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Europ. J. Agronomy 42 (2012) 11– 21

Contents lists available at SciVerse ScienceDirect

European Journal of Agronomy

jo u r n al hom epage: www.elsev ier .com/ locate /e ja

ight–nitrogen relationships within reproductive wheat canopy are modulatedy plant modular organization

essica Bertheloota,b,c, Bruno Andrieub,c,∗, Pierre Martred,e

INRA, UMR 1345 Institut de Recherche en Horticulture et Semences, SFR 149 QUASAV, Beaucouzé F-49071, FranceINRA, UMR 1091 EGC, Thiverval-Grignon F-78 850, FranceAgroParisTech, UMR 1091 EGC, Thiverval-Grignon F-78 850, FranceINRA, UMR 1095 Genetic, Diversity and Ecophysiology of Cereals, 234 Avenue du Brezet, Clermont-Ferrand F-63 100, FranceBlaise Pascal University, UMR 1095 Genetic, Diversity and Ecophysiology of Cereals, Aubière F-63 177, France

r t i c l e i n f o

rticle history:eceived 14 July 2011eceived in revised form 9 March 2012ccepted 12 March 2012

eywords:nternodeeaf laminaeaf sheathitrogen distributionlant structureheat (Triticum aestivum L.)

a b s t r a c t

Within dense canopies, the spatial distribution of nitrogen between leaves in relation to the local lightenvironment is an important and widely investigated adaptive response of plant carbon and nitrogeneconomy. However a general picture of how nitrogen distribution in photosynthetic tissues, not onlyin leaf laminae, is structured at plant scale and is affected by the topology and light environment ofplant modules (i.e. lamina, sheath, internode) is missing. We investigated the spatial patterns of nitrogendistribution in relation with plant botanical structure for wheat (Triticum aestivum L.) culms. Nitrogendistribution between and within laminae, sheaths and internodes was quantified at anthesis and duringgrain filling, for two cultivars with contrasted leaf posture grown in the field under low and high Nfertilization. We found that independently of leaf posture, specific nitrogen mass (i.e. nitrogen mass perunit surface area) was homogeneous within individual laminae and sheaths, although they spanned asignificant depth in the canopy. Sharp changes in nitrogen specific mass at module boundaries wereobserved. At the canopy level, vertical nitrogen gradients resulted from a decrease of mean specificnitrogen mass of individual plant modules with their position along the culm, and laminae and sheathsspecific nitrogen mass decreased linearly with the distance from the top of the canopy. There was no

significant gradient of N concentration on a dry mass basis within and between the enclosed internodes,only the distal part of the ear peduncle, which was exposed to the light, showed a strong N gradient. Thisstudy gives important information to better understand the phenotypic plasticity of nitrogen distributionin wheat and to build a process-based model of N distribution within wheat culms during the post-anthesis period. It strongly supports the idea that processes should be formalized at the module scaleand that a similar formalization can be used for individual laminae and sheaths.

. Introduction

In monocarpic species such as wheat (Triticum aestivum L.), dur-ng the grain-filling period, nitrogen (N) dynamics within the plantre at the core of complex interactions determining grain yieldnd protein concentration. On the one hand, up to 75% of reduced

in cereal leaves are involved in photosynthetic processes and

pecific N mass (i.e., N mass per unit surface area) of the vegeta-ive tissues determines their photosynthetic capacity (Evans, 1989;reccer et al., 2000). On the other hand, the remobilization of N

∗ Corresponding author at: INRA, UMR 1091 EGC, Thiverval-Grignon F-78 850,rance. Tel.: +33 130 815 527; fax: +33 130 815 563.

E-mail addresses: jessica.bertheloot@angers.inra.fr (J. Bertheloot),runo.andrieu@grignon.inra.fr (B. Andrieu),ierre.martre@clermont.inra.fr (P. Martre).

161-0301/$ – see front matter © 2012 Elsevier B.V. All rights reserved.oi:10.1016/j.eja.2012.03.005

© 2012 Elsevier B.V. All rights reserved.

from the vegetative tissues to the grains, which accounts for 30–90%of mature grain N (Mi et al., 2000; Kichey et al., 2006), decreasesleaf photosynthetic capacity and the photosynthetic surface area(Dreccer et al., 1998). The spatial distribution of N between leavesis an important and widely investigated component of these inter-actions.

Many authors have studied the vertical distribution of specificleaf lamina N mass (SLN) between canopy horizontal layers inrelation with the photosynthetic photon flux density (PPFD) atten-uation down the canopy or the cumulative leaf area index (LAI)or green area index (GAI) from the top of the canopy (e.g., Hiroseand Werger, 1987; Hirose et al., 1988; Anten et al., 1995a; Drecceret al., 2000; Bertheloot et al., 2008a). In these studies, leaf laminae

spanning several vertical layers are implicitly treated as clustersof independent tissues acclimated to their local light environment.The correlation between SLN and PPFD within the canopy has beenreported for a number of species during both the vegetative and

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eproductive phases and allows for a higher canopy carbon gainompared with a uniform SLN distribution (Sadras et al., 1993;rindlay, 1997; Drouet and Bonhomme, 1999; Dreccer et al., 2000;ertheloot et al., 2008a). Field (1983) and Hirose and Werger (1987)roposed that within dense vegetative canopies the variation ofLN with cumulated LAI is exponential and that SLN and PFFD dis-ributions are parallel (i.e. nitrogen and light extinction coefficientsre equal), which corresponds to the optimum distribution of N forhotosynthesis (Sands, 1995). However, several works (Kull, 2002;in et al., 2003; Johnson et al., 2010) showed that the vertical distri-ution of leaf N is more uniform than predicted by the optimizationheory.

The optimization theory lets aside two questions. Firstly, planttructure includes both an organization in discrete modules (i.e. inhytomers, each consisting of a lamina, a sheath, and an internode)nd continuous aspects (e.g. the network of vascular elements),hich can be expected to result in correlations between properties

f adjacent tissues, even if submitted to different local environ-ents. Secondly, altogether 35% or more of plant photosynthetic

is distributed in other modules than laminae, such as sheaths,nternodes, and chaffs (Grindlay, 1997), and contributes nearly aalf to crop photosynthesis in the post-anthesis period (Thorne,959; Simpson et al., 1983; Araus and Tapia, 1987; Maydup et al.,010).

Few studies have dealt with N distribution along individual lam-nae in reproductive canopies and frequently no SLN gradient haseen observed despite the existence of PPFD gradients. For exam-le, Bertheloot et al. (2008a) observed no SLN gradient in the flag

eaf laminae for bread wheat canopies after anthesis. Drouet andonhomme (1999) and Hirel et al. (2005) did not observe a clearattern in the SLN distribution along the large lax laminae of fieldrown maize (Zea mays L.) plants after silking. Similarly, Pons et al.1993) reported the lack of SLN gradient along the erect laminae of a

ixture of vegetative and reproductive plants of tor grass (Brachy-odium pinnatum L.) grown both in the field and in growth cabinets,lthough the laminae spanned up to 20 cm of a 1-m-high vege-ation. However, the same authors found positive SLN gradientsrom the base to the tip of leaf laminae of maize plants (Drouet andonhomme, 1999) and of plants of Carex acutiformis Ehrh. (Ponst al., 1993) at the vegetative stage. Similarly, for the lamina of thehird leaf of Italian ryegrass (Lolium multiflorum Lam.), which hasn erect posture, Prioul et al. (1980b) observed a decrease of theuantities of Rubisco and chlorophyll per unit leaf area as well as

decrease in the rate of light-saturated photosynthesis from theip to the base of the leaves. Taken together, these studies suggesthat there is a SLN gradient within individual laminae of monocotlants the vegetative stage, but not at the reproductive stage.

Very few studies have investigated the N content of photosyn-hetic modules other than leaf laminae. Bertheloot et al. (2008a)ave shown, for bread wheat culms during the reproductive stage,hat the N concentration on a dry mass basis (%N) of the individ-al sheaths and internodes was positively related to their positionlong the culms. Within a given phytomer, there was also a sys-ematic ranking between the three types of modules: the %N wasigher for the lamina than for the sheath, and it was higher forhe sheath than for the internode. This is consistent with the rel-tive vertical position of these modules, as the lamina of a givenhytomer is located above its sheath, and the sheath is above theorresponding internode. These results suggest that, as for lami-ae, the vertical distribution of N between sheaths and between

nternodes is determined by their local light environment.The objective of this study was to clarify the pattern of N distri-

ution within wheat canopies during the grain-filling period (butefore the onset of the phase of rapid canopy senescence), at bothhe intra and inter module scale. We focused on wheat culms afternthesis, that is when the architecture, and thus the distribution

onomy 42 (2012) 11– 21

of light is stable and some kind of equilibrium can be expectedfor N distribution. Specifically we addressed the following ques-tions: does N distribution along a plant module follow the PPFDgradient, or is it integrated at the scale of the module? How doPPFD–N relations in the sheaths and internodes compare to thosein laminae? Answering these questions is required to determinethe spatial scale at which N dynamics should be described in afunctional–structural plant model and whether the same frame-work applies independently of the type of module considered. Adetailed picture of N distribution may also give hints on the pro-cesses responsible for the N gradient with depth in the canopyand whether morphological characteristics of a genotype (e.g. leafsize, leaf-to-stem ratio) may interact with N functioning. To answerthese questions, two bread wheat cultivars characterized by con-trasted leaf postures, which affect the pattern of PPFD with depthin the canopy and along individual modules (Angus et al., 1972),were grown in the field under two N fertilization treatments. Theexperiment was repeated during two growing seasons, N distri-bution within main culms was determined each year at anthesisand during the grain-filling period. Specific N mass distributionwas quantified within green tissues of individual laminae, sheaths,internodes, and within the ear peduncle. The vertical distributionsof the mean specific N mass of individual leaf laminae and sheathswere related to their distance from the top of the canopy in orderto approximate the PPFD environment in which they were located.

2. Material and methods

2.1. Plant material and growing conditions

Crops were grown at Grignon, France (48◦51′ N, 1◦58′ E, 70 melevation), on a deep silty loam soil for 2 years. The first year(hereafter 2006), the winter bread wheat (T. aestivum L.) cultivarsSoissons (So) and Caphorn (Ca) were sown on 27 October 2005 ata density of 250 seeds m−2. These two cultivars have contrastedleaf posture, erected for Caphorn and more horizontal for Sois-sons. In addition, ears of Soissons bear awns while those of Caphorndo not. Each cultivar was submitted to two contrasted N fertiliza-tion treatments, denoted N0 and N+. In the N0 treatment, no Nwas applied during the crop growth cycle, while in the N+ treat-ment, 50 and 70 kg N ha−1 were applied as ammonium nitrate attillering and beginning of stem elongation, respectively. Mineralsoil N at the end of winter (31 February 2006) was 90 kg N ha−1 inthe 0.9-m-deep soil profile. In the second year (hereafter 2008), Ndistributions were characterized for Soissons grown under high Nfertilization only. Plants were sown on 12 November 2007 at a den-sity of 228 seeds m−2. Mineral soil N at the end of winter (29 January2008) was 70 kg N ha−1, and 50 and 80 kg N ha−1 were applied asammonium nitrate at tillering and beginning of stem elongation,respectively.

For each cultivar and N treatment, crops were sown in fiveparallel 30 m by 1.6 m plots, with a spacing of 0.2 m between adja-cent plots. The row spacing was 0.175 m. An irrigation system wasinstalled to complement natural rainfall when needed. Crops werekept disease free by chemical treatment. In the first year, a growthretardant (Cycocel C3, BASF Agro, Germany) was applied at a rate of2.0 L ha−1 at the beginning of stem elongation (4 April 2006). In bothgrowing seasons, air temperature was recorded by a weather sta-tion adjacent to the field plots. Thermal time (◦Cd) was calculatedby summing hourly averaged temperatures above 0 ◦C.

In both years, anthesis occurred at the end of May (Table 1)

and, in the first year, was synchronous in both N treatments andwithin 2 days for Soissons and Caphorn. Light and temperature con-ditions differed between years (Table 1), but were not significantlydifferent from long term averages at that site (data not shown).

J. Bertheloot et al. / Europ. J. Agronomy 42 (2012) 11– 21 13

Table 1Sowing date, anthesis date, average air temperature from sowing to anthesis and from anthesis to sowing, and average diurnal photosynthetic photon flux density (PPFD)during the week before plant sampling. Plant were sampled at 95 and 65 ◦Cd after anthesis in 2006 and 2008, respectively (sampling 1), and at 350 and 500 ◦Cd after anthesisin 2006 and 2008, respectively (sampling 2).

Growing season Sowing date Anthesis date Average air temperature (◦C) Average PPFD (mol m−2 day−1)

Soissons Caphorn From sowing to anthesis From anthesis to end of grain filling Sampling 1 Sampling 2

2005–2006 27 October 29 May 31 May 6.3 17.2 30 51

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.2. Plant sampling procedure for N quantification

Plants were sampled shortly after anthesis (95 and 65 ◦Cd afternthesis in 2006 and 2008, respectively), during the grain-fillingeriod, before the phase of rapid canopy senescence (350 and00 ◦Cd after anthesis in 2006 and 2008, respectively, with at leasthree leaves consisting mostly of green tissues), and after grain fill-ng in 2006 (around 850 ◦Cd after anthesis). For each sampling date,ear, cultivar, and N treatment, 0.2 m2 samples (2 rows × 50 cm)ere taken from three central plots in order to get three indepen-ent replicates. The two outer rows on each side of the plots wereot considered. For each replicate, five culms were selected fol-

owing a procedure aiming at identifying median individuals. First,ulms were separated, counted, individually weighed and all culmsith a fresh mass differing by more than 10% from the average massere disregarded. Second, five median culms were selected from

he remaining sample by rejecting the more extreme individualsased successively on the length of the three upper laminae and on

visual assessment of the percentage of necrotic tissues.Selected culms were dissected as described below, according to

heir organization in the following modules: lamina, sheath, intern-de, and the peduncle (Fig. 1). The three upper leaf laminae (Ln,n−1, Ln−2, n being the phytomer number counted from the top),hich were the only ones with a dominant proportion of green tis-

ues at anthesis, and the three upper leaf sheaths (Sn, Sn−1, Sn−2)ere separated from each other and from the rest of the culm. For

he second sampling date in 2006, the four upper internodes (In,n−1, In−2, In−3) were also separated and, for both sampling datesn 2008, the two upper internodes and the ear peduncle (P) wereeparated.

Each lamina was subdivided into segments so as to character-

ze N gradients within the non-senescent tissues. The positionsorresponding to one third and two thirds of the entire laminaength were marked, then non-green tissues were removed andhe remaining tissues were cut into segments according to the

ig. 1. Structure of a wheat culm after flowering. Dashed lines show the way culmodules were dissected into segments in 2008.

16.7 28 50

marks. Therefore, the number of laminae segments kept for fur-ther analysis depended on the length of necrotic tissues at thedistal end of the laminae. Sheaths were devoid of necrotic tis-sues and, in 2006, they were divided in three segments of equallength. In 2008 the procedure was refined. The basal part of thesheaths (ca. 4 mm length) surrounding the stem nodes and con-sisting of tissues thicker and greener than the rest of the sheathswas analyzed separately; the rest of the sheaths was subdividedinto three segments of equal lengths. For the true stem, only theear peduncle and the upper internode (In) were partly exposed tolight, the others internodes were enclosed within the sheaths ofthe subtending phytomer. The ear peduncle (in 2008) and the upperinternode (in both years) were first divided into their enclosed (Penc,Iencn ) and light exposed (Pexp, Iexp

n ) parts, then Pexp was divided inthree segments while Iexp

n was left intact. Each enclosed part ofan internode or fully enclosed internode was divided in a numberof segments depending on its length. Penc and Ienc

n were dividedinto three segments of equal length, In−1 was subdivided into two(in 2006) or three (in 2008), In−2 into two and In−3 was keptintact.

2.3. Determination of leaf lamina curvature, surface areas,lengths, and distances from the top of the ear peduncle

Leaf lamina curvature was determined using the silhouettemethod (Udagawa et al., 1968; Bonhomme and Varlet-Grancher,1978). At anthesis, 10 median culms in each replicate were ran-domly selected among the ones not used for N quantification. Thepseudo stem (sheaths plus internodes) was cut at approximatelymid-distance between each leaf collar and was secured on a verticalboard so that phytomers were in their natural position but laminaelaid in the same plane. Then, photographs were taken with a digitalcamera (D70, Nikon Corp. Tokyo, Japan) and the Cartesian coordi-nates of 10–20 points along each lamina midrib were determinedby image analysis using Optimas v. 6.5 for Windows (Optimas Corp.,Seattle, Washington, USA).

At each sampling date, the laminae, sheaths, Pexp, Iexpn of the

five culms dissected for N determination were scanned (RGB colorimages, 300 dpi resolution) using a Perfection 2400 Photo scanner(Seiko Epson Corp., Nagano, Japan) after having marked the positionof the future segments. The length and the projected areas of eachsegment were determined by image analysis. The developed areaof sheath or internode segments was calculated as the projectedarea multiplied by �. In addition, the lengths of Penc, Ienc

n , In−1, In−2,In−3 were measured with a ruler.

Since PPFD decreases with the distance from the top of thecanopy (Monsi and Saeki, 2005), we calculated for each lamina,sheath, internode, or ear peduncle segment the vertical distancefrom its mid-point to the base of the ear. Calculations were done

from the measured lengths of each internode, sheath and lamina,approximating the stem to a vertical structure. For laminae, we con-sidered the mid-height between the leaf collar and the uppermostpart of the lamina determined from the silhouettes.

14 J. Bertheloot et al. / Europ. J. Agronomy 42 (2012) 11– 21

Table 2(i) Number of culms per unit soil area, N mass, dry mass, N concentration in the three upper vegetative phytomers and green area index of the three upper leaves (GAI*) ofmedian culms, culm total N and dry mass (DM) per unit soil area at the first sampling date before grain filling; (ii) N and DM accumulation in grains and loss from vegetativeparts between the first sampling date and the last one after grain filling. The bread wheat cultivars Soissons and Caphorn were grown in the field at low (N0) and high (N+) Nsupplies during the 2005–2006 growing season, and Soissons at high N supply during the 2007–2008 growing season. Data are mean ± 1 SE for n = 3 independent replicates.P-values for N, cultivar, and year effects are also shown.

2005–2006 2007–2008 Main effects

Soissons Caphorn Soissons N Cultivar Year

Before grain fillingNumber of fertile culms (culm m−2) N0 262 ± 28 197 ± 41 – 0.046 0.008 0.066

N+ 365 ± 25 284 ± 56 308 ± 54

GAI* N0 1.98 ± 0.34 2.18 ± 0.45 – 0.004 0.150 0.303N+ 3.69 ± 0.16 2.92 ± 0.38 4.08 ± 0.37

N mass per culm (mg N culm−1) N0 7.11 ± 1.09 12.00 ± 2.05 – <0.001 0.001 <0.001N+ 14.73 ± 0.46 20.03 ± 1.89 25.92 ± 0.77

Dry mass per culm (g DM culm−1) N0 1.36 ± 0.031 1.88 ± 0.025 – <0.001 <0.001 <0.001N+ 1.28 ± 0.04 1.62 ± 0.014 1.49 ± 0.088

N concentration (×100 g N g−1 DM) N0 0.52 ± 0.071 0.62 ± 0.107 – <0.001 0.43 <0.001N+ 1.15 ± 0.069 1.62 ± 0.014 1.74 ± 0.122

Total N mass (g N m−2) N0 4.16 ± 0.57 5.01 ± 1.17 0.70 0.02N+ 9.88 ± 0.69 10.14 ± 0.52

Total dry mass (g DM m−2) N0 481 ± 47 535 ± 102 0.55 <0.001N+ 675 ± 45 667 ± 38

After grain fillingN mass in grains (g N m−2) N0 2.98 ± 0.34 4.47 ± 0.57 <0.001 0.002

N+ 7.51 ± 0.27 9.00 ± 0.31

Dry mass in grains (g DM m−2) N0 224 ± 23 308 ± 46 <0.001 0.06N+ 410 ± 19 436 ± 28

N lost by vegetative parts (g N m−2) N0 1.85 ± 0.41 2.35 ± 0.60 <0.001 0.42N+ 5.66 ± 0.48 6.14 ± 0.50

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N0 177 ± 29

N+ 238 ± 45

.4. Determination of dry mass and N concentration, andalculation of specific N and dry mass

The dry mass of each sub-sample was determined after ovenrying at 80 ◦C until constant mass. The samples were then groundnd their N concentration (total N mass per unit dry mass, %N) wasetermined by the Dumas method using a NA 1500 CN analyzerFisons Instruments, France). The specific N mass of green laminand sheath segments was calculated as their %N times their dryass divided by their surface area. Specific dry mass of green lamina

nd sheath segments was calculated as their dry mass divided byheir surface area.

.5. Statistics

All statistical analyses were done using R for Windowshttp://www.r-project.org). The existence or not of %N, specific N

ass or specific dry mass gradients within and between individ-al modules was tested using a complete linear model. A complete

inear model was first developed using, as predictors, the samplingate, the treatment, the rank, the module segment position. Thisomplete model was then compared to a nested one without theredictor whose effect had to be tested (phytomer or module posi-ion) by using an F-test. A similar method was used to compare thelopes of the linear relationships linking the distance from the topf a canopy of a module and its specific N mass between sampling

ates, N treatments or module type (sheath or lamina). Differences

n canopy architecture between treatments and in SLN of the flageaf lamina were also tested in a similar manner. Parameters of theinear models were estimated using the least square method.

35 ± 42 0.12 0.1492 ± 18

3. Results

3.1. Experimental treatments created contrasted canopystructures and N contents

Experimental treatments produced contrasted canopy struc-tures. In 2006, for both cultivars, the number of culms per squaremeter was about 40% higher for N+ than for N0, and Soissons had30% more culms per square meter than Caphorn (Table 2). In addi-tion, N fertilization significantly increased the length of the threeupper laminae (P-value < 0.001), sheaths (P-value = 0.005), and ofthe ear peduncle (P-value < 0.001; Fig. 2), but not the length ofthe internodes (P-value = 0.74). The response to N fertilization waslarger for Soissons than for Caphorn so that Soissons had shorterlaminae and equivalent sheaths than Caphorn under N0 but longerlaminae and sheaths under N+. However, the mean distance from agiven module to the top of the canopy did not significantly dif-fer between cultivars or N treatments (data not shown). Meangreen area index of the three upper leaves (GAI*) was 86% and34% higher for N+ than for N0, for Soissons and Caphorn, respec-tively. Under N0 treatment, GAI* was similar for both cultivars;under N+ treatment GAI* was 26% higher for Soissons than forCaphorn.

In 2008, the culm density of Soissons was 16% smaller thanin 2006. On the other hand, the laminae, sheaths, and the earpeduncle were 32% longer, consistent with the absence of growthretardant (P-value = 0.001) and with the warmer conditions in the

pre-anthesis period in 2008 compared to 2006. This led to a higherdistance between the mean height of each module and the top ofthe canopy in 2008 than in 2006. On the contrary, GAI* was almostsimilar between 2006 and 2008.

J. Bertheloot et al. / Europ. J. Agr

Fig. 2. Length of the three upper laminae (a), sheaths (b), internodes (n, n − 1, n − 2,counted from the top) and of the ear peduncle (P) (c) for crops of the bread wheatcultivars Soissons (So) and Caphorn (Ca) grown at low (N0) and high (N+) N suppliesin 2006 and Soissons grown under high N supply in 2008. Plants were sampled at9 ◦

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5 and 65 C d after anthesis in 2006 and 2008, respectively. Data are means ± 1 SEor n = 3 independent replicates.

Experimental treatments also produced canopies with different content at anthesis, as indicated by N mass and concentration

%N) in the upper three vegetative phytomers of the median culmsnd by culm total N mass per square meter (Table 2). These vari-bles were ca. two-folds higher for N+ than for N0, in 2006 for bothultivars. Dry mass of the upper three vegetative phytomers wasa 10% lower for N+ than for N0; on the contrary, total dry masser square meter was ca 30% higher for N+ than for N0, mainly dueo a higher number of fertile culms per square meter under N+.

hen comparing Caphorn and Soissons in 2006, the N mass in the

pper three phytomers was 41% and 26% higher for Caphorn thanor Soissons, for N0 and N+, respectively. The differences in N massetween the two cultivars were mainly due to differences in dryass, while the %N was similar for the two cultivars. At the scale of

onomy 42 (2012) 11– 21 15

the crop, no significant differences were observed for total N anddry masses per square meter, due to a higher number of culms persquare meter for Soissons than for Caphorn. In 2008, N mass forSoissons was 43% higher than in 2006, due to higher %N and drymass.

For all treatments in 2006, the first sampling date was char-acterized by low grain N and dry mass (data not shown), whichconfirms that this date was before grain filling. At the end of grainfilling, grains had accumulated ca. 4.4 g N m−2 more for N+ than forN0 and ca. 1.6 g N m−2 more for Caphorn than for Soissons. Thiswas accompanied by a N loss in the vegetative parts ca. 3.8 g N m−2

higher for N+ than for N0. Grains had accumulated ca. 160 g DM m−2

more for N+ than for N0 and ca. 50 g DM m−2 more for Caphorn thanfor Soissons.

Altogether the treatments produced a contrasted range ofculm density, plant size, GAI*, N status, and N remobilization tograins.

3.2. Specific N mass was homogeneous along individual laminaeand sheaths despite a significant gradient of specific dry mass

For the three upper leaves, specific N mass, specific dry mass and%N distributions were quantified along the green tissues of eachlamina and sheath. At the first sampling date, green tissues repre-sented more than two thirds of the length of any lamina. Resultswere essentially the same for the two sampling dates and years andonly results for the first sampling (95 ◦Cd after anthesis) in 2006 arepresented in detail below (Fig. 3).

For all cultivar/N treatment/sampling date combinations, spe-cific N mass (i.e. N mass per unit area) did not show significantvariations along the laminae (P-value = 0.95), although each laminaspanned 8.6–20 cm of the canopy height and was thus submit-ted to a heterogeneous light environment (Fig. 3a, d, g, j). SpecificN mass of the sheaths did not vary (P-value = 0.55) between theupper two segments, but in 2006 it was on average 46% higherin the basal segment. In 2008 the basal part of the sheath enclos-ing the nodes were analyzed separately which showed that thehigher specific N mass of the basal sheath segments in 2006was due to a short (∼4 mm) region of thicker tissues at the verybase of the sheath (data not shown). These tissues had similar%N compared to the rest of the sheath but ca. two-times higherspecific N mass than the rest of the sheath, due to a high spe-cific dry mass. Specific N mass was homogeneous along sheathsin which these tissues were removed (P-value = 0.91; data notshown).

There was a significant gradient of specific N mass betweenlaminae and the specific N mass of laminae decreased with lowerphytomer rank (P-value < 0.001). Similarly, there was a significantgradient of specific N mass between sheaths of different phytomers(P-value < 0.001). For all cultivar/N treatment/sampling date com-binations, there was a clear discontinuity in specific N mass withinthe leaves between the sheath and the lamina. The specific N massof the distal part of the sheath was 40–70% lower than that of thebasal part of the lamina.

For both cultivars and N treatments, a significant decrease ofspecific dry mass (P-value < 0.001) was observed at all samplingdates between the base and the tip of the laminae and of thesheaths (Fig. 3b, e, h, k). These gradients were steeper along thesheaths than along the laminae. In addition, the gradient of spe-cific dry mass was much steeper in the basal part of the sheathdue to the region of thicker tissue. The mean specific dry mass of

laminae and sheaths decreased (P-value < 0.001) down the canopy.However, differences in mean specific dry mass were smaller thandifferences in specific N mass. In 2006, mean lamina and sheathspecific dry mass decreased down the canopy by 21% and 33%,

16 J. Bertheloot et al. / Europ. J. Agronomy 42 (2012) 11– 21

Fig. 3. Distance from the leaf collars versus lamina (above the horizontal dotted lines) and sheath (below the horizontal dotted lines) specific N mass (a, d, g, and j), specificd for thc +) N su

rt

t

ry mass (b, e, h, and k) and N concentration (c, f, i, and l) at 95 ◦Cd after anthesisultivars Soissons (So) and Caphorn (Ca) grown in the field at low (N0) and high (N

espectively, and their specific N mass by 53% and 68%, respec-ively.

The homogeneity of specific N mass from the base to the tip ofhe laminae and sheaths despite a decreasing gradient of specific

e three upper leaves (n, n − 1, and n − 2; n being the flag leaf) of the bread wheatpplies in 2006. Data are mean ± 1 SE for n = 3 independent replicates.

dry mass means that an opposite gradient (P-value < 0.001) of %Nexisted (Fig. 3c, f, i, l). However, between successive phytomers, thegradient of %N and specific dry mass were parallel and both con-tributed to the vertical gradient of specific N mass. In 2006, mean

J. Bertheloot et al. / Europ. J. Agronomy 42 (2012) 11– 21 17

Fig. 4. Distance from the top of the ear peduncle versus N concentration of the enclosed (closed symbols) and exposed (open symbols) parts of the ear peduncle (P) and oft read wa issono licates

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he upper four internodes (In , In−1, In−2, and In−3) at 350 ◦Cd after anthesis for the bnd high (N+) N supplies in 2006 and at 65 and 500 ◦Cd after anthesis for cultivar Sor ear peduncle are linked by a line. Data are mean ± 1 SE for n = 3 independent rep

amina and sheath %N decreased down the canopy, by respectively1% (P-value < 0.001) and 51% (P-value < 0.001), from phytomers no n − 2.

.3. N mass per unit dry mass was homogeneous for the hiddenarts of all internodes, while it increased along the exposed part ofhe upper internode and the ear peduncle

The distribution of N along the true stem was analyzed shortlyfter anthesis (in 2008, 65 ◦Cd after anthesis), during the early lin-ar stage of grain filling (in 2006, 350 ◦Cd after anthesis) and latern during grain filling (in 2008, 500 ◦Cd after anthesis; Fig. 4). Thehree lower elongated internodes (i.e. In−3, In−2, In−1) were hid-en by the sheaths and did not show gradient of specific N masslong their length (P-value = 0.77), except for In−1 in 2008, whichhowed higher %N in the upper segment. Similarly, no gradientas observed along the enclosed part of the upper internode (P-

alue = 0.17) or of the ear peduncle (P-value = 0.89). In 2008, at5 ◦Cd after anthesis the %N of the enclosed part of the ear pedun-le was significantly higher (P-value < 0.001) than that of the lowernternodes. At 350 and 500 ◦Cd after anthesis, %N of the enclosedart of the upper internode and the ear peduncle were not sig-ificantly different from that of the lower internodes. The generalicture appears to be a fairly homogeneous %N through all thenclosed parts of the stem, except around anthesis, when the hid-en part of the recently-grown ear peduncle had a higher %N thanhe lower internode. Beside, the %N of all the internodes differedetween genotypes and N treatments and decreased with time. Onverage it was 1.3-folds higher (P-value < 0.001) for Caphorn thanor Soissons, two-folds higher (P-value < 0.001) for N+ than for N0nd two-folds higher at 65 ◦Cd after anthesis than at 500 ◦Cd afternthesis.

Along the part of the stem exposed to light, %N showed strong vertical gradient. For the upper internode (Iexp

n ), theradient ranged from 6 × 10−4 g N g−1 DM cm−1 (for CaN0) to8 × 10−4 g N g−1 DM cm−1 (for SoN+). The %N gradient was smalleror the ear peduncle (3 × 10−4 g N g−1 DM cm−1) than for the upper

nternode. Finally, in all cases except for the ear peduncle at5 ◦Cd after anthesis, a strong gradient existed between the uppernclosed segment and the lower exposed segment of a samenternode. Besides, mean %N differed between genotypes and N

heat cultivars Caphorn (Ca; a) and Soissons (So; b) grown in the field at low (N0)s grown under high N supply in 2008 (c). Symbols belonging to the same internode.

treatments with trends similar to those observed for the enclosedpart of the stem.

3.4. Mean specific N mass of individual laminae, sheaths andinternodes were linearly related to their distance from the top ofthe canopy

For all cultivar/N treatments/sampling date combinations, themean specific N mass of the three laminae was negatively corre-lated with the distance from the top of the canopy (all r2 > 0.95;and P-value < 0.001; Fig. 5). Similarly, the mean specific N mass ofthe three sheaths decreased linearly with the distance from the topof the canopy (all r2 > 0.95 and P-value < 0.001) and the mean spe-cific N mass decreased between the exposed parts of the peduncleand In (i.e. the true stem; P-value < 0.001). The vertical gradients ofspecific N mass were higher for the laminae than for the sheaths(P-value < 0.001) but similar for sheaths and the exposed part ofthe true stem at the first sampling date in 2008 (P-value = 0.91). Inaddition, at a given distance from the top of the canopy, the spe-cific N mass of laminae was always higher than that of sheaths andlower than that of internodes, the differences being larger in 2006for Soissons than for Caphorn and for the first sampling date thanfor the second sampling date.

For both cultivars, the vertical gradients increased with N avail-ability (P-value < 0.001). Between anthesis and mid-grain filling,specific N mass had decreased for every laminae, every sheaths, andfor In and the peduncle, and the variations between phytomer rankshad been reduced for laminae and sheaths (P-value < 0.001). No dif-ference was observed in vertical specific N mass gradient betweenthe two sampling dates in 2008 for the upper internode and the earpeduncle (P-value = 0.65).

4. Discussion

In dense canopies, N distribution follows that of PPFD (Hiroseand Werger, 1987; Terashima et al., 2005). The model NEMA(Bertheloot et al., 2008b, 2011a,b) explains PPFD–N relationship

by the turnover of photosynthetic proteins: N content of a pho-tosynthetic organ is the result of the balance between a proteindegradation rate and a synthesis rate dependent on local light envi-ronment. During grain filling, high N demand by grains results in

18 J. Bertheloot et al. / Europ. J. Agr

Fig. 5. Distance from the top of the ear peduncle versus mean specific N mass of thethree upper leaf laminae and sheaths at 95 ◦Cd (open symbols) and 350 ◦Cd (closedsymbols) after anthesis for the bread wheat cultivars Soissons (So) and Caphorn (Ca)grown in the field at low (N0) and high (N+) N supplies in 2006 season, and at 65(open symbols) and 500 ◦Cd (closed symbols) after anthesis for cultivar Soissonsgrown under high N supply (N+) in 2008. In 2008, specific N masses of the upperinternode and the peduncle are also shown. Data area mean ± 1 SE for 3 independentreplicates. Lines are linear regressions.

onomy 42 (2012) 11– 21

low availability of amino-acid for protein synthesis and this createsan unbalance as protein synthesis rates become lower than degra-dation rates; however both model and measurements showed thatduring that phase, the distribution of N still follows that of PPFD.Previous studies do not document whether changes in N con-tent with PPFD take place continuously or by discrete transitionsbetween plant modules. In this study, N distributions along andbetween leaf laminae, sheaths and internodes were analyzed forwheat culms just after anthesis and during the early linear grain-filling period before the phase of rapid canopy senescence. Fourcanopies contrasted for their N content and structure (i.e. numberof culms per unit of ground area, individual leaf surface area, lengthand posture, green area index) which affects PPFD extinction withinthe canopy, were analyzed.

4.1. Mean specific N mass of individual laminae and sheathsdecreased with the depth in the canopy

The mean specific N mass, specific dry mass and %N of individuallaminae decreased down the canopy. Similar decreases have beenreported in a number of species (see reviews by Grindlay, 1997;Dreccer et al., 1998; Hirose, 2005). The vertical gradient of SLN wassteeper than that of specific dry mass, and resulted of the cumula-tive effects of the gradients of %N and specific dry mass, similarlyto what has been reported for other annuals such as lucerne (Med-icago sativa L.; Lemaire et al., 1991, 1992) and sunflower (Helianthusannuus L.; Sadras et al., 1993). These gradients reflect an acclimationof the structural characteristics of the leaf and of its photosyn-thetic apparatus to the local light environment (Prioul et al., 1980b).Strong evidence exists that, in annuals, PPFD affects N content of aleaf through changes in the transpiration rate (Pons and Bergkotte,1996; Pons et al., 2001; Boonman et al., 2007). The rate of transpi-ration determines the amount of nitrate and cytokinins importedby a leaf, which are both involved in the synthesis of proteins ofthe photosynthetic apparatus (Pons and de Jong-Van Berkel, 2004;Aloni et al., 2005; Boonman et al., 2007).

Few studies have analyzed the vertical distribution of N in othertissues than leaf laminae. We showed that specific N mass, specificdry mass and %N of individual sheaths decreased down the canopy.As for laminae, the gradient of specific sheath N mass resultedfrom the cumulative effects of the gradients of %N and specificdry mass. Do sheaths acclimate to their local PPFD environmentsimilarly to laminae? For laminae, N content acclimation can beseen as an adjustment of the amount of photosynthetic apparatusto local light conditions. Sheaths also are photosynthetic organs,so that similar processes are likely to explain N content for bothlaminae and sheaths. However, differences in the degree of acclima-tion may exist due for example to differences in light–transpirationrelations; Araus and Tapia (1987) indeed reported a stomatal con-ductance three times lower for sheaths than for laminae. Our resultsshowed that sheaths had a lower specific N mass than had lam-inae at the same depth in the canopy. This difference existed forboth cultivars, independently of the leaf posture. However, even foran erectophile canopy, light intercepted per unit developed sheatharea is expected to be less than intercepted per unit single-sidedlaminae area at a same depth (Lang, 1991) so that a difference in Nis expected even if PPFD–N relations are the same for laminae andsheaths. Accurate calculation or measurements of light and tran-spiration rate distribution would be required to establish if PPFD–Nrelationships are identical or not for laminae and sheath. This dif-ference in specific does not seem to impact the general dynamic ofN after anthesis, as in a previous study, we have shown that these

dynamics were similar for laminae and sheaths (Bertheloot et al.,2008a). It is interesting to note that the different average light envi-ronment perceived by the sheath and the lamina of a leaf, togetherwith the homogenization of specific N mass within each module

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mplies a sharp change of specific N mass at the sheath/laminaoundary, as observed in this study (Fig. 3).

.2. Specific N mass was homogeneous along individual laminaend sheaths

There was no gradient of specific N mass along individual lami-ae or sheaths, although they were subjected to large light gradientince each module spanned about 25% of the canopy height. Ingreement with our results, no significant trend in SLN distributionas observed along the long, lax leaves of maize plants at silking

Drouet and Bonhomme, 1999) or within the erect leaves of a mix-ure of reproductive and vegetative tor grass (Pons et al., 1993). SLNas also been reported to remain homogeneous along the greenissues of the wheat flag leaf lamina during the whole grain-fillingeriod; although, the PPFD decreased by 40% from the tip to thease of the flag leaf lamina (Bertheloot et al., 2008a). Together withhe present results, this supports the idea that in grasses during theeproductive stage, the response of specific N mass to PPFD is notontinuous along individual grass leaf laminae and sheaths, but isntegrated at the scale of the whole module.

This homogeneity of specific N mass within laminae and sheathsas observed despite a significant increase in specific dry mass

oward the base of each module. This was also reported for theeaves of tor grass (Pons et al., 1993). A higher specific dry massoward the base of leaf laminae is a common observation for mono-ot species and is interpreted as fulfilling the increase in the amountf structural tissue (midribs, sclerenchymae) required for mechan-cal support of the borne tissues (Gastal and Lemaire, 2002).

The physiological process which would integrate environmen-al signals at the scale of individual leaf laminae and sheaths andesult in the observed homogeneous distribution of specific N masss unclear. N–PPFD relationships are usually analyzed by express-ng N mass per unit area, but no study has directly established if its the specific N mass that is driven by PPFD distribution or ratherhe %N. In our study, %N increased acropetally along laminae andheaths. Consequently, specific N mass homogeneity within lam-nae and sheaths could be the coincidental consequence of twopposite gradients: a basipetal gradient of specific dry mass andn acropetal gradient of %N, the latter resulting from the PPFD gra-ient. In our view, this interpretation is unlikely because specific Nass homogeneity was observed for a range of species, and in our

tudy, a wide range of leaf N status, orientations, and area indicesand thus light gradient), and for both leaf laminae and sheaths.ven if the physiological processes are unclear, it is interesting toote that the xylem and phloem vessels that irrigate/drain the api-al regions of a grass leaf transit through the more shaded basalegions and thus may affect the behavior of basal regions. The wellrdered basipetal progress of tissue death during leaf senescences a related example of such putative control. Consequently, it isuite expected that some balance must exist between the differentarts of a same module. While the processes behind specific N massomogeneity remain to be clarified, this result suggests that longrect laminae plus short internodes may result in a lower ability forhe plant to adjust tissue N content with the local light environmentnd it would be worth to further investigate possible consequencesor radiation use efficiency of cultivars combining dwarfism plusrectophile traits.

.3. Pattern of N along the stem shows a more complex situation

The stem showed more complex patterns of N distribution, as

ost of its length was fully enclosed by the sheaths. SLN of the

nclosed part of the stem was low and show little variation withinr between phytomers. The light exposed part of the stem (i.e.he top of the upper vegetative internode and the upper half of

onomy 42 (2012) 11– 21 19

the ear peduncle) had a higher %N than the subtending enclosedinternodes. Because the upper internode and the ear peduncle con-tinued to extend during approximately a week after anthesis, it islikely that our first sampling date occurred before their N contentwas acclimated to their local environment, which would explainthe high %N observed in the enclosed part of the ear peduncle.At 350 and 500 ◦Cd after anthesis a sharp transition of %N at theenclosed/exposed boundaries of the ear peduncle was observedwith a strong gradient in the lower few centimeters of the exposedsegment. The length of the upper internode exposed to light wasonly ca. 4 cm, no significant difference in PPFD could have existedalong it and the gradient of N here likely reflected the transition in%N from the enclosed to the exposed parts. The exposed length ofthe ear peduncle was long enough so that the two upper segmentsshowed little difference in %N. Specific ear peduncle N mass washigher than that of the upper sheath and similar to that of the flagleaf lamina. This is consistent with previous reports showing thatat mid-grain filling the rate of light saturated photosynthesis of theear peduncle is higher than that of flag leaf sheath and is about 75%of that of the flag leaf lamina (Wang et al., 2001).

The sharp gradients of specific N mass and %N observed at theenclosed/exposed boundaries of the ear peduncle contrasted withthe homogeneity observed along the laminae and sheaths. Com-pare to light exposed tissues, hidden tissues perceive extremelydifferent conditions in term of light amount, light quality, andgaseous concentration which impair the development of functions(this was illustrated by the abrupt change from green to whitishcolor at the transition between exposed and hidden tissues; datanot shown). So, it is not unexpected that this inhibits the processesthat usually lead to homogeneity, and this does not appear spe-cific to internodes tissues. Localized response to full shading wasfor instance described for leaves of Arabidopsis thaliana (L.; Weaverand Amasino, 2001).

The absence of SLN gradient along leaf laminae has beenreported for several Gramineae species at the reproductive stage(Pons et al., 1993; Drouet and Bonhomme, 1999; Bertheloot et al.,2008a); however, it might not be the case at the vegetative stage.For example, Pons et al. (1993) and Drouet and Bonhomme (1999)observe positive SLN gradients along leaf laminae of vegetativeplants of C. acutiformis and maize, respectively. Similarly, Prioulet al. (1980b) observed, for the third leaf of Italian raygrass, adecrease in Rubisco quantity and chlorophyll per unit leaf area aswell as a decrease in the rate of light saturated photosynthesis fromthe tip to the base of the lamina. In Gramineae, expanding leavesshow clear developmental gradient along their length (Gastal andNelson, 1994). Since acclimation of N content to light takes severaldays (Prioul et al., 1980a; Irving and Robinson, 2006), such develop-mental gradients may still be observed for several days after the leafhas reached its final size. Moreover, during the vegetative period,the light environment of each leaf is continuously changing due tothe production of new leaves at the top of the canopy. Therefore,during the vegetative period, leaves may not have time to acclimatetheir N content to their local light environment. For wheat, thereis about 300 ◦Cd (i.e., ca. 15 days under our experimental condi-tions) between the appearance of the flag leaf collar and anthesis(Brooking et al., 1995), which is probably enough time for the leavesto acclimate to a more constant light environment.

5. Conclusion

This study clearly showed that, for wheat plants after anthesis,

there were abrupt changes in specific N mass between successivelaminae and sheaths according to height in the canopy, while itwas homogeneous within each module. Consequently, the botani-cal organization of wheat plants after anthesis appears – together

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ith light attenuation within the canopy – a strong determinantf N distribution. This study highlights the importance to take intoccount plant modular organization when analysing vertical N gra-ients observed at canopy scale. Homogeneity of specific N massas demonstrated within laminae but also within sheaths, whichave often been neglected in past studies. Both types of modulesppeared to acclimate to light.

There is a need for models that formalize processes at aner scale than that of the whole crop: to this end existingrop models are adapted to distinguish different horizontal lay-rs, and there is an increasing development of individual-based,unctional–structural, plant models (FSPM) that explicitly rep-esents plant botanical organization (Vos et al., 2010). Resultsrovided by this study give important information to integrate

dynamics in a FSPM of wheat. During the reproductive period,rocesses governing N content in response to PPFD should beormalized at the scale of each botanical module and similar for-

alization could probably be used for photosynthetic tissues ofifferent modules (Bertheloot et al., 2008a, 2011a), although differ-nces may exist in the values of parameters. This provides a frame tontegrate photosynthesis calculation that depends on tissue N con-ent, such as LEAFC3-N model (Müller et al., 2005), and to accountor other organs than leaf laminae. The existence of homogeneousones of N content has direct consequences for phenotyping N dis-ribution in plants: compared with the stratified sampling schemehat is usually followed to obtain detailed distributions, samplingy botanical modules is not only easier but also more adapted toescribe the N distribution during the reproductive period. On thether hand modeling N dynamics during the vegetative stage wouldrobably need to formalize the processes at a finer scale.

cknowledgments

This work was supported by the French Ministry of Research andechnology and by the doctoral school ABIES. We thank Emmanuelovart, Fabrice Duhamel and Florence Lafouge for their skillful tech-ical assistance.

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