ORIGINAL ARTICLE

HVP10 encoding V-PPase is a prime candidate for the barleyHvNax3 sodium exclusion gene: evidence from fine mappingand expression analysis

Yuri Shavrukov • Jessica Bovill • Irfan Afzal •

Julie E. Hayes • Stuart J. Roy • Mark Tester •

Nicholas C. Collins

Received: 16 October 2012 / Accepted: 5 December 2012

� Springer-Verlag Berlin Heidelberg 2012

Abstract In cereals, a common salinity tolerance mecha-

nism is to limit accumulation of Na? in the shoot. In a cross

between the barley variety Barque-73 (Hordeum vulgare

ssp. vulgare) and the accession CPI-71284 of wild barley

(H. vulgare ssp. spontaneum), the HvNax3 locus on chro-

mosome 7H was found to determine a *10–25 % differ-

ence in leaf Na? accumulation in seedlings grown in saline

hydroponics, with the beneficial exclusion trait originating

from the wild parent. The Na? exclusion allele was also

associated with a 13–21 % increase in shoot fresh weight.

The HvNax3 locus was delimited to a 0.4 cM genetic

interval, where it cosegregated with the HVP10 gene for

vacuolar H?-pyrophosphatase (V-PPase). Sequencing

revealed that the mapping parents encoded identical HVP10

proteins, but salinity-induced mRNA expression of HVP10

was higher in CPI-71284 than in Barque-73, in both roots

and shoots. By contrast, the expression of several other

genes predicted by comparative mapping to be located in the

HvNax3 interval was similar in the two parent lines. Previ-

ous work demonstrated roles for V-PPase in ion transport

and salinity tolerance. We therefore considered transcrip-

tion levels of HVP10 to be a possible basis for variation in

shoot Na? accumulation and biomass production controlled

by the HvNax3 locus under saline conditions. Potential

mechanisms linking HVP10 expression patterns to the

observed phenotypes are discussed.

Keywords HvFT � HvVRT2 � Na? exclusion � Salinity

tolerance � Vacuolar H?-pyrophosphatase

Abbreviations

CAPS Cleaved amplified polymorphic sequence

HVP Hordeum vacuolar H?-pyrophosphatase

MFS Major facilitator superfamily

q-RT-PCR Quantitate reverse transcriptase polymerase

chain reaction

ORF Open reading frame

SAM Sterile alfa motif

V-PPase Vacuolar H?-pyrophosphatase

Introduction

Salinity is a major abiotic stress limiting the production of

agricultural plants in Australia and elsewhere (Flowers and

Yeo 1995; Munns 2007). In cereals, certain genotypes are

able to more effectively limit the amount of Na? accumu-

lating in their shoot tissue, and this trait has often been found

to be positively correlated with salinity tolerance, at least for

particular conditions and germplasm (Yeo and Flowers 1986;

Chhipa and Lal 1995; Zhu et al. 2001; Munns and James

2003; Poustini and Siosemardeh 2004; Garthwaite et al.

2005; Colmer et al. 2006; Chen et al. 2007; Shavrukov et al.

2010a). For example, the presence of the Na? exclusion gene

TmHKT1;5-A (Nax2), encoding a Na?-selective transporter,

increased grain yield in field trials on saline soils in durum

wheat by up to 25 % (Munns et al. 2012). Therefore, the

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00425-012-1827-3) contains supplementarymaterial, which is available to authorized users.

Y. Shavrukov (&) � J. Bovill � I. Afzal �J. E. Hayes � S. J. Roy � M. Tester � N. C. Collins

Australian Centre for Plant Functional Genomics,

School of Agriculture, Food and Wine, University of Adelaide,

Urrbrae, SA 5064, Australia

e-mail: yuri.shavrukov@acpfg.com.au

I. Afzal

Department of Crop Physiology, University of Agriculture,

Faisalabad 38040, Pakistan

123

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DOI 10.1007/s00425-012-1827-3

identification of further loci controlling Na? exclusion from

the shoot may provide molecular markers for use in salinity

tolerance breeding. Cloning and characterization of the

underlying genes would also help shed light on the molecular

mechanisms controlling Na? transport.

Members of sub family 1 of the high-affinity potassium

transporter (HKT) group of proteins have been identified at

loci for Na? exclusion and salinity tolerance in wheat,

Triticum spp. (Huang et al. 2006; James et al. 2006; Byrt

et al. 2007; Munns et al. 2012) and rice, Oryza sativa (Ren

et al. 2005). In barley, the HvCBL4 gene cosegregated with

the HvNax4 Na? exclusion locus on chromosome 1H

(Rivandi et al. 2011). HvCBL4 was considered to be a

plausible candidate for the HvNax4 locus, because it

encoded a protein similar to salt overly sensitive 3

(SOS3)—a calcineurin B-like protein that contributes to

salinity tolerance and sodium exclusion in wild-type Ara-

bidopsis (Liu and Zhu 1997, 1998).

Previously our group identified the HvNax3 Na?

exclusion locus on barley chromosome 7H, using popula-

tions derived from crosses between selection-73 of the

Australian feed cultivar Barque and accession CPI-71284

of wild barley (Shavrukov et al. 2010a, b). This locus

controlled *10–25 % differences in seedling leaf Na?

concentrations in plants grown hydroponically in 150 mM

NaCl, with CPI-71284 contributing the desired exclusion

trait. HvNax3 was mapped to a 1.3 cM marker interval, and

comparisons to the sequenced rice and Brachypodium

genomes predicted that this barley interval contained genes

for 16 different classes of proteins. Genes for five classes of

proteins were regarded as potential candidates for the

HvNax3 gene, based on their similarity to proteins with

previously reported roles in ion transport and/or salinity

tolerance (Shavrukov et al. 2010a).

In the current study, we advanced our search for the

HvNax3 gene, using further fine genetic mapping, along

with comparisons to sequenced grass genomes, barley BAC

clone sequencing, allele sequencing, and mRNA expression

analysis. On the basis of our findings, and the literature,

the HVP10 gene encoding vacuolar H?-pyrophosphatase

(V-PPase) was identified as the best candidate for the

observed shoot Na? phenotype, with differences in mRNA

expression levels between the mapping parents potentially

explaining the functional difference between alleles.

Materials and methods

Growth in supported hydroponics and measurement

of Na? concentration in shoots

Mapping populations were derived from crosses between

Australian barley cultivar Barque, selection 73 (Hordeum

vulgare L. ssp. vulgare) and accession CPI-71284 of wild

barley (H. vulgare L. ssp. spontaneum), and were

described earlier (Shavrukov et al. 2010a, b). Seeds were

provided by Dr. Jason Eglinton (Barley Breeding Lab)

and Dr. Ken Chalmers (Molecular Marker Lab) from the

University of Adelaide, Australia. Plants were grown in

PVC tubes filled with black polycarbonate beads that

were periodically flooded and drained with nutrient

solution, as described previously (Shavrukov et al.

2010a). Growth was in a greenhouse during the Adelaide

summer, with natural light (*16 h day) and day/night

temperatures of around 26/16 �C. At around the time of

third leaf emergence (*10 days after sowing), the con-

centration of NaCl in the solution was increased by

25 mM twice a day for three days to reach a final con-

centration of 150 mM NaCl. With each addition of NaCl,

supplementary CaCl2 was also added to maintain the

calcium activity of the solution at 0.98 mM. After

10 days of further growth, the fourth leaf was collected

for DNA extraction and molecular marker analysis, and

the third leaf was collected for Na? determination by

flame-photometry. Na? was quantified in terms of con-

centration in the sap (tissue water basis), as described

previously (Shavrukov et al. 2010a). After a further

20 days of growth in 150 mM NaCl (plants one month of

age), the fresh weight of the entire shoot was measured

for each plant. In a separate experiment under similar

conditions, and at the same time-point, roots of the par-

ents only were harvested, rinsed in 10 mM CaCl2 to

remove traces of salts, blotted on paper-towel, and used

for flame-photometry (in addition to the third leaves).

Molecular markers

Barley orthologues of genes located in the corresponding

HvNax3 marker interval in rice and Brachypodium

(between markers AK374980 (HYI) and AK371206 (NOD))

were used as a basis for designing six new cleaved

amplified polymorphic sequence (CAPS) markers for the

barley genome, using procedures previously described

(Shavrukov et al. 2010a). A similar procedure was applied

to make a marker for the HvVRT2 gene, located near the

middle of barley chromosome 7H (Szucs et al. 2006).

Markers for the HvFT, AK374980 (HYI), and AK371206

(NOD) genes were as described previously (Shavrukov

et al. 2010a). Details of new markers are shown in Table 1.

Platinum High-Fidelity Taq polymerase (Invitrogen) was

used to score the AK250270 marker. Otherwise, procedures

for plant genomic DNA extraction, and CAPS fragment

amplification, digestion, and electrophoresis were as

described by Shavrukov et al. (2010a).

Copy number and chromosome arm designation of

barley genes were determined by searching the shotgun

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sequence assemblies of cv. Morex flow sorted chromosome

arms (Mayer et al. 2009, 2011) using ViroBLAST (Deng

et al. 2007) at http://webblast.ipk-gatersleben.de/barley/

index.php, while cDNA sequences of barley genes were

identified by searching full-length cDNA sequences (Mat-

sumoto et al. 2011) at the same site. Genes in rice and

Brachypodium were identified using annotations provided

in the genome browsers at http://rice.plantbiology.msu.edu/

cgi-bin/gbrowse/rice/ and http://gbrowse.brachypodium.org/

cgi-bin/gbrowse/brachypodium-gbrowse/ and by BLAST

searches of the genomic sequences.

Genetic mapping

From a previous screen of 960 Barque-73 9 CPI-71284 F2

plants (Shavrukov et al. 2010a), we identified 16 recom-

binants for the HvNax3 marker interval AK374980 (HYI) to

AK371206 (NOD). These 16 lines were scored for the six

new intervening CAPS markers to create a framework

molecular marker map of the interval. To locate HvNax3

on the map, HvNax3 genotypes of four F2 recombinants

were determined, by testing F3 progeny of each F2

recombinant in saline hydroponics. For each F3 family,

80–100 individuals were grown in an independently irri-

gated hydroponics unit that also included six plants each of

Barque-73 and CPI-71284 as controls. Every plant was

phenotyped for Na? accumulation in sap of the third leaf,

and for shoot fresh weight. In each F3 family, individual

plants were also scored for segregating molecular mark-

ers in the HvNax3 chromosome region, to identify

plants homozygous for each of the two chromosome

types inherited from the F2 parent (recombinant and

non-recombinant chromosome types). Within each F3

family, the means for the two homozygous classes were

compared by Student’s t test (unpaired, equal variance) to

determine if the corresponding F2 recombinant was

homozygous or heterozygous for HvNax3. The HvFT and

HvVRT2 markers were mapped by scoring them in the

entire population of 960 F2 plants.

Genomic sequencing

To obtain genomic sequence from the barley HvNax3

region, the 3.7 9 genome-coverage barley cv. Morex BAC

library HVVMRXALLeA (Schulte et al. 2011) was ini-

tially screened for BAC clones containing the HVP10 gene.

The library was screened by PCR from the BAC clone

DNA super-pools and matrix-pools constructed by

Amplicon Express (Ariyadasa and Stein 2012; http://

ampliconexpress.com/), using the two primer pairs: PYR-

31F ? PYR-32R (455 bp product) and PYR-33F ? PYR-

34R (567 bp product) (Table 2). The single positive clone

identified, 0262H05, was kindly provided by Nils Stein

(IPK, Gatersleben, Germany). The BAC clone DNA was

purified using Large-Construct Kit (Qiagen) following the

manufacturer’s protocol, and sequenced at the Australian

Genome Research Facility (Brisbane, Australia) using a

454 GSFLX analyzer. Sequence contigs were assembled

using Newbler software (Roche Diagnostics) and used in

BLAST searches of NCBI-databases to identify genes.

Five primer pairs were designed to amplify and

sequence overlapping genomic fragments spanning the

entire HVP10 gene, from 103 bp upstream of the

start-codon to 384 bp downstream of the stop-codon:

Table 1 Details of new CAPS markers used to fine map the HvNax3 interval

Barley genea Primer sequence (50–30) Restriction

enzyme

Digestion product size (kbp)

Barque-73 CPI-71284

AK251117 (F)-GACCGTTTAGACCTATTGGTGG

(R)-GATTACGTTCCAAACGGAGTCT

TaiI 1.0 0.8 ? 0.2

AK251445 (F)-CAAGGACACCACAACTGTGAGCCTG

(R)-AAGTAGGCCATGTTTAGCATATGTG

PflMI 1.2 1.0 ? 0.2

AK360851 (F)-CTGACCAGCTTCGAGAACTCGTTCG

(R)-AGGTGTGTTACTGCAGCTAGTGCTG

HaeII 0.5 ? 0.2 0.7

AK250270 (F)-TCAAGAGGAACACCTTCCGTGGCTC

(R)-TCCACCATTCAGGTTCATACCATTG

N/Ab 2.0 2.3

AK250655 (F)-GAGCCGGTATGTGATCGCTGAGCAC

(R)-CAGTGGCTCAATCTCCTTCTCAGGG

DrdI 0.7 ? 0.5 1.2

AK250247 (HVP10) (F)-GTCATCAGCTGGTTGGCTCTTCCAG

(R)-TCCAACACTCTTCATGGTCATGGCA

HincII 1.1 0.9 ? 0.2

AK356695 (HvVRT2) (F)- CAGTATGAACGAGATCATTGACAAG

(R)-TACTATAGGATCATTCCAGTCAGCC

DdeI 1.0 0.8 ? 0.2

a Defined by full-length cDNA sequences (Genbank)b Not applicable. The PCR fragments from the two parents differed for an insertion/deletion

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PYR-7F ? PYR-35R, PYR-13F ? PYR-2R, PYR-1F ?

PYR-10R, PYR-9F ? PYR-14R, and PYR-22F ? PYR-8R

(Table 2). These were used to determine the HVP10 coding

sequences in Barque-73 and CPI-71284.

Real-time RT-PCR

Barque-73 and CPI-71284 plants were grown in supported

hydroponics and NaCl applied as described for the F3 fam-

ilies, except that controls without any NaCl were also

included. At 0, 1, 2, 3, 5, and 10 days after the time of first

salt application, shoots and roots were sampled. For each

genotype, treatment, and time-point, tissue was collected

separately from three individual plants, making three bio-

logical replicates. Tissue samples were immediately frozen

in liquid nitrogen and stored at -80 �C until extracted.

Tissue was ground to a fine powder using 9-mm ball bear-

ings in a Heldolph Multi Reax mixer (John Morris Scientific,

Chatswood, NSW, Australia) or IKA-Vortex Genius 3

(Crown Scientific, Minto, NSW, Australia). RNA was

extracted using a modified method (Chomczynski and Sac-

chi 1987) with a single extraction buffer containing 38 %

equilibrated phenol, 1 M guanidine thiocyanate, 1 M

ammonium thiocyanate, 0.1 M sodium acetate, and 5 %

glycerol similar to TRIzol reagent (Invitrogen). Concentra-

tions were determined by absorbance at 260 nm using a

NanoDrop ND-1000 spectrophotometer (Biolab, Mulgrave,

VIC, Australia) and the RNA integrity was checked by

electrophoresis through a 2 % agarose gel.

A SuperScript III Reverse Transcriptase kit (Invitrogen)

was used to construct first-strand cDNA reactions which

included 1 lg of each RNA sample, oligo(dT)20 primer, and

RNaseOUT ribonuclease inhibitor. cDNA samples were

used for quantitative reverse transcriptase (q-RT) PCR

analysis of the genes AK367830 and AK377134 (encoding

oxoglutarate-Fe(II) oxygenases), AK359427 (encoding beta-

glucuronidase), and HVP10, using the primer pairs OFeO-

2-5F ? 6R, OFeO-3-4F ? 2R, b-GUS-3F ? 2R, and

PYR-9F ? 12R, respectively (Table 2). A Rotor-Gene-6000

real-time PCR machine (Corbett Research, Sydney, Austra-

lia) was used. HvGAP transcript levels were used in nor-

malization, to account for variation in cDNA concentrations,

as described by Burton et al. (2008). Means of normalized

transcript levels for various genotype/time/treatment com-

binations were compared using Student’s t tests.

Results

Fine mapping

The gene colinearity between barley and the sequenced

rice and Brachypodium genomes enabled the targeted

generation of six new CAPS markers in the previously

Table 2 Primers used for BAC

library screening, HVP10sequencing, and q-RT-PCR

Name Primer sequence (50–30) Purpose

PYR-31F TGTTCATATTTTATGTTATTGTGCCTT BAC clone library screening

PYR-32R TCTGGATGTGGCAAAAATCA BAC clone library screening

PYR-33F TGATTATGCTTTCTTGATTTTTCA BAC clone library screening

PYR-34R AAGGAGTTGCCCAACAAATG BAC clone library screening

PYR-7F TGCAGGNAATTCGGCACGAGGCCCG HVP10 PCR sequencing

PYR-35R CCATGAACATACCAACGTACTG HVP10 PCR sequencing

PYR-13F ATGGCGATCCTCGGGGAGCTCG HVP10 PCR sequencing

PYR-2R CTGGAAGAGCCAACCAGCTGATGAC HVP10 PCR sequencing

PYR-1F CTCTTTAGCACTGCATCTTTCTTGC HVP10 PCR sequencing

PYR-10R CTTAGCATGCCAAGAGCAGCCATTG HVP10 PCR sequencing

PYR-9F GGTCTGTGGGCTGGTCTGATTATTG HVP10 PCR sequencing and q-RT-PCR

PYR-14R GATGTACTTGAACAGCAGACCTCCG HVP10 PCR sequencing

PYR-22F TGCCATGACCATGAAGAGTGTTGGA HVP10 PCR sequencing

PYR-8R AGCAAGAAACAAGATAAGAATTTAGCAC HVP10 PCR sequencing

PYR-12R GCTGACGTAGATGCTGACAGCAATAG q-RT-PCR

OFeO-2-5F TCTACACGAGTACGCATTGAAGT q-RT-PCR

OFeO-2-6R GAATCTAGCAAAAGTGGGGGCGT q-RT-PCR

OFeO-3-4F TGCATGAGTACACACTGAAGAGC q-RT-PCR

OFeO-3-2R ATCTAGCATAAGTGAGAGCCCTG q-RT-PCR

b-GUS-3F TGTTGACACGCATCTGGTTGAGA q-RT-PCR

b-GUS-2R TCCTGCGGACTTCAGTATCCCCT q-RT-PCR

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defined HvNax3 interval, between the genes AK374980

(HYI) and AK371206 (NOD) (Fig. 1). A *40 kb section

of this interval is tandemly duplicated in rice but not in

Brachypodium (Shavrukov et al. 2010a) (Fig. 1). Searches

of the barley cDNA and chromosome arm shotgun

sequence databases and our PCR/sequence analysis of

barley gene fragments indicated that this gene block was

not duplicated in barley (data not shown). Apart from the

lack of the duplication in the barley genome, the new

markers mapped in the same linear order in barley as the

corresponding genes in the rice and Brachypodium

genomes.

To locate HvNax3 on the high-resolution map, HvNax3-

genotypes of four of the F2 recombinants were determined

by evaluating F3 progeny families in saline hydroponics.

This allowed the HvNax3 locus to be placed proximal of

the AK250655 gene (Fig. 1). Previous work located

HvNax3 one recombinant distal of the AK371206 gene

(Shavrukov et al. 2010a). Considered together, these data

placed the HvNax3 locus in the 0.4 cM interval between

genes AK250655 and AK371206, where it cosegregated

with gene AK250247 (Fig. 1).

Interestingly, F3 plants carrying the CPI-71284 allele of

HvNax3 also showed an average of 15.5 % greater shoot

fresh weight than those plants containing the Barque-73

allele, one month after exposure to 150 mM NaCl (Fig. 1),

suggesting that the HvNax3 locus can also influence plant

growth under saline conditions.

Fig. 1 Genetic mapping of the HvNax3 region on the short arm of

barley chromosome 7H. The barley genetic map is shown at the top,

and includes the number of recombinants identified for each interval

from the population of 960 Barque-73 9 CPI-71284 F2 plants. The

three recombination events to either side of HvNax3 (top of figure)

represent recombinants whose HvNax3 genotypes were determined in

this and our previous study (Shavrukov et al. 2010a). The middle

portion of the figure represents Na? accumulation assays performed

on F3 progeny families of four F2 recombinants (4 M, 6 M, 8 M and

10 M). The bars represent the two chromosomes from each F2

recombinant, with white and black indicating segments derived from

Barque-73 and CPI-71284, respectively. Na? accumulation in third

leaf and shoot fresh weight (mean ± standard error) of F3 plants

homozygous for the respective chromosomes and for parents are

indicated to the right. In each F3 family, a Student’s t test showed

where the homozygous groups were significantly different, or not

(n.s.). These results were used to determine the HvNax3 genotype of

each recombinant and the position of the HvNax3 locus. For each

family 18–31 plants were sampled for each chromosome type, and six

plants were analyzed for each of the two parents. The bottom portion

of the figure illustrates the locations of the corresponding genes in the

rice and Brachypodium genomes, and includes the tandemly dupli-

cated segment in rice. Only the mapped genes are represented outside

the HvNax3 interval in rice and Brachypodium

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As expected, parental controls that were assayed

alongside the F3 families showed lower third leaf Na?

concentrations in CPI-71284 than in Barque-73 (Fig. 1). In

a separate experiment under similar conditions, the parents

only were analyzed for Na? content in the roots as well as

in third leaves. Root Na? concentrations were higher in

CPI-71284 than in Barque-73 (238.8 ± 4.1 mM vs.

214.2 ± 3.0 mM, respectively), while differences in con-

centrations of third leaves were in the expected direction

(216.5 ± 5.1 mM vs. 256.4 ± 4.5 mM, respectively),

suggesting that HvNax3 may influence leaf and root Na?

content in opposite directions.

The putative flowering time genes HvFT and HvVRT2

are also located near the middle of wheat and barley Tri-

ticeae group 7 chromosomes (Szucs et al. 2006; Yan et al.

2006; Kane et al. 2005, 2007). There were no obvious

flowering time or gross morphological effects controlled by

this chromosome region in the Barque-73 9 CPI-71284

cross (data not shown). In addition, HvFT and HvVRT2

genes were mapped 6.35 cM distal and 2.4 cM proximal of

HvNax3, respectively (Fig. 1), making it very unlikely that

any developmental effects controlled by these genes might

be the basis for HvNax3 Na? accumulation or shoot fresh

weight phenotypes.

Interval gene content

The two genes defining the smallest HvNax3 genetic

interval, AK250655 and AK371206 (NOD), encode a pre-

dicted 2-oxoglutarate-Fe(II) oxygenase and a protein

showing similarity to the major facilitator superfamily

(MFS) of proteins, respectively. In rice/Brachypodium, the

orthologues of these genes occur in clusters. BLAST

searches against the barley full-length cDNA and chro-

mosome arm shotgun sequence databases indicated that the

barley genes probably also occur in clusters of at least five

and two genes, respectively (Table 3). Therefore, it is

possible that as yet unmapped genes of either of these two

classes may be present in the defined HvNax3 genetic

interval. The HvNax3 cosegregating gene AK250247 is the

previously described HVP10 gene (Tanaka et al. 1993;

Fukuda et al. 2004) encoding a vacuolar inorganic H?-

pyrophosphatase (V-PPase). The colinearity with rice and

Brachypodium predicts the presence of two more genes in

the barley HvNax3 interval, encoding a putative beta-

glucuronidase, and a member of the sterile alpha motif

(SAM) domain containing family of proteins (Table 3).

On the basis of this information, the HvNax3 genetic

interval could contain up to eight genes, encoding five

classes of proteins.

The SAM domain family of proteins interacts with

proteins and RNAs to facilitate a wide range of biological

functions (Kim and Bowie 2003). To our knowledge,

neither SAM proteins nor beta-glucuronidases have been

implicated to play a role in ion transport or salinity tolerance.

Members of the MFS group of proteins function as trans-

porters for a range of solutes, and Saier et al. (1999) have

defined at least 29 families based on both sequence affinity

and substrate. One of these families (the anion:cation sym-

porters) includes inorganic-anion/Na? symporters, but

members of this family are most likely absent in plants (Pao

et al. 1998). The best matches to the barley proteins in the

Transporter Classification Database: http://www.tcdb.org/

(Saier et al. 2006), was to the OxlT, oxalate:formate anti-

porter from the bacterium Oxalobacter formigenes (Abe

et al. 1996), although the similarity was only 49 %. Among

characterized MFS proteins from plants (Tsay et al. 1993;

Sauer and Stolz 1994; Stolz et al. 1994; Muchhal et al. 1996;

Smith et al. 1997; Vincill et al. 2005; Haydon and Cobbett

2007), the two barley MFS proteins showed the most simi-

larity to the nodulins GmN70 from soybean and LjN70 from

Lotus japonicus, which are anion transporters with a pref-

erence for nitrate (Vincill et al. 2005), although the similarity

to GmN70 and LjN70 (29–30 %) was much lower than to

other uncharacterized proteins from rice or Arabidopsis

(84–90 % and 71–71 %, respectively). Based on this evi-

dence, the barley MFS proteins encoded by the AK371206 or

AK369020 genes appeared unlikely to be involved in Na?

transport. By contrast, V-PPases have known activities

relating to ion transport and have been used to engineer

salinity tolerance in transgenic plants (Gaxiola et al. 2001;

Bao et al. 2009; Li et al. 2010; Pasapula et al. 2011).

Therefore, at this point the V-PPase gene HVP10 was

regarded as the best candidate for HvNax3.

To explore the possibility that there may be other genes

in the barley HvNax3 interval not disclosed using rice or

Brachypodium, a barley cv. Morex BAC clone containing

HVP10 (HVVMRXALLeA-0262H05) was identified and

sequenced. The sequence of the clone was assembled into

four contigs that together totalled 117.3 kb (Supplemental

Table S1). A full-length copy of HVP10 was found to be

present in the 29.7 kb contig, but none of the other

sequences showed similarity to any known proteins or

barley cDNA sequences. Therefore, sequencing of this

BAC clone, which partially spanned the HvNax3 interval,

provided no evidence for the presence of any additional

genes in the interval.

HVP10 sequence analysis

The 2,774 bp coding region of HVP10 was sequenced from

Barque-73 and CPI-71284, revealing seven SNPs between

the two genotypes (Supplemental Fig. S1). None of these

SNPs change the predicted amino-acid sequence. Hence,

no protein sequence differences were observed between the

HVP10 alleles from Barque-73 and CPI-71284 which could

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potentially explain control of the HvNax3 phenotypes by

HVP10.

Comparisons of the Barque-73 and CPI-71284 sequen-

ces to the cv. Morex sequence and the published HVP10

ORF sequences from cv. Kashima (accession D13472)

(Tanaka et al. 1993) revealed three additional SNPs

(Supplemental Fig. S1). None of these alter the amino-acid

sequence of the protein. Together, these data indicate that

the HVP10 protein sequence has a high level of conser-

vation between barley genotypes.

mRNA expression analysis

Selected genes shown or predicted to be in the HvNax3

chromosome region were investigated for functional

polymorphism by q-RT-PCR (Fig. 2). Application of salt

resulted in a transient increase in the abundance of HVP10

transcripts in the shoots and roots of both genotypes, with

maximal levels of induction seen at three days after the first

addition of NaCl (P \ 0.001–0.05). In addition, at day

three, levels of HVP10 transcript in NaCl-treated plants

were higher in CPI-71284 than in Barque-73, in both roots

(P \ 0.05) and shoots (P \ 0.01) (Fig. 2). By contrast, the

other genes examined, including two members of the

oxoglutarate-Fe(II) oxygenase gene family (AK367830 and

AK377134) and the beta-glucuronidase gene (AK359427),

showed no genotypic differences in expression, although in

both genotypes, NaCl exposure significantly suppressed or

induced expression of AK367830 and AK377134 in specific

tissues and at specific time-points (Fig. 2). Transcript

quantification therefore revealed allelic differences

between the mapping parents, only for the HVP10 gene. In

the absence of protein sequence differences between the

mapping parents, these genotypic differences for expres-

sion in roots and shoots could potentially account for

control of the HvNax3 phenotype by HVP10.

Discussion

In our previous study (Shavrukov et al. 2010a), we iden-

tified five possible classes of candidate genes for HvNax3.

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to be located in the HvNax3 region (AK251117 and

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proteins, respectively) have now been mapped in barley

and were separated from HvNax3 by recombination

(Fig. 1). A third candidate (encoding a predicted DELLA

protein) has a copy in the corresponding interval in Brac-

hypodium (Bradi1g47900) but not in rice (Shavrukov et al.

2010a), and searches of the barley chromosome arm shot-

gun sequence database revealed that the barley HvNax3

interval also lacks a copy of this gene (data not shown).Ta

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Planta

123

Fig. 2 Quantitative reverse transcriptase PCR (q-RT-PCR) analysis

of genes mapped to the HvNax3 interval (HVP10, AK367830 and

AK359427), and predicted to be located nearby (AK377134). Tran-

scripts from each gene were quantified in roots and shoots of the

barley mapping parents Barque-73 (squares) and CPI-71284 (circles),

for up to 10 days after the addition of salt stress (filled shapes), or in

unstressed plants of the same age (open shapes). Each data point

represents the mean ± SE of three biological replicates

Planta

123

The fourth candidate encodes a MFS transporter. While

this gene (AK371206; NOD) was mapped one recombina-

tion event proximal to HvNax3 (Fig. 1), it is possible that

the second (unmapped) copy of this gene in barley

(AK369020) may be present in the HvNax3 interval, based

on the presence of tandemly duplicated copies of this gene

in Brachypodium (Table 3). However, based on the results

of database and literature searches, the chances that this

particular MFS protein transports Na? appeared to be rel-

atively slim.

The remaining gene in the interval is HVP10 encoding

a vacuolar inorganic H?-pyrophosphatase (V-PPase).

V-PPase represents one of two classes of tonoplast-resident

transporters in plants (the other being H?-ATPase) that

establish an electrochemical H?-gradient across the tono-

plast by pumping protons into the vacuole. In the case of

V-PPase, this process is energized by the hydrolysis of

inorganic pyrophosphate (PPi) to phosphate ions (reviewed

in Maeshima 2000). Such a proton gradient provides the

driving force required for antiporters, such as the vacuolar

Na?/H? antiporter NHX, to transport Na? ions into the

vacuole (Blumwald and Gelli 1997; Gaxiola et al. 2002).

This process reduces cytoplasmic Na? concentration and

minimizes the toxic effect Na? can otherwise have on

important metabolic processes (Vasekina et al. 2005; Bayat

et al. 2010). Overexpression of V-PPase in transgenic

plants has also been shown to enhance salinity tolerance in

several species (Gaxiola et al. 2001; Gao et al. 2006; Guo

et al. 2006; Zhao et al. 2006; Bao et al. 2009). In the

current study, HVP10 cosegregated with the HvNax3

sodium exclusion locus (Fig. 1). Furthermore, in CPI-

71284, which carries the Na? excluding HvNax3 allele,

HVP10 showed a higher level of mRNA induction in

response to salt, both in the roots and in shoots, than the

HVP10 allele in Barque-73. Complete sequencing across

the barley HvNax3 interval may yet reveal the presence of

other candidate genes in the interval. However, on the basis

of available information, HVP10 would appear to be an

excellent candidate for the HvNax3 gene, with mRNA

expression differences between barley genotypes poten-

tially providing the functional difference between HVP10

alleles.

We observed NaCl-induced HVP10 expression in both

roots and shoots (Fig. 2), whereas Fukuda et al. (2004)

reported it in roots but not in shoots. In part, this discrep-

ancy might be explained by the fact that HVP10 expression

in roots and shoots peaks at between two and five days after

the beginning of salt stress (Fig. 2), and because sampling

for the latter study finished much earlier, at 24 h after the

addition of NaCl. Another explanation may be provided by

the genotype-dependence of salt-induced HVP10 expres-

sion in the shoots (Fig. 2), i.e., like Barque-73, the barley

cv. Kashima used by Fukuda et al. (2004) may have carried

an HVP10 allele whose shoot expression was relatively

unresponsive to salt stress.

Barley has another V-PPase gene HVP1, which has

80 % identity to the HVP10 coding sequence (Tanaka et al.

1993; Fukuda et al. 2004). HVP1 transcripts may also be

up-regulated by NaCl stress in roots or shoots (Fukuda

et al. 2004; Ueda et al. 2006; Ligaba and Katsuhara 2010).

Expression of HVP1 was higher in the shoots than roots

(Ligaba and Katsuhara 2010), whereas HVP10 is more

highly expressed in the roots than shoots (Fig. 2; Fukuda

et al. 2004), hinting that these genes may play reciprocal

roles in different tissues.

Salinity tolerance conferred by transgenically over-

expressing V-PPase has usually been accompanied by

elevated Na? levels in the roots and shoots (e.g., Gaxiola

et al. 2001; Bao et al. 2009; Li et al. 2010). Most likely,

this effect arises because increased sequestration of Na? in

the vacuoles of all cells makes the tissues a stronger sink

for Na? accumulation. However, the net effect is still

anticipated to be a reduction of Na? and its associated

toxicity in the cytoplasm, which would explain the salinity

tolerance effect. Because of the enhanced accumulation of

Na? and other cations, the tissues of transgenic plants

overexpressing V-PPase also exhibit a more negative

solute water potential and a higher relative water content

than those of untransformed plants, leading to enhanced

drought tolerance (Gaxiola et al. 2001; Bao et al. 2009;

Li et al. 2010).

The enhanced Na? accumulation resulting from trans-

genically overexpressing PPases contrasts with the finding

in the current study that reduced shoot Na? concentrations

were associated with higher expression of HVP10. How-

ever, this potential discrepancy can perhaps be explained

by expression patterns. Invariably, V-PPases have been

overexpressed using the CaMV 35S promoter, which pro-

vide high expression in all tissue types. By contrast,

expression of the native HVP10 gene is approximately ten-

fold higher in roots than in shoots (Fig. 2), suggesting that

regulation of native HVP10 in the root will have a far

greater impact on Na? transport than its regulation in the

shoot. In roots, the higher expression levels of HVP10 in

CPI-71284 than in Barque-73 may result in more Na?

being sequestered in the root vacuoles, making less Na?

available for translocation to the shoot in the former

genotype. The differences in HVP10 expression in the roots

(*19 % by three days after salt application; significant at

P \ 0.05) are admittedly small, but so too are the effects of

HvNax3 on shoot Na? accumulation (*10–25 %). Our

finding that CPI-71284 had higher root Na? concentrations

than Barque-73 is consistent with our hypothesis, although

further work will be needed to test whether this difference

in the roots is attributable to the HvNax3 locus rather than

an unlinked gene. The higher expression of the CPI-71284

Planta

123

HVP10 allele in the shoots may also lead to a greater

proportion of shoot Na? being sequestered in the vacuoles.

Salinity tolerance conferred by the CPI-71284 HVP10

allele could therefore derive from two effects—a reduction

of transfer of Na? from the roots to shoots and an increased

compartmentalization of shoot Na? into the vacuoles.

Evidence for the aforementioned hypotheses is still

circumstantial and needs further support. For example, the

proposed differences in root and shoot V-PPase activities

between the parental genotypes need to be tested using

biochemical methods. Indeed, the work of Krebs et al.

(2010) suggests that manipulation of trans-tonoplast proton

pumping activity in the range that is close to or below that

of normal (wild-type) levels cannot impact Na? compart-

mentalization, at least in Arabidopsis. Arabidopsis muta-

tions that eliminated the activity of the other vacuolar H?

pump (V-ATPase) raised the root cell vacuole pH from 5.9

to 6.4, but did not significantly affect salinity tolerance (as

measured by root growth) or plant Na? accumulation levels

(Krebs et al. 2010). Furthermore, V-PPase has other

important plant functions, such as the facilitation of glu-

coneogenesis (Ferjani et al. 2011), auxin transport, and

normal shoot and root development (Li et al. 2005). Con-

ceivably, any change to these major processes arising from

variation in V-ATPase activity could have far-reaching

consequences, including alterations to Na? transport or

salinity tolerance.

In a mapping population derived from a cross between

two cultivated barley genotypes YYXT and Franklin, Zhou

et al. (2012) identified a QTL controlling salinity-induced

mortality and leaf chlorosis in 7-week-old potted plants,

located in the 10.5 cM interval between the markers bPb-

1209 and Ebmatc0016. The HvNax3 locus segregating in

the Barque-73 9 CPI-71284 cross was located between

these same two markers (Shavrukov et al. 2010a). The

implications are that variation for HvNax3 may also exist

between cultivated barleys, and that it may also control

salinity tolerance in soil. In light of the transient nature of

the mRNA expression difference observed between the

HVP10 alleles and the modest differences in Na? accu-

mulation in shoots (10–25 %) and in roots (11.5 %), and

shoot fresh weight (13–21 %) controlled by HvNax3, it

would be prudent to test whether the HvNax3 phenotype is

expressed under other conditions. Accordingly, we are

evaluating the agronomic potential of HvNax3 by testing

near-isogenic barley lines for their performance at field

sites having different levels of salinity.

We conclude that transcription level of HVP10 is a

possible basis for the control of Na? accumulation in both

shoot and root, and biomass production, controlled by the

HvNax3 locus under saline conditions, although further

work will be required to confirm this.

Acknowledgments This work was supported by funding to the

ACPFG from the ARC, GRDC, and the South Australian government.

We gratefully acknowledge Jason Eglinton (Barley Breeding Lab)

and Ken Chalmers (Molecular Marker Lab) for the genetic resources

used as the basis for this project.

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