Journal of Experimental Botany, Vol. 60, No. 12, pp. 3443–3452, 2009doi:10.1093/jxb/erp180 Advance Access publication 10 June, 2009This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

RESEARCH PAPER

A comparative analysis of phenylpropanoid metabolism, Nutilization, and carbon partitioning in fast- and slow-growingPopulus hybrid clones

Scott A. Harding1,2,*, Michelle M. Jarvie2, Richard L. Lindroth3 and Chung-Jui Tsai1,2,4

1 School of Forestry and Natural Resources, University of Georgia, Athens, GA 30602, USA2 School of Forest Resources and Environmental Science, Michigan Technological University, Houghton, MI 49931, USA3 Department of Entomology, University of Wisconsin, Madison, WI 53706, USA4 Department of Genetics, University of Georgia, Athens, GA 30602, USA

Received 12 December 2008; Revised 23 April 2009; Accepted 13 May 2009

Abstract

The biosynthetic costs of phenylpropanoid-derived condensed tannins (CTs) and phenolic glycosides (PGs) are

substantial. However, despite reports of negative correlations between leaf phenolic content and growth of Populus,

it remains unclear whether or how foliar biosynthesis of CT/PG interferes with tree growth. A comparison was made

of carbon partitioning and N content in developmentally staged leaves, stems, and roots of two closely related

Populus hybrid genotypes. The genotypes were selected as two of the most phytochemically divergent from a seriesof seven previously analysed clones that exhibit a range of height growth rates and foliar amino acid, CT, and PG

concentrations. The objective was to analyse the relationship between leaf phenolic content and plant growth, using

whole-plant carbon partitioning and N distribution data from the two divergent clones. Total N as a percentage of

tissue dry mass was comparatively low, and CT and PG accrual comparatively high in leaves of the slow-growing

clone. Phenylpropanoid accrual and N content were comparatively high in stems of the slow-growing clone. Carbon

partitioning within phenylpropanoid and carbohydrate networks in developing stems differed sharply between

clones. The results did not support the idea that foliar production of phenylpropanoid defence chemicals was the

primary cause of reduced plant growth in the slow-growing clone. The findings are discussed in the context ofmetabolic mechanism(s) which may contribute to reduced N delivery from roots to leaves, thereby compromising

tree growth and promoting leaf phenolic accrual in the slow-growing clone.

Key words: Cellulose, chemical defence, development, lignin, poplar, root, salicortin, vascular.

Introduction

Despite their fast growth rates, Populus species exhibit

a propensity toward the accumulation of large foliar

reserves of non-structural phenylpropanoid derivatives

(NSPs) (Lindroth and Hwang, 1996). NSPs are metaboli-cally costly to synthesize, relatively stable, and less available

for growth than more labile carbon (C) forms (Lindroth

and Hwang, 1996; Kleiner et al., 1999; Ruuhola and

Julkunen-Tiitto, 2000; Kandil et al., 2004). In the context

of overall tree fitness, putative metabolic costs (to tree

growth) of NSPs are regarded as a potential trade-off in

favour of enhanced fitness in stressful environments (Lin-

droth and Hwang, 1996; Koricheva, 2002; Glynn et al.,

2007). Whether or how foliar NSP biosynthesis comprises

a trade-off to growth in Populus or other taxa of fast-growing, early-successional tree species remains largely

unresolved (Hamilton et al., 2001; Stamp, 2003; Glynn

et al., 2007). In annual crop species where secondary

metabolite levels often do not exceed a few percent of leaf

dry weight, the cost to growth is estimated to be negligible

(Foyer et al., 2007). However, the flavonoid-derived

* To whom correspondence should be addressed. E-mail: sharding@uga.eduª 2009 The Author(s).

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

condensed tannins (CTs) and the salicin-containing pheno-

lic glycosides (PGs) commonly comprise 1 to >25% of leaf

dry weight in Populus (Lindroth and Hwang, 1996; Harding

et al., 2005), and therefore may represent a significant cost

to growth.

The regulation of NSP accumulation has not been

reported for Populus, but in herbaceous taxa such as

tobacco, foliar NSP concentrations increase as a functionof photosynthetic carbon fixation and diminishing cellular

nitrate-N levels (Matt et al., 2002; Fritz et al., 2006). In

accordance with the tobacco findings, foliar NSP accrual is

promoted in Populus grown under high light, low N, or

elevated carbon dioxide levels (Bryant et al., 1983; Agrell

et al., 2000; Osier and Lindroth, 2001, 2006). Because

Populus are perennial species, they must cope with periodic

changes in a range of environmental factors that affectgrowth. As they mature, they exhibit a number of ontoge-

netic, adaptive changes in growth rate, shoot–root ratio,

and leaf chemistry (Wullschleger et al., 2005; Donaldson

et al., 2006b). Due to their lengthy and complex growth

cycle, a number of factors are expected to contribute to the

regulation of growth and chemical defence, and a number

of rationalizing hypotheses have been developed (reviewed

in Stamp, 2003).The most widely cited theories that have been used to

model NSP accumulation and growth in Populus and

related taxa (Bryant et al., 1983; Herms and Mattson, 1992;

Jones and Hartley, 1999) differ largely in their emphasis on

the importance of N and C use in controlling the partition-

ing of assimilate between NSP and growth. According to

the ‘Protein Competition Model’ (PCM) of Jones and

Hartley (1999), phenylalanine is the N currency shared byphenylpropanoid and protein biosynthesis. At the cellular

level, however, tissue nitrate concentration is now known to

be an important regulator of phenylpropanoid synthesis

(Fritz et al., 2006). In addition, the amino N of phenylala-

nine can be recovered for other uses (vanHeerden et al.,

1996; Singh et al., 1998), making it unclear whether phenyl-

propanoid synthesis would interfere with, or be suppressed

by, protein synthesis.Other reports are in support of a less prominent role for

phenylalanine, and illustrate the importance of C fixation

and use as posited in the ‘growth–differentiation balance

hypothesis’ (GBDH) of Herms and Mattson (1992).

According to the GBDH, NSP synthesis depends on the

availability of labile carbohydrates in relation to overall

growth demand (Mattson et al., 2005; Glynn et al., 2007).

Indeed, biosynthesis of NSP is metabolically costly in termsof C use, and requires substantial sucrose catabolism

following localized induction in Populus (Arnold et al.,

2004). C from acetate groups is sufficient to sustain

flavonoid biosynthesis even when the phenylalanine ammonia-

lyase (PAL) enzyme is inhibited by chemical means in

Salix (willow) (Mattson et al., 2005; Keski-Saari et al.,

2007). Similarly, in Populus, foliar CT content was found to

be correlated with flavonoid pathway gene expression, whileexpression of the upstream PAL gene remained relatively

constant (Harding et al., 2005). Overall, phenylpropanoids

appear to be more directly linked to growth via a trade-off-

based mechanism than do other C-rich secondary metabo-

lites that are not derived from N-containing precursors (e.g.

terpenoids; Honkanen et al., 1999)

The present investigation employed a comparative,

whole-plant approach for the analysis of phenylpropanoid

metabolism and its possible effects on growth of two

Populus genotypes. Genotypic differences in the control ofleaf N supply appeared to be important to foliar NSP

accrual. N concentration was lower in leaves of the slow-

growing clone, although it was similar in roots and actually

higher in stems compared with the fast-growing clone. A

hypothetical scenario is presented by which stem effects on

N delivery to expanding leaves could modulate leaf NSP

accrual and plant growth.

Materials and methods

Plant materials

The two cottonwood clones compared in this study

originate from a suite of P. fremontii 3 angustifolia hybrids

that have been reported on previously (Schweitzer et al.,

2004; Harding et al., 2005; Fischer et al., 2006; Woolbright

et al., 2008). The subject clones, one slow-growing (SG)with relatively high foliar NSP concentrations, and the

other fast-growing (FG) with lower NSP levels, have been

maintained by vegetative propagation at Michigan Techno-

logical University (Houghton, MI, USA). FG is an F1

hybrid and SG is a hybrid backcrossed with the P.

angustifolia parent (TG Whitham, personal communica-

tion). For this study, rooted cuttings, 10–15 cm in height,

were transferred into aerated nutrient solution formulatedfor temperate tree species and modified for Populus

(Harding et al., 2005). Individual plants were maintained in

perlite-filled pots (12 cm38 cm38 cm) bathed in a continu-

ous flow of nutrient solution which received a daily

augmentation of 0.36 mmol l�1 N (2.5 mmol l�1 per week)

with a molar ratio of nitrate-N to ammonium-N of 4.

Distilled water was added daily to the nutrient solution

reservoir as needed to maintain a constant volume. Nutrientsolution was replaced weekly, and a pH of 5.860.3 was

maintained by daily additions of 2 N KOH or 2 N HCl.

Plant growth monitoring and tissue processing

At the start of the growth monitoring period, 15 FG and 17

SG plants ;1 m in height were distributed into four large

(1 m2 surface area) ebb and flow hydroponics tubs. The

selection of similar sized plants was achieved by the use of

staggered plantings of the populations from which experi-

mental plants were selected. Established cuttings of SG

plants were 4–6 weeks older than FG plants at the time ofselection. Approximately 30 cm separated neighbouring

plants in each tub, and each tub contained only one

genotype. Tubs were positioned 30 cm apart and 3.2 m

below a bank of 1000 W, ventilated, metal halide lamps

providing supplemental lighting of 600 lmol m�2 s�1 at

3444 | Harding et al.

plant tops. A photoperiod of 16 h was maintained. Daytime

temperatures in the greenhouse ranged between 25 �C and

32 �C, and night-time temperature was 22 �C. Humidity

was not controlled. Plant height from the base of the stem

to leaf plastochron index (LPI)-0, of the youngest unfurled

leaf, 3 cm in length (Larson and Isebrands, 1971), was

recorded, and the petiole of LPI-0 was marked. Two of the

tubs, containing seven and eight plants of FG and SG,respectively, were harvested 4 weeks after the start of the

experiment. Those plants were used for chemical analysis

only. The remaining plants were harvested 8 weeks after the

start of the experiment for both chemical and biomass

measurements.

Harvesting took place between late morning and mid-

afternoon, and conditions were sunny on both the 4 week

and 8 week dates. Prior to processing, stem lengths aboveand below the marked petiole were measured, and the

number of leaves above the mark recorded. Stem internodes

above the mark (new growth) and in the subjacent mid-stem

region were split with a razor and vacuum-dried. Root

tissue aliquots were snap-frozen in liquid nitrogen for

storage at –80 �C. The remaining root tissue was rinsed,

blotted, and vacuum-dried. Dried roots were divided into

two fractions. The ‘elongating’ root fraction combinedsmall lateral roots, <3 cm in length, with the distal 3–4 cm

of longer lateral roots and primary roots. The ‘coarse’ root

fraction comprised the remaining root tissue, excluding the

basal 3–5 cm. As defined, elongating roots comprised ;30–

50% of total root mass. Leaves at LPI-3 (expanding) and

LPI-6 (source) were snap-frozen in liquid N and stored at

–80 �C. Powder aliquots were later freeze-dried for chemical

analysis. Leaves for biomass determinations were weighedand vacuum-dried.

Chlorophyll fluorescence monitoring

Estimates of variable fluorescence (Fv#¼Fm#–Ft, where Fm#is the maximum fluorescence in the light-adapted state and

Ft is the basal fluorescence in the light-adapted state), and

photosystem II (PSII) quantum yield (QY¼Fm#/Fv#) were

obtained using a hand-held Fluorpen� (Qubit Systems Ltd,

Canada). For each determination, four readings werecollected from each leaf and averaged. LPI-1 to LPI-5 of

three plants of each genotype were monitored every third

day over a 9 d period for the analysis.

Chemical analysis

Dried leaf and stem internode samples were ground through

a Wiley mill to pass a 20-mesh (fibre analysis) or 40-mesh

sieve. N concentrations were measured with a Thermo

Finnigan Flash 1112 elemental analyser. The PGs salicin,

salicortin, and hydroxycyclohexenone (HCH)-salicortinwere measured via high-performance thin-layer chromatog-

raphy (HPTLC) according to Lindroth (1993). PGs purified

via flash chromatography served as standards. Total PG

concentration was determined as the sum of the three

constituent PGs. CTs were assayed using a modified

butanol-HCl method (Porter et al., 1986). CTs purified

from P. angustifolia via adsorption chromatography served

as the standard. Sugar (hexose and sucrose) and starch

levels were determined via the dinitrosalicylic acid method

as modified by Lindroth (2002). Glucose served as the

reference standard. Glucose released from salicin, salicortin,

and HCH-salicortin was determined separately, and the

value subtracted from total sugar. Acid-digestible fibre(ADF; used to estimate cellulose content) and Klason lignin

were estimated gravimetrically using the Ankom 200 Fiber

Analyzer, following sequential extractions in hot acid–

detergent solution (100 �C for 1 h) and incubation in 72%

H2SO4 (3 h).

Statistical analysis

Because only small amounts of tissue from young stem

(internodes 1–6 of both clones) and LPI-3 (SG in particular)

were available per plant for the entire suite of chemicalanalyses, samples from several individual plants were

pooled for the assays that required larger amounts of tissue.

As a result of pooling, the number of replicates was small in

some cases, as specified in the figure legends.

It was clear from preliminary leaf disc screening that

clonal differences in sugar, CT, and PG were essentially the

same at both the 4 week and 8 week dates. Therefore, all 4

week and 8 week data were combined for statistical analysisand simplified presentation. Mixed-effects, nested analysis

of variance (ANOVA) was performed using PROC MIXED

(SAS 9.0), with genotype, position (organ), and (genoty-

pe3position) as fixed effects, and individual tree as a ran-

dom effect in the following model: Yijk¼l+ai+bj+cij+bk(i)+eijk. In this model, l represents the overall means;

ai represents the effect due to genotype i; bj represents the

effect due to position j; cij represents interaction betweengenotype i and position j; bk(i) represents random tree k

effect nested within genotype i; and eijk represents error

factors. Significance testing of linear correlations, and of

selected means comparisons between clones, was performed

by the two-sample t-test (data with normal distribution),

and the Mann–Whitney rank sum test (data with non-

normal distribution) using SigmaStat 3.5.

Results

Height and biomass

During the 8 week monitoring period, shoot incremental

height gain was larger in FG than SG (Table 1). Biomass of

terminal shoot organs emerging during the monitoring

period was greater in FG, primarily due to more rapid leaf

growth. In general, leaf expansion was more rapid and of

shorter duration in FG (Fig. 1A and Supplementary TableS1 available at JXB online). Photosynthetic chlorophyll

fluorescence parameters were measured several times over

a 9 d period to compare the development of photosynthetic

competence in the clones (Fig. 1B and Supplementary Table

S2). The increase in PSII quantum yield and variable

Phenylpropanoid metabolism and N use in Populus | 3445

fluorescence with leaf development was similar for both

clones (Fig. 1B). Dry mass of lower stem internodes did not

differ between clones (Table 1), although it is important to

note that this was expected since lower stems of SG were 4–

6 weeks older than those of FG. The stem biomass trends

are consistent with the interpretation that, relative to SG,

FG exhibited more rapid height growth in elongating

internodes, and more rapid or more sustained radial growthin mature internodes. Root biomass and the root-to-shoot

ratio calculated from averaged root and shoot biomass data

were higher in FG than SG (Table 1).

Phenylpropanoid and carbohydrate concentrations

Non-structural components: In all organs analysed, NSPswere more abundant in SG than FG (Fig. 2; Supplementary

Tables S3, S4 at JXB online). In leaves, sharply higher CT

concentrations accounted for most of the NSP difference

Fig. 1. Leaf expansion and chlorophyll fluorescence. (A) Dry

masses of fully expanded leaves that occupied LPI positions +1,

–1, and –3, denoted as pre(+1), pre(–1) and pre(–3), respectively,

at the start of the 8 week experiment, and of expanding leaves

that occupied LPI positions 2, 4, 5, 8, and 10 at the time of

harvest. Data represent the means and SD of eight plants.

(B) Quantum yield of PSII and variable fluorescence of expanding

leaves. Data represent the means and SD of three plants

for each LPI position, each derived from the mean of 36

instrument readings.

A

B

0

4

8

16

20

Phen

olic

gly

cosi

des

(% d

ry w

t)

SG FG0

2

4

6

8

10

Con

dens

ed ta

nnin

s (%

dry

wt)

LPI-3 LPI-6Int 1-6Int 7-8Int 9-13elongating rootcoarse root

******

***

***

***

******

*****

**

12

Fig. 2. Tissue concentrations of NSP constituents, PG and CT, in

leaves, roots, and stem internodes of the two clones. (A) Phenolic

glycosides. (B) Condensed tannins. Shown are data (% dry weight)

from expanding leaf (LPI-3), fully expanded source leaf (LPI-6),

upper stem internodes near expanding leaves (Int 1–6), upper

stem internodes along a developmental gradient of maturing

source leaves (Int 7–8 and 9–13), and elongating root and coarse

root fractions. Internode number increases basipetally. Replicates

(n) ranged from 11 to 15 plants for each LPI, from 7 to 11 plants

for each subset of internodes, and from 15 to 16 plants for each

root fraction. Comparisons between clones were made for each

organ fraction shown. The two-sample t-test was used to

determine significance of differences between clone means,

indicated by asterisks above the SG histogram bars (*P <0.05;

**P <0.01; ***P <0.001).

Table 1. Height and biomass accrual of hydroponically grown

plants

Means and standard deviations of biometric data were determinedusing n¼8 plants of each genotype. Lower stem leaf biomassincluded twigs. Statistical significance of the difference betweenclone means was determined using the two-sample t-test forparametric data, and the Mann–Whitney rank sum test for non-parametric data (*).

SG FG t df P

Height

Initial height (IH, cm) 10668.8 118610.3 – – –

New height growth (NH, cm) 7863.6 10367.5 8.38 14 <0.001

Incremental change (NH/IH) 0.7460.06 0.8860.1 3.28 14 0.005

Upper stem (new growth) biomass

Leaf (g) 6.460.9 11.962.2 * * <0.001

Stem (g) 7.661.4 7.460.9 –0.46 14 0.656

Total (g) 14.162.1 19.262.9 4.09 14 0.001

Lower stem biomass

Leaf (g) 23.468.1 18.167.9 –1.27 14 0.224

Stem (g) 28.466.8 34.064.6 1.94 14 0.073

Root biomass

Root (g) 12.263.3 19.264.7 3.59 15 0.003

Root-to-shoot ratio 0.18 0.24 – – –

3446 | Harding et al.

between the two clones (Fig. 2B). In upper internodes,

concentrations of both CT and PG were 3- to 5-fold higher

in SG than in FG (Fig. 2). Soluble sugar concentrations

were generally similar in source leaves (LPI-6) of both

clones, but were higher in expanding leaves (LPI-3) of FG

than SG (Fig. 3A; Supplementary Tables S3, S4). In

contrast, soluble sugar contents were lower in the upper

stem internodes of FG than SG (Fig. 3A). Foliar starchconcentrations were higher in leaves and upper internodes

in SG than FG (Fig. 3B, C). NSP and non-structural

carbohydrate concentrations in elongating and coarse root

fractions, though substantial, were smaller than in

leaves, and differed little between clones (Figs 2, 3). Starch

concentrations were nearly identical in the elongating root

fraction of both clones, but were lower in the coarse root

fraction of FG than SG (Fig. 3B, C; Supplementary TablesS3, S4).

Structural components: Lignin content reached a sustained

maximum with respect to stem biomass much earlier in FG

than in SG (Fig. 4A). A histological approach was used to

compare lignification and vascular development of pri-

mary–secondary transitional internodes 3–6. UV autofluor-

escence of stem cross-sections revealed that fibredevelopment and lignification were comparatively slow in

xylem, but not phloem, of SG relative to FG (Fig. 5). The

production of lignifying cells, based on the width of the

autofluorescing zone in the xylem at both stages of

internode growth, was more rapid in the FG clone (Fig. 5,

arrows). Together, the Klason lignin and histological data

were consistent with slowed xylem development relative to

diameter growth of young internodes in SG. Lignin

concentration increased between internode ;10 and mid-stem internodes 20–25 in SG, but not FG (Fig. 4A).

At all internodes analysed, cellulose was more abundant

than lignin, and exhibited a more uniform accrual trajectory

than did lignin (Fig. 4B). However, cellulose content was

substantially lower in stem internodes of SG compared with

FG. Of potential relevance to cellulose biosynthesis and

accrual was the observation that starch concentrations were

higher in stems and leaves of SG than FG (Fig. 3). Leaftissues were used to investigate the possibility of a clonal

difference in the metabolic relationship between starch and

cellulose biosynthesis (Fig. 6). Due to sample limitation (see

Materials and methods), leaf tissues, but not stem tissues,

offered sufficient replication to conduct the correlation

analysis. Strong individual plant entrainment of starch

metabolism throughout leaf expansion was observed in

***

0

4

8

12

16

20

Solu

ble

suga

rs (%

dry

wt)

LPI-3 LPI-6Int 1-6Int 7-8Int 9-13elongating rootcoarse root

A

B

0

2

4

6

8

10

Star

ch (%

dry

wt)

********

***

**

** ****

SG FG

Fig. 3. Tissue concentrations of (A) soluble sugars and (B) starch.

Shown are data (% dry weight) from expanding leaf (LPI-3), fully

expanded source leaf (LPI-6), upper stem internodes near

expanding leaves (Int 1–6), and upper stem internodes along

a developmental gradient of maturing source leaves (Int 7–8 and

9–13), and elongating root and coarse root fractions. Replicate

numbers and significance testing are as in Fig. 2.

A

B

0

5

10

15

20

25

Lign

in (%

dry

wt)

SG

FG

0

10

20

30

40

50

Int 1-6 Int 7-8 Int 9-10 Int 11-12 Int 20-25

Cel

lulo

se (%

dry

wt)

Fig. 4. Lignin and cellulose in developing stems. (A) Klason lignin

and (B) ADF cellulose (% dry weight). Each of the upper internode

data points (Int 7–8, Int 9–10) represents the mean and SD of 4–6

determinations. The mid-stem internode data points (Int 20–25)

represent the means of eight (SG) and 16 (FG) determinations. In

several cases, SD bars are present but are smaller than the data

symbol. Exceptions are the Int 1–6 and Int 11–12 data points

which represent single determinations from pooled stems with no

SD. Internode replicates (n) differ from those in Figs 2 and 3

because pooling of stem internodes necessary for lignin and

cellulose determinations differed from that for other stem internode

assays.

Phenylpropanoid metabolism and N use in Populus | 3447

both clones (Fig. 6). Source leaf starch and cellulose content

were negatively correlated in FG (–0.908) but not in SG

(Table 2). Clonal differentials in cellulose, lignin, and NSP

were smaller in roots than in shoots (Figs 2, 7). A largeincrease in cellulose and a decrease in starch during the

maturation of elongating into coarse roots were observed in

both clones (Figs 3, 7).

Total nitrogen and carbon

After N is taken up, it is transported to expanding leaves

through root and stem tissues where NSP, lignin, sugar, andcellulose metabolism differed between the two clones. Not

surprisingly, the plant-wide distribution of N from roots to

leaves differed between the clones (Table 3). Total N of

elongating and coarse roots differed little between clones,

but was slightly lower in SG than FG (Table 3). Nitrogen

concentrations in mid-stem (internodes 20–25) and upper

stem internodes subtending the source leaves (internodes 7–

13) were substantially higher in SG than FG (Table 3, Figs

2, 4). The N concentration difference between clones in

young internodes (1–6) was less striking, although the

tendency for higher %N in SG persisted. In contrast, foliar

N concentrations were substantially lower in SG than in FG

(Table 3). When the leaf and internode %N data wereexpressed in the form of a leaf:internode N ratio, the ratio

was higher in FG, for both expanding and recently

expanded (source) leaves (Table 3). The N ratio illustrates

the comparative reduction in N distribution to leaves of SG.

Total C was slightly higher in expanding and source

leaves of SG than FG (Table 4). The C:N ratio also was

higher in SG leaves. Thus, the decrease in C:N ratio

between source and expanding leaves of SG was not due toa decrease in total C.

Discussion

Analysis of the six major carbohydrate and phenylpropa-

noid pools (soluble sugar, starch, CT, PG, lignin, and

Fig. 5. Lignin UV autofluorescence of primary–secondary transitional stem internodes. Shown are internode 3 from FG (A) and SG (B),

and internode 6 from FG (C) and SG (D). Images were obtained from 75 lm vibratome sections using an excitation wavelength of

365 nm. Scale bar¼500 lm. The arrow placed across the xylem is to facilitate a comparison of secondary xylem width which was wider

in FG than in SG. pf, phloem fibre; xy, xylem.

Table 2. Correlation analysis of starch and cellulose concentra-

tions in leaves

Parameter R P

LPI-8 cellulose versus LPI-6 starch

SG, n¼11 –0.449 0.16

FG, n¼15 –0.908 <0.001

LPI-3 starch versus LPI-6 starch

All, n¼26 0.938 <0.001

y = 0.91x R2 = 0.88

0

2

4

6

8

10

100 2 4 6 8 LPI-3 starch (% dry wt)

LPI-6

sta

rch

(% d

ry w

t) FG SG

Fig. 6. Regression analysis of starch concentrations between

expanding (LPI-3) and fully expanded source (LPI-6) leaves. Starch

data were collected from leaves of 26 plants for the analysis.

3448 | Harding et al.

cellulose) in developing leaves, stems, and roots of two

hybrid lines was undertaken as a systems approach to

determine whether foliar NSP biosynthesis causes, or di-

rectly contributes to, growth reduction in Populus. A trade-

off would be expected based on published data that growth

often correlates negatively with foliar NSP accrual in

Populus (Lindroth and Hwang, 1996). A metabolic basis

for such correlations has been offered in Salix (willow),where application of PAL-specific inhibitors reduced NSP

accrual while enhancing plant growth (Ruuhola and

Julkunen-Tiitto, 2003).

Recently it has become evident that negative effects of

high foliar NSP on Populus growth may only be significant

under conditions of limiting external N (Donaldson et al.,

2006a). This finding is in line with a central tenet of GBDH

that when growth is limited, and C is not, NSP accrual isfavoured (Herms and Mattson, 1992). The results showed

further that higher rates of foliar NSP accrual can indeed

pose a greater cost to growth when N becomes limiting

(Donaldson et al., 2006a). Based on the present findings,

GBDH can also be used to rationalize the foliar NSP

differences between genotypes, in this case SG and FG,

under N-replete conditions. Total leaf %C was slightly

higher in SG (Table 4), as were the contents of metaboli-cally active sugar and starch (Fig. 2). Together, the C and N

results are consistent with the interpretation that leaf

growth was comparatively N-limited in SG. In nearby stem

internodes, neither biomass growth nor %N was reduced in

SG, relative to FG, during the same period. Because it is

not clear how C availability limited SG leaf growth, the

results potentially provide a degree of validation for the

PCM tenet that phenylpropanoid competition for N withinexpanding SG leaves directly led to their reduced growth

rate (Jones and Hartley, 1999). However, molecular evi-

dence supports nitrate-N sensing rather than consumption

of phenylalanine-N as the basis for phenylpropanoid

stimulation in higher plants (Fritz et al., 2006).

It is worth noting the adaptive benefit that may be linked

with how foliar N supply, foliar NSP accrual, leaf growth,

and overall plant growth are integrated in SG. Foliar CTabundance is a characteristic that appears to vary with soil

N mineralization rates in the ancestral habitats of SG, FG,

and related hybrid genotypes (Schweitzer et al., 2004;

Fischer et al., 2006). This pattern may be of genetic

significance in that CT and other phenolics can affect soil

N cycling, and promote habitation of leach-prone environ-

ments by N-demanding plants (Northup et al., 1995;

Chapman et al., 2006). In such environments, slower plantgrowth may be favoured in exchange for improved soil N

sequestration. In other words, reduced provision of N to

leaves would slow plant growth, coupling growth rate with

the rate of soil N mineralization. In response to lower leaf

N, CT becomes a quantitatively important leaf C sink. In

turn, the CT-enriched detritus of such leaves continues to

promote soil N sequestration and to modulate soil N

mineralization into leach-susceptible forms (Northup et al.,1995; Chapman et al., 2006). Because nitrate reductase

activity is generally low in roots of Populus (Black et al.,

Table 3. Leaf, internode and root total N concentration and

internode-to-leaf ratios in new growth

Total N (% dry weight) data were pooled from both harvests of FGand SG plants. Means and standard deviations were obtained fromat least n¼8 SG and n¼15 FG plants. Statistical significance of thedifferences between clone means was determined using the two-sample t-test for parametric data, and the Mann–Whitney rank sumtest for non-parametric data (*).

SG FG t df P

Expanding leaf 3.560.30 4.260.46 –4.10 24 <0.001

Expanding leaf internodes (1–6) 2.360.24 1.960.49 * * 0.04

Leaf-to-internode ratio 1.5 2.1 – – –

Source leaf 2.960.19 3.960.45 –7.12 24 <0.001

Source leaf internodes (7–13) 2.160.30 1.660.44 3.89 31 <0.001

Leaf-to-internode ratio 1.4 2.4 – – –

Mid-stem internodes 1.660.2 1.260.2 4.42 21 <0.001

Elongating root 3.160.24 3.360.3 –2.29 29 0.03

Coarse root 2.260.15 2.460.2 –1.92 29 0.06

Table 4. Total leaf C content and C:N ratio

Data were pooled from both harvests of FG and SG plants. Meansand standard deviations were obtained from n¼8 (SG) or n¼15 (FG)plants. As determined by two sample t-test, differences betweengenotypes were significant (P <0.001) for all comparisons, and theC:N ratio differed significantly between expanding and source leaf inSG (P <0.001).

SG FG

Total C

Expanding leaf 46.7160.29 45.8860.67

Source leaf 46.6360.97 45.4260.51

C:N ratio

Expanding leaf 13.3161.04 11.0761.25

Source leaf 16.2561.08 11.7661.27

0

10

20

30

40 %

Dry

wei

ght

elongating root coarse root elongating root coarse root

Lignin Cellulose

******

**

SG FG

Fig. 7. Lignin and cellulose concentrations in root fractions. (A)

Klason lignin of elongating and coarse root fractions and (B) ADF

cellulose of elongating and coarse root fractions (both in % dry

weight). Histogram means and SD were determined from n¼14–15

replicates. The two-sample t-test was used to determine signif-

icance of differences between clone means, indicated by asterisks

above the SG histogram bars (**P <0.01; ***P <0.001).

Phenylpropanoid metabolism and N use in Populus | 3449

2002), nitrate-N is an important N transport form in these

species (Siebrecht and Tischner, 1999). In turn, variation in

nitrate availability due to soil/climate conditions could be

an important modulator of phenylpropanoid metabolism

and, perhaps, biomass accrual during Populus ontogeny.

The observations that soluble sugar concentrations were

not reduced and that both starch and CT were elevated in

SG source leaves argue against the idea that leaf demandfor N in that genotype was limited by low rates of C

fixation. This is supported by data showing that the C:N

ratio of SG leaves was ;20–40% higher than that of FG

leaves (Table 4). Chlorophyll fluorescence parameters used

to gauge clonal differences in the efficiency of light

utilization according to Genty et al. (1989) did not provide

any indication that photosynthetic development followed

a different trajectory in SG than in FG (Fig. 1). For bothgenotypes the increase in variable fluorescence (Fv) with leaf

maturation was significant, but there was not a significant

effect of genotype on Fv (Supplementary Table S2 at JXB

online). The quantum yield of PSII did not differ by leaf age

or genotype. From the chlorophyll fluorescence data it does

not appear that the genotype differences in leaf C partition-

ing resulted from, or fundamentally altered, light harvesting

and photosynthesis.A decrease of soluble sugars in the expanding leaf

compared with the source leaf was observed in SG but not

FG (Fig. 2). The sugar concentration gradient observed

between source and sink leaves of SG could be due to

a combination of slowed secondary xylem development in

the connecting internodes (Figs 4, 5) and/or enhanced levels

of sugar-demanding metabolic activities there. Mass trans-

port of water through the xylem is an important facilitator

of phloem sugar transport in taxa such as Salix and Populus

where phloem loading is passive (Munch, 1930; Turgeon

and Medville, 1998). Therefore, a reduced rate of secondary

xylem growth in younger internodes could affect the phloemtransport of sugars. In addition, source leaf sugars destined

for expanding leaves were routed through a region of

developing internodes that was highly NSP enriched in SG.

Primary internodes are one likely site of PG biosynthesis in

Populus and related taxa (Clausen et al., 1989) and,

therefore, the internodes could exhibit enhanced sink

strength relative to developing leaves. The predominant PG

in both clones is salicortin (Rehill et al., 2005) which is 42%glucose by weight. Therefore, the relatively high PG content

of young SG internodes (;12% dry weight versus ;3% in

FG) may reflect a substantial demand for soluble sugars, in

addition to the basal C demand for phenylpropanoid

skeletons. The high level of stem NSP biosynthesis could

thus affect growth by competing with younger, more sink-

like leaves, for soluble sugars in the transport stream. It is

also possible that demand by expanding SG leaves forimported sugars was low due to an effect of slower xylem

development on N delivery.

A unifying and interpretive scenario of the present

findings is shown in Fig. 8. A cumulative effect of some

Fig. 8. Proposed phenylpropanoid effects on N distribution and vascular development in SG and FG. Enhanced lignification in roots and

lower stems of SG is proposed to reduce N distribution, represented by the term ‘flux’, into upper internodes. A decrease in foliar %N

results, which favours starch and CT accrual at the expense of cellulose deposition in developing vascular traces. With less cellulose

scaffolding, lignification is reduced and NSPs such as the PGs become the predominant phenylpropanoids. Ultimately, both reduced

vascular development and high sugar demand for NSP biosynthesis interfere with the provision to expanding leaves in SG.

3450 | Harding et al.

metabolic process(s) in roots and stems on N distribution is

posited to reduce the provision of N to expanding leaves

while promoting pooling of N in SG stems. While starch

and CT were both elevated in leaves of SG, consistent with

a perceived N limitation, cellulose accrual was lower than in

FG. In contrast to FG, there was not a correlation between

foliar starch and cellulose contents in SG. The absence of

a correlation could reflect strong interference by competingsynthetic activities for starch-derived glucose in SG. Under

the circumstances of lower foliar N in SG, CT synthesis

could be an important alternative destination for starch

glucose. Because cellulose precedes lignin deposition during

secondary cell wall growth (Mellerowicz et al., 2001),

reduced cellulose synthesis could have interfered with

lignification of vascular traces, explaining the reduced xylem

fluorescence in elongating internodes of SG (Figs 4, 5). Aloop would thereby be constituted in which reduced

vascular growth in upper stems further impeded N delivery

to leaves. In general, the scenario illustrates how the

relatively low amino acid concentrations commonly ob-

served in leaf metabolic profiles of slower growing, high

foliar NSP clones (Harding et al., 2005) could reflect a foliar

N limitation, due to an inefficient transport system and/or

an N-sequestering process in secondary stems and roots.The use of labelled N will be necessary to determine

unequivocally the nature of N flux differences between SG

and FG referred to in Fig. 8. Besides phenylpropanoid

synthesis, important determinants of variable N flux in trees

also include N remobilization from storage reserves, and

reallocation of N between roots and shoots according to

seasonal growth demands (Millard, 1996; Grassi et al., 2003,

and references therein). The present findings that nutrientdistribution to leaves may be conditioned by metabolic

processes related to stem N use are partially analogous to

findings of others that vascular architectural heterogeneity

affects leaf provision and growth (Orians et al., 2002, and

references therein). Integration of gene networks that control

vascular architecture and chemistry remains a relatively

unexplored topic for future investigation.

Supplementary data

Results of the mixed-model, nested ANOVA for CT, PG,

soluble sugars, and starch are available in Supplementary

Tables S1–S4 at JXB online

Acknowledgements

The authors express their gratitude to Adam Gusse for

excellence in tissue chemical analysis, and to Professor

Thomas Whitham (Northern Arizona University) for pro-viding the cottonwood genotypes. This research was sup-

ported by the US Department of Energy’s Biological and

Environmental Research Program DE-FG02-05ER64112

and by the National Science Foundation Plant Genome

Program DBI-0421756.

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