N remobilisation from vegetative to generative
plant parts (Figure 6.5B) (Balint and Rengel 2011b). Further, genotype Wesway had a
higher 15
N atom excess in seeds than Westar at both harvests under deficient N supply
(Figure 6.5B) (Balint and Rengel 2011b). Therefore, it could be speculated that differences
in N remobilization could indeed contribute to genotypic differences in N efficiency.
In relation to our 34
S soil- and leaf-labelling studies, the results revealed that
genotype Surpass 402 CL was more efficient in using available S compared to Karoo in the
vegetative stage at deficient S supply, as it produced higher dry matter of leaves with lower
tissue concentrations of S than genotype Karoo (Figure 6.6A) (Balint and Rengel 2011b).
Further, 34
S atom excess in leaves and stems of Surpass 402 CL at maturity was higher
than in Karoo, suggesting lower 34
S remobilization from leaves to siliques and seeds in
Surpass 402 CL (Figure 6.6B) (Balint and Rengel 2011b). As a consequence, greater 34
S
losses via dropped leaves in the maturity stage in Surpass 402 CL contributed to this
genotype being less efficient than Karoo. Differential S remobilization from the vegetative
170
(leaves and stems) to the generative plant parts (siliques and seeds) were reported by Malhi
et al. (2007) in four oilseed cultivars. Therefore, genotype Surpass 402 CL appeared to be
more efficient than Karoo in taking up S during the vegetative and particularly during the
maturity stage, but was less efficient than Karoo in remobilizing endogenous S from leaves
and stems to siliques and seeds at maturity.
8.2 Suggestions for further work
In the light of the above discussion several directions for future research are suggested:
T negative correlation between the leaf N concentration and the leaf size at maturity
was reported in some earlier studies on N efficiency in canola genotypes. Further
work is recommended to identify whether genotypes with large leaves are more
efficient at N mobilization than small-leaved ones, and whether this feature
contributed to differential N efficiency in canola genotypes.
The yellow-coated genotype IB 1368 had the highest S concentrations in the shoots
and in the xylem sap in the vegetative stage, indicating a higher capacity to take up
S from the soil compared to other genotypes. At maturity, beside IB 1368, two
more yellow-coated genotypes (IB 1337 and IB 1363) accumulated higher amounts
of S in seed than other genotypes. These genotypes (designated IB) are Brassica
juncea (Indian mustard). Further work is needed to elucidate potentially differential
processes/regulation of xylem loading as well as S accumulation in seed in B.
napus and B. juncea.
Expression of two sulphate transporter genes, Sultrl;3 and BnSultr4;1, essential for
sulphur redistribution in the vegetative stage in canola plants may play an important
role during the maturity stage. Further work is required to ascertain whether
differential expression of these two genes contributed to differential S efficiency of
genotypes under limited S supply.
Higher glutamate concentration in the phloem sap of S-inefficient genotype at
deficient S supply indicated the poor capacity of this genotype to convert glutamate
to glutathione. Further research could investigate this phenomenon.
The negative correlation between nutrient utilisation efficiency rating of genotypes
and the amino acid concentrations of glutamine and asparagine in the xylem sap
171
(for N efficiency) and concentrations of methionine and glutamate in the xylem sap
(for S efficiency) could be further characterised.
Field experiments on N- and/or S-deficient soils should further investigate
differential N and S efficiency of selected canola genotypes. Further work would
include producing doubled-haploid mapping populations from crosses of N and/or
S efficient with N and/or S inefficient genotypes to identify QTLs (Quantitative
Trait Loci) and molecular markers for use in breeding genotypes with increased N
and/or S efficiency.
172
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