quantitative contributions of blue light and par

RESEARCH PAPER

Quantitative contributions of blue light and PAR tothe photocontrol of plant morphogenesis inTrifolium repens (L.)

Angelique Christophe*,†, Bruno Moulia‡ and Claude Varlet-Grancher

Unite d’Ecophysiologie des Plantes Fourrageres, INRA, Lusignan, F-86600, France

Received 17 October 2005; Accepted 22 March 2006

Abstract

Shade-avoidance is a major adaptive response of

plants, and is usually considered to be controlled by

phytochromes through the perception of changes in

the red:far red light ratio. However, few studies on the

effects of blue light (BL) and of light intensity [photo-

synthetically active radiation (PAR)] on light-grown

plants have been conducted, especially concerning

changes in PAR at constant BL. The objective here was

to quantify the photocontrol of aerial morphogenesis

by BL and PAR. Experiments were conducted varying

BL and PAR independently, with three BL levels (4, 38,

and 83 lmol m22 s21) at constant PAR (300 lmol

m22 s21) and three PAR levels (338, 705, and 163 lmol

m22 s21) at constant BL (36 lmol m22 s21). Effects on

morphogenetic processes were analysed as quantita-

tive modulations of ontogenic trends and response

curves were produced. White clover (Trifolium repens

L.) was used, as it is a typical shade-avoider display-

ing the whole syndrome of shade-avoidance in a

purely vegetative stage. Morphological responses

were strongly controlled by both BL and PAR changes,

through antagonist effects on leaf appearance rate and

additive effects on petiole elongation. All the other

responses appeared to be the indirect consequences

of changes in the leaf appearance rates. BL acted as

a light signal for plant morphogenesis. However, the

PAR control probably implicates two distinct mech-

anisms, such as a trophic effect and a signal. Both

PAR and BL actions involved organ-specific differ-

ences, which are central in the control of the shade-

avoidance responses.

Key words: Blue light, leaf appearance rate, PAR, petiole

growth, photomorphogenesis, plasticity, shade-avoidance,

Trifolium repens L.

Introduction

Plants have evolved a high level of plasticity in response tolight conditions. In many species, light-grown plants reactto canopy shade by a suite of morphogenetic responses,involving (i) an enhancement of stem and petiole elonga-tion and an associated reduction in lamina expansion, (ii)an increase in main axis development compared with thatof existing branches, and (iii) a reduction in the numberof branches (Smith and Whitelam, 1997). This suite ofphotomorphogenetic responses has been called the shade-avoidance syndrome (Grime, 1981) and is interpreted as anadaptive light-foraging behaviour (for reviews see deKroon and Hutchings, 1995; Ballare et al., 1997). How-ever, the way that these responses are controlled by shadingis not yet fully understood.

Many questions remain unanswered, such as, whichspectral components of light are involved and to whatextent? Generally, studies of the shade-avoidance syndromeare primarily concerned with the perception of the changesin the red far:red light ratio (R:FR) through phytochromes.This has now been well characterized (for reviews seeSmith and Whitelam, 1997; Smith, 2000; Franklin andWhitelam, 2005). However, it is known that approximatelyhalf of the total shade-avoiding responses can also be ob-served under neutral shading at a constant R:FR ratio, thusinvolving other photocontrolling mechanisms ( Lotscher andNosberger, 1997; Stuefer and Huber, 1998). More recently,

* To whom correspondence should be addressed. E-mail: [email protected] Present address: Laboratoire d’Ecophysiologie des Plantes sous Stress Environnementaux, UMR INRA – ENSAM, place Viala, F-34060 Montpelliercedex 1, France.z Present address: UMR PIAF, INRA, Site de Crouel, 234 avenue du Brezet, F-63039 Clermont-Ferrand cedex 02, France.

Journal of Experimental Botany, Vol. 57, No. 10, pp. 2379–2390, 2006

doi:10.1093/jxb/erj210 Advance Access publication 23 June, 2006

Published by Oxford University Press [2006] on behalf of the Society for Experimental Biology.

by on 18 October 2009 http://jxb.oxfordjournals.orgDownloaded from

http://jxb.oxfordjournals.org

manipulation of light conditions during plant developmentand the use of various photoreceptor mutants have indeedrevealed the specific role of the blue light (BL) fluence ratein the shade-avoidance syndrome, acting through crypto-chromes (Ballare et al., 1991; Kozuka et al., 2005).However, despite significant advances in the understand-ing of the molecular mechanisms involved in BL perception(Lin and Shalitin, 2003) and in the BL control of seedlingmorphogenesis (Ahmad et al., 2002), there is still littleinformation about the effects of BL on light-grown plants.And even fewer studies have been conducted on light-grownplants to separate BL fluence rate from changes in phyto-chrome photoequibrium and in photosynthetically activeradiation (PAR) (Ballare et al., 1991; Gautier et al., 1997,1998; Dougher and Bugbee, 2001a).

Additionally, there is evidence indicating that PAR isalso implicated in the control of plant morphogenesis. Forexample, light-grown plants displayed different responsesin stem elongation or growth under two levels of PAR withno reduction in BL and in the R:FR ratio (Ballare et al.,1991; Dougher and Bugbee, 2001a). More recently,interactions between the BL responses and the sugar-sensing pathway (which is likely to be PAR-dependent)were also observed by using a genetic approach onArabidopsis using sugar-insensitive mutants (Kozukaet al., 2005). However, the effects of PAR are still rarelydocumented and no quantitative insights have been givenon the relative importance of BL and PAR responses ina given amount of shading. Indeed, most of studies on theeffects of PAR on plant morphogenesis are focused uponthe so-called ‘neutral shading’, which reduces light homo-geneously in the 400–700 nm waveband with a constantphytochrome photoequilibrium but inducing a proportionalreduction of BL. Interpretation of the effects of PAR andBL on plant morphogenesis in these studies is complicatedbecause the light sources used and the BL:PAR ratios varyconsiderably. Plants may respond simultaneously, but indifferent ways, to changes in BL and in PAR so thesestudies can only yield circumstantial conclusions aboutthe photocontrol of shade-avoidance responses.

Another central question concerns the photocontrol ofthe different, and possibly antagonistic responses, of theplant parts involved in the adaptive shade-avoidancesyndrome, for example, stimulation of stem elongationand inhibition of lamina expansion (Kozuka et al., 2005).Several hypotheses can be proposed to explain suchdifferences. The simplest one is based on the fact that plantgrowth during ontogenesis is generally allometric ratherthan isometric. So an effect of light on the rate of plantdevelopment would change the absolute growth rates oforgans, whereas the allometric relationships would be keptconstant (Wright and Mc Connaughay, 2002). This poten-tial confusion between plasticity in growth rates and inallometries could be overcome by taking plant ontogenyinto account (through an index of developmental stages)

when studying adaptive responses of plant morphology toshading (Huber and Stuefer, 1997). Alternatively, growthallometries could be directly controlled by light-inducingcontrasted plastic responses of the organs (Wright andMcConnaughay, 2002). In that case, the contrasted plasticresponses of the different organs could be explained by twohypotheses: (i) the photoperception mechanisms are thesame, but the selection of morphogenesis responses isorgan specific; or (ii) the photoperception sensitivity isorgan-dependent (the different organs being more or lesssensitive to the different spectral components of light). Anapproach to test these hypotheses is to consider lightfluence-rate response curves. Indeed, if the hypothesis oforgan-specific selection holds true, then all the responsesthat are selected in all the organs should display fluence-rateresponse curves with similar shapes (as they depend on thesame process of photoperception). By contrast, if thephotoperception itself is organ-dependent, then differentfluence-rate response curves should be produced. However,until now, few BL response curves have been character-ized (Wheeler et al., 1991; Dougher and Bugbee, 2001a),and possible changes in developmental rates caused byshading were not considered. Moreover, no PAR responsecurves independent of BL have ever been produced, as faras is known.

The aim of this study was to identify and quantify therelative contributions of BL and PAR in the photocontrol ofaerial morphogenesis of a typical shade-avoiding species(Trifolium repens L.). This species presented the advantagethat the analysis of its shade-avoidance syndrome is notcomplicated by flowering time. Although this work re-quired controlled environmental conditions, light condi-tions were selected to mimic those found in natural shade,in the range in which PAR and BL responses are likely tobecome significant (Ballare et al., 1991). Developmentalontogenic trends were quantified to investigate howmorphogenetic processes are plastically affected by shad-ing, separating responses of the rate of ontogenic de-velopment from specific plastic responses of organ growth.Additional light conditions were also studied to producePAR and BL fluence-rate response curves to determine howeach spectral component of light controls the differentmorphogenetic responses for each organ.

Materials and methods

Defining ecologically relevant artificial light treatments

Each artificial light treatment was controlled to simulate the PAR andBL irradiances experienced by a plant that is shaded by a white clovercanopy of a given leaf area index (LAI) in natural conditions.The PAR incident in the growth chambers was fixed to produce atypical natural incident light. The levels of PAR and BL irradiancesto be applied in the artificial treatments were determined using therelationship between the fraction of transmitted light in the visible

2380 Christophe et al.



range and the LAI for a natural white clover canopy [PARtransmitted/PARincident=exp(–0.594LAI); data compiled from Sinoquet et al.,1990] in order to correspond to that of natural shading (see below fordetails on the shade conditions retained). The fraction of lighttransmitted in the BL waveband was assumed to be similar to thatof incident light, as the BL:PAR ratio has been shown to beindependent of the amount of shading (Holmes, 1981; Messier andBellefleur, 1988).

As the aim was to vary independently BL and PAR fluence rates,each light treatment was designated as BLx_PARy where x is thefraction of light transmitted in the BL range and y that for PAR. Whenx and y are equal, the treatment corresponds to a neutral shadetreatment that could be achieved under a natural canopy.

Experimental design and artificial light treatments

In the growth chambers, the incident light was fixed to simulatea typical day at the beginning of the main growing season for clover(early spring in central France; Simon et al., 1989), i.e. a mean dailyincident PAR of 25.4 mol m�2 d�1, a mean daily BL of 7.6 mol m�2

d�1, and a 10 h photoperiod (10 year means, INRA Lusignan,France, 0.078E, 46.38N, meteorological data).

Two neutral shade treatments, BL45_PAR45 and BL20_PAR20,were applied to study the effects of neutral shading. These fractions oftransmitted light in BL and PAR correspond to ranges of naturalshading (LAIs from 1.4 to 2.8) in which (i) PAR and BL responsesare likely to become significant (Ballare et al., 1991) and (ii) shade-avoidance responses have been observed in natural conditions(Simon et al., 1989). A third treatment, BL20_PAR45, was used tomeasure quantitatively the separated contributions of PAR and BLreductions in this neutral shading (Fig. 1). The contribution of BLreduction at a constant PAR was assessed by comparing BL45_PAR45 and BL20_PAR45 treatments. That of PAR reduction ata constant BL was assessed by comparing BL20_PAR45 andBL20_PAR20.

Two additional light treatments, BL20_PAR100 and BL2_PAR45(Fig. 1), were defined to establish PAR and BL fluence-rate responsecurves. BL20_PAR100, with no PAR reduction (i.e. with the samePAR level as that of the incident light) was preferred to a deepPAR reduction, to prevent the plants from being under the point ofcompensation of photosynthesis. BL2_PAR45 treatment, corres-ponding to 4 lmol m�2 s�1 BL in the present conditions, wasretained as the effects of BL were sometimes found maximum forphoton fluxes lower than 40 lmol m�2 s�1 BL (Wheeler et al.,1991; Dougher and Bugbee, 2001a). Moreover it is similar to theBL (–) treatment used by Gautier et al. (1997) in white clover.Fluence-rate response curves to BL at constant PAR could thenbe established considering BL45_PAR45, BL20_PAR45, andBL2_PAR45, and that of PAR at constant BL consideringBL20_PAR20, BL20_PAR45, and BL20_PAR100.

Each light treatment was applied in a separate growth chamber(Phytotron, Froid et Mesure, Angers, France), using a ceiling ofmetallic halide lamps (HQI, 400 W; Osram, France). In theBL45_PAR45 treatment no filter was used. In the other lighttreatments, PAR and BL irradiances required were obtained froma similar ceiling but using one layer of a filter (Lee Filters,ATOHAAS France SA Argenteuil, France; transmission curves ofthe filters are shown in Fig. 2A) on top of the plants. BL2_PAR45was obtained by adding a deep straw filter (Lee Filter HT015);BL20_PAR100 by adding an amber-yellow filter (Lee filter 102);BL20_PAR45 by adding a straw tint filter (Lee Filter HT013), andBL20_PAR20 by adding a perforated reflector (Lee Filter 270). Thelight spectrum at plant level (Fig. 2B) was measured for eachtreatment using an LI-1800 spectroradiometer (Li-Cor Inc., Lincoln,NE, USA) and relevant actinic light fluxes were then computed forall the treatments (Table 1). For PAR, the standard definition of

the PPFD (photosynthetic photon flux density at 400–700 nm) wasused. For BL, fluence rate was measured as the photon fluence ratein the 350–500 nm waveband (Ahmad et al., 2002; Lin and Shalitin,2003). In order to eliminate the confusion between BL and photo-synthetic effects, the photosynthetic efficiency (light spectrum timesthe action spectrum of the leaf photosynthetic yield; Sager et al.,1988) was computed for all the light treatments and controlled to bedecorrelated with BL (Table 1).

In all treatments, the phytochrome photoequilibrium was main-tained at its maximal ‘unshaded’ values (i.e. slightly higher thanthat of incident natural solar radiation), the UV fluence rate wasnegligible (Fig. 2) and the photoperiod was 10 h.

Note that, from the commercially available filters, it was notpossible to produce the levels of BL and PAR fluence rates matchingexactly the targeted percentage of light transmitted. The greatestdeviation from the values was 6% (Table 1).

Plant material and growth conditions

Cuttings of a single clone of white clover (Trifolium repens L. cv.Huia) were grown in individual pots (2031238 cm) filled withsterilized sand (Gautier et al., 1997). During the pre-experimentperiod, all the cuttings were grown together and subjected to theBL45_PAR45 treatment (Table 1). After 2 weeks, plants wereselected for uniformity of (i) internode and petiole lengths and (ii)unfolding foliar stage using the decimal scale proposed for whiteclover by Carlson (1966) (10 stages from 0.1 when the leaf firstbecomes visible to 1 when the leaf is fully unfolded). Eight plantswere then randomly assigned to each light treatment. Pots werewatered liberally by excess solution eight times a day, using acomplete nutrient solution containing nitrogen (Gastal and Saugier,1986) to avoid any water or nutrient limitation. Relative air humidity

BL45_PAR45

PARreduction Neutral

shading

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20

45

45

BL reduction BL20_PAR45

BL20_PAR20

BL2 PAR45

100

100

BL20_PAR100

transmitted light in the PAR (%)

transmitted light in the BL (%)

Fig. 1. Experimental design. Each treatment was designated byBLx_PARy where x is the percentage light transmission in the BL rangeand y that for PAR. When x and y are equal, the treatment was describe as‘neutral’. The comparison between treatments with different x wasdescribed as ‘BL reduction’, whereas that between treatments withdifferent y was described as ‘PAR reduction’.

BL and PAR photocontrols of aerial plant morphogenesis 2381



was maintained constant at 80%. Air temperature was measured atplant height using six thermocouples per growth chamber and main-tained at 23 8C day/20 8C night. A more complete description ofthe growth conditions is presented in Table 1. Note that with a 10 hphotoperiod all the plants in all the treatments remained vegetative.

Plant measurements

Plant morphology: White clover (Trifolium repens L.) is a clonalstoloniferous plant with plagiotropic axes (main axis and lateralbranches) and orthotropic petioles bearing leaves (a leaf consists ofthree leaflets). An axis can be viewed as a succession of phytomerscomprising a node, an internode, an axillary bud, a leaf, and twonodal root primordia (Bell, 1984).

Number of leaves: The number of leaves and their unfolding stage(using the Carlson decimal scale; Carlson, 1966) were recorded every3 d, on each axis of the cutting, for eight plants per treatment. These

data were used to calculate the usual indices of development rep-resenting the number of leaves of a given axis (cumulated Carlsonstage; Gautier et al., 1997) and of the whole plant (plant cumulatedCarlson stage; Christophe et al., 2003), in decimal units. Note that thedevelopment of the whole plant depends on both (i) the rate of leafappearance on the axes and (ii) the rate of branch production.

Rate of leaf appearance: In each treatment, the rates of leafappearance (RLA; leaf 8C�1 d�1) on the whole plant and on eachaxis were estimated by fitting the number of leaves (in Carlsondecimal units) that were visible on the whole plant and on each axisagainst thermal time. Thermal time was calculated as the cumulativedegree-days from emergence, assuming a base temperature of 0 8C(Simon et al., 1989; Gautier et al., 1997).

The balance between the rate of leaf appearance on the lateralbranches and that on the main axis was estimated by the slope of therelationship between the number of leaves of each branch (in Carlsondecimal units) and the number of leaves of the main axis (in Carlsondecimal units) produced at the same time.

Rate of branch production per bud: The rate at which newly formedaxillary buds were produced on new lateral branches was estimatedby the slope of the relationship between the number of branches andthe number of leaves of the whole plant produced over the same timeinterval (in Carlson decimal units), as proposed by Davies (1974)(and also called the site filling).

Plant growth: For each plant, the lengths of (i) all the internodes and(ii) the midribs and the petioles of all the leaves were measured every3 d using callipers. For each treatment, the individual leaf area wasestimated from an allometric relationship between the leaflet area andthe midrib length determined from a sample of leaves using an Li-Corplanimeter (Li-3100, Li-Cor Inc.). In order to study plastic growthresponses, the plant axis length and the plant leaf area were comparedbetween treatments at the same plant developmental stage (Christopheet al., 2003). Thus the rates of the plant axis elongation and of theplant leaf expansion were estimated by fitting the plant axis length andthe plant leaf area, respectively, against the number of leaves of thewhole plant (in Carlson decimal units). Lengths of petioles werecompared between treatments at their final stage of leaf development(when maximal length is achieved). Only the petiole lengths of leaves3 and 4 of the main axis are given, as they were the only petioles thatcompleted the entire visible growth during the light treatments.

Statistical analysis

Final plant measurements were compared using a one-way analysisof variance (general linear model with treatment as a fixed factor;procedure GLM in SAS).

The regression and analysis of variance of the various ontogenic-dependent variables were determined using a mixed general linearmodel, with the mixed procedure (Proc Mixed) in SAS version 6.12(Littell et al., 1996). The effects of individual plants (nested withinthe treatment) were added as random effects in the error of the model,using the repeated option in Proc Mixed. The variance–covariancematrix of the error was specified by a ‘spatial’ power structure [SP(Pow)]. SP (Pow) takes into account uneven intervals in the datasets. For the present data sets, it displayed the highest Akaike’sinformation criterion (AIC) and Schwarz’ Bayesian criterion (SBC)(as defined in SAS version 6.12; Littell et al., 1996). For the study ofdevelopmental kinetics, kinetics were fitted using a linear or poly-nomial regression model. The light treatments were first tested asfixed effects on the heterogeneity of the parameters of this model(slopes, higher order coefficients). If this test was significant, theparameters were compared using the estimate statement; otherwisea covariance analysis was performed. When the common slope wasnot different from zero, the model was reduced to a (mixed) analysisof variance, with the light treatment effects as fixed effects.

Wavelength (nm)300 400 500 600 700 800

Ligh

t tra

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Lee Filter 270

Lee Filter HT013

Lee Filter HT015

Lee Filter 102

BLPAR

BL45_PAR45

BL20 PAR100

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BL20_PAR20BL20_PAR45

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Phot

on fl

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Fig. 2. (A) Light transmission curves of the filters used in theexperimental design. Continuous line, Lee Filter 270; dashes, LeeFilter HT015; dots, Lee Filter 102; dash-dot-dots, Lee Filter HT013.(B) Spectral photon distribution of incident radiation in the differ-ent treatments. Continuous line, BL20_PAR45; long dashes,BL20_PAR100; dots, BL20_PAR20; dash-dots, BL45_PAR45; dash-dot-dots, BL2_PAR45.




Whenever a statistically significant effect was found for at leastone light treatment, fluence-rate response curves were constructedusing means values (and standard error).

Results

Morphology of shaded plants

At the end of experiment, the effects of neutral shading onplant morphology were tested by comparing plants grownunder the two neutral shade treatments (Table 2). Shadedplants had produced about half the number of leaves andbranches than plants grown under high-light conditions.Shaded plants also presented a reduction in the numberof leaves on the branches and in the total leaf area. Bycontrast, the shaded plants produced petioles much longerthan plants under high-light conditions. The number ofleaves on the main axes and the total stem length were notaffected by shading (Table 2).

Neutral shading induced changes in both plant develop-ment and growth, leading to important differences in plantmorphology. Morphogenetic processes were thus studiedduring ontogeny in order to separate responses of the rateof the ontogenic development and their possible in-direct consequences on organ growth from direct plas-tic responses of organs. Then, for each morphogenetic

process, the effects of the neutral shading and the contri-bution of the effects of PAR and BL shading were analysedby quantifying modulations of these ontogenic trends.

Ontogenic development trends

In all the treatments, the plants had a similar initialdevelopmental stage and their number of leaves increasedexponentially during development. As a consequence, therate of the ontogenic development can be easily quantifiedby estimating the slope of the changes in the number ofleaves on a natural log scale against thermal time (Fig. 3A).By contrast, the rate of leaf appearance on the main axis(RLA, slope of the curve in Fig. 3B) was constant overtime for the period considered (23 d). The balance betweenthe rate of leaf appearance on the primary branches and thaton the main axis (i.e. the slope of the curve in Fig. 3C) wasalso constant over the time. Additionally, as there was nosignificant difference in this ratio between the first fourbranches, the data were pooled. In all the treatments, thisratio was below 1, indicating that the production of leavesof the main axis was always superior to that of the primarybranches. The relationship between the total number ofleaves and the total number of branches was complex andexhibited a cubic pattern when the plant had <10 branches(Fig. 3D). This suggests that the rate of branch production

Table 1. Environmental conditions

Characteristics of light treatments were determined at plant height, using a spectroradiometer (Li-1800, Li-Cor, Lincoln, NE, USA). The phytochromeequilibrium state (/c) and the photosynthetic efficiency (light spectrum multiplied by photosynthetic yield) were calculated as described by Sager et al.(1988). Mean air temperature was monitored at plant height. Each treatment was designated as BLx_PARy where x is the percentage light transmission inthe BL range and y that for PAR. The exact fractions of transmitted light were calculated afterwards from the exact percentage transmission throughfiltering.

Treatments BL2_PAR45 BL20_PAR100 BL20_PAR45 BL45_PAR45 BL20_PAR20

Mean airtemperatureday/night(and standarddeviation)

Tair (8C) 22.860.5/20.160.3 23.060.2/20.060.2 23.160.9/20.160.3 22.760.6/2060.1 23.060.4/20.060.1

Relative humidity % 80Light source HQI+a Lee filter

HT015HQI+a Lee filter 102 HQI+a Lee filter

HT013HQI HQI+a Lee filter 270

Photoperiod h d�1 10Photosyntheticefficiency(350–750 nm)

y lmol m�2 s�1 317 646 334 287 141

Photosyntheticphoton flux density(400–700 nm)

PPFD lmolm�2 s�1

338 705 301 333 163

Mean daily PAR mol m�2 d�1 12.2 25.4 10.8 12 5.9Blue light fluencerate (350–500 nm)

BL lmolm�2 s�1

4 36 38 83 36

Mean daily BL mol m�2 d�1 0.14 1.3 1.37 3 1.3Phytochromephotoequilibrium

/c 0.78 0.78 0.78 0.77 0.77

Transmitted lightin the PARa

(% of the incidentlight)

48 100 42.5 47 23

Transmitted lightin the BLa

(% of the incidentlight)

1.8 17 18 39 17

a The ‘incident light’ reference corresponded to the natural incident light during a typical day in early spring, with a mean daily PAR=25.4 mol m�2

d�1, a mean daily BL=7.6 mol m�2 d�1.




(i.e. the inverse of the slope of this curve) was not constantover the developmental range investigated (up to sixbranches). This rate increased rapidly during the early

stage of plant development, reaching a peak when the planthad two branches, and then declined continuously (data notshown). Considering growth of the whole plant, the total

Table 2. Comparison of the morphology of plants grown under two neutral shade treatments BL45_PAR45 and BL20_PAR20, after23 d (i.e. 786 degree-days)

Values are means of eight plants (6 standard deviation). Data were analysed by a one-way ANOVA and the asterisks indicate significant differencesbetween treatments (P <0.05).

Light treatment BL45_PAR45 BL20_PAR20 Probability value P

Total no. of leaves (decimal units) 29.768.2 16.7*62.6 0.0001Total no. of branches 7.863.2 4.8*60.8 0.0045No. of leaves in the main axis (decimal units) 8.261.3 8.160.8 0.8335No. of leaves in the first four primary branches (decimal units) 3.760.8 2.1*60.9 0.0001Total leaf area (mm2) 11714.861310.9 5612.3*61327.9 0.0027Total stem length (mm) 198.86106.2 99.3638.4 0.065Length of petiole 4 (mm) 80.265.9 113.9*613.7 0.0004

A B

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Fig. 3. Characteristics of leaf production and branching. Each point represents an individual plant. Open squares, BL45_PAR45; closed circles,BL20_PAR45; closed triangles,, BL20_PAR20. The lines are the mixed linear regression. (A) Changes in the total number of leaves (in Carlsondecimal units) with thermal time. Dashed line: BL20_PAR20, log(y)= –0.516(60.148)+0.0041(60.0002)x; continuous line: BL45_PAR45,BL20_PAR45, log(y)= –0.872(60.091)+0.0054(60.0001)x. (B) Changes in the number of leaves on the main axis (in Carlson decimal units)with thermal time. Continuous line: BL20_PAR45, y= –2.058(60.201)+0.0145(60.0003)x; dashed line: BL45_PAR45, BL20_PAR20, y=�2.058(60.201)+0.0122(60.0004)x. (C) Changes in the number of the first four primary branches plotted as a function of the number of leaves onthe main axis (in Carlson decimal units). Continuous line: BL20_PAR45, y= –3.341(60.121)+0.768(60.021)x; dashed-and-dotted line: BL45_PAR45,y= –3.341(60.121)+0.880(60.05)x; dashed line: BL20_PAR20, y= –3.341(60.121)+0.695(60.05)x. (D) Changes in the total number of leaves (inCarlson decimal units) with the total number of branches. Continuous line: y=3.168(6 0.186)+2.627(60.415)x–0.430(60.197)x2+0.08(60.02)x3.




leaf area increased proportionally with the number of leavesof the whole plant (Fig. 4A), whereas the plant axis lengthincreased as a three-quarters power law function of the totalnumber of leaves (Fig. 4B). Thus a three-quarter powerallometry between total internode length and total leaf areaper plant was maintained during development.

Despite significant differences in the final petiole lengthof leaves 3 and 4 (Fig. 4C) there was no significantinteraction between shade treatments and leaf rank(P=0.3944) and all the petioles on all the branches weresimilarly affected, even when correcting for total leafnumber (data not shown).

Effects of neutral shading and PAR and BL reductionson development of the whole plant

The neutral shading significantly slowed down the relativerate of the ontogenic development of the plant (by –0.0013,–24%, Fig. 3A; Table 3), and this was shown to be causedentirely by the reduction in PAR (Table 3). In the presentconditions, the rate of branch production was not signifi-

cantly affected by either neutral shading or by BL and PARreductions (Fig. 3D). Only the rate of leaf appearance(RLA) on the different axes was differentially affected bythe treatments (Fig. 3B, C; Table 3).

Under the neutral shading, the RLA on the main axis wasnot significantly modified (Table 3) and its mean value was0.0122 leaf 8C�1 d�1. However, individually, both BL andPAR reductions affected the RLA on the main axis (Table3). BL reduction significantly increased RLA by +0.002leaf 8C�1 d�1 whereas, on the contrary, PAR reductiondecreased RLA by –0.002 leaf 8C�1 d�1. This explains theabsence of neutral shading effects.

The neutral shading significantly reduced the ratio of therates of leaf appearance on the main axis and primarybranches by 21% (Table 3). This was the consequence ofa reduction of the RLA on the primary branches (–0.002leaf 8C�1 d�1, �20%). BL reduction decreased this ratio by13% (Table 3), which was due to a greater increase of theRLA on the main axis (three times more) than that on thebranches (+0.0004 leaf 8C�1 d�1, +4%). PAR reduction

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Fig. 4. Characteristics of plant growth. Each point represents an individual plant. Open squares, BL45_PAR45; closed circles, BL20_PAR45; closedtriangles, BL20_PAR20. The lines are the mixed linear regression. (A) Changes in the total leaf area per plant as a function of the total number of leaves(in Carlson decimal units). Continuous line: y=351.93(610.70)x. (B) Changes in the total internode length per plant as a function of the total number ofleaves (in Carlson decimal units). Continuous line: loge(y)=1.326(60.03)loge(x)+0.867(6 0.07). (C) Final length of petioles 3 and 4 of the main axis.




also decreased this ratio by 10.5% (Table 3) but, in thiscase, it was due to a greater reduction of the RLA on thebranches (–0.003 leaf 8C�1 d�1, –23%) than that on themain axis.

Effects of neutral shading and PAR and BL reductionson plant growth

No significant modification in the rate of the plant leafexpansion and of the plant axis elongation was observed inresponse to neutral shading, BL and PAR reductions (Fig.4; Table 3). By contrast, the final petiole length of themature petioles was significantly increased by the neutralshading (Fig. 4C; Table 3). Neutral shading induceda significant increase of 28 mm of the petiole length ofleaves 3 and 4 of the main axis (corresponding to anincrement of 35%). BL reduction increased the petiolelength of leaves 3 and 4 by 12.5 mm (corresponding to anincrement of 16%). PAR reduction increased the petiolelength of leaves 3 and 4 by 15.5 mm (corresponding to anincrease of 17%).

PAR and BL fluence-rate response curves

The morphogenetic parameters shown to be significantlyaffected by at least one light treatment were studied furtherby the analysis of the fluence-rate response curves, in-cluding the two additional light treatments BL20_PAR100and BL2_PAR45. Plant axis length and branching pro-duction were not affected under the present treatments(P=0.2691 and P=0.074, respectively).

The shape of the fluence-rate response curves variedbetween responses (Fig. 5A, B). Two response curves toPAR (the rate of leaf appearance on the main axis and thepetiole length; Fig. 5A) displayed no significant changesbetween 300 and 750 lmol m�2 s�1 despite a 2.5-folddifference in irradiance, whereas they displayed clear andopposite responses under 300 lmol m�2 s�1. These tworesponses thus probably tend to saturate for irradiance over300 lmol m�2 s�1 or, at least, reach a maximum between300 and 750 lmol m�2 s�1. Surprisingly, the ratio between

the rates of leaf appearance on the main axis and on thebranches did not show such a saturating pattern, although itseemed to display damped increments between 300 and 750lmol m�2 s�1. Lateral axes probably displayed a differentrange and shape of PPFD sensitivity than the main axis.Also surprising, the rate of leaf expansion was neversignificantly affected by the levels of PAR in the conditions(P=0.2395), contrary to the petiole.

The rate of leaf appearance on the main axis increased formoderate BL reductions (Fig. 5B) and then levelled off withBL fluence rates lower than 38 lmol m�2 s�1, or at leastreach some maximum for a deep BL reduction. Theresponse of the ratio between the rates of leaf appearanceon the main axis and on the branches was distinct from thatof the rate of leaf appearance on the main axis, as noted forPAR response, but to a higher extent. Indeed a deep BLreduction decreased very significantly the ratio on the leafappearance rates. Contrary to the PAR responses, BLreduction increased both the rate of leaf expansion andthe petiole final length to similar extents of, respectively,+35% and +40% (between extreme treatments).

Discussion

Varying BL and PAR independently within ecologicallyrelevant ranges

The action spectra of phytochromes, cryptochromes, andphotosynthetic efficiency largely overlap (Schafer et al.,1983). Therefore, uncoupling experimentally blue light(BL) and photosynthetically active radiation (PAR) withina realistic broad-waveband light without affecting phyto-chrome photoequilibirum (/c) is not simple. The experi-mental design reported here is the first as far as is knownthat was constructed (i) to quantify the relative contribu-tions of BL and PAR in the photocontrol of plantmorphogenesis, and (ii) to control the level of artificiallight irradiance in BL and PAR ranges to correspond to thatof natural shading (as defined with a relationship betweenthe fraction of transmitted light and the leaf area index

Table 3. Parameters of the plant ontogenic processes under the three light treatments BL45_PAR45, BL20_PAR45, and BL20_PAR20

For each ontogenic parameter, the effects of neutral shading were assessed by comparing BL45_PAR45 and BL20_PAR20, the effects of correspondingBL reduction were assessed by comparing BL45_PAR45 and BL20_PAR45 and those of corresponding PAR reduction by comparing BL20_PAR45and BL20_PAR20. The values correspond to the estimated parameters (mean 6standard error). The letters indicate significant differences betweentreatments (P <0.05).

Parameters of plant ontogenic development Light treatments

BL45_PAR45 BL20_PAR45 BL20_PAR20

Relative rate of the plant ontogenic development (leaf leaf�1 8C�1d�1) 0.005560.0002 a 0.006060.0002 a 0.004260.0002 bRate of leaf appearance on the main axis (leaf 8C�1 d�1) 0.012160.0006 a 0.014560.0003 b 0.012360.0005 aRatio of the rates of leaf appearance on the main axis and on the firstfour primary branches

0.8960.05 a 0.77760.02 b 0.69560.05 c

Rate of the total leaf area expansion (mm2 leaf�1) 356.64641.6 a 347.85622.30 a 376.14617.8 aMean petiole length of the phytomers 3 and 4 on the main axis (mm) 78.662.0 a 91.062.0 b 106.562.8 c




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Fig. 5. Fluence-rate response curves of morphogenetic processes: the rate of leaf appearance on the main axis, the ratio of the rates of leaf appearance onthe branches and on the main axis, the rate of the leaf expansion and the final length of petiole 3 of the main axis. Each point is the mean of eight plantsand the vertical bars represent the standard error of the mean. The different letters indicate significant differences between treatments (P <0.05). (A)Effects of three PAR levels: 163 lmol m�2 s�1 (BL20_PAR20); 301 lmol m�2 s�1 (BL20_PAR45); 705 lmol m�2 s�1 (BL20_PAR100). (B) Effects ofthree BL levels: 4 lmol m�2 s�1 (BL2_PAR45); 38 lmol m�2 s�1 (BL20_PAR45); 83 lmol m�2 s�1 (BL45_PAR45).




(LAI; see Materials and methods). It is a generalization ofthe method of ‘light equivalence’ designed to analyse theBL effects at constant /c using two monochromatic lights(Schafer et al., 1983). Dougher and Bugbee (2001a)produced BL variations for two levels of PAR bycombining different light sources and filters in a waysimilar to the one used in the present study. But, in theirexperimental design, the relative contribution of the effectsof BL and of PAR in shading could not be fully analysed.Note also that the present approach can be conducted overthe whole range of plant development (the light treatmentscan be applied at different times during plant development)and the whole range of possible shade. Indeed, it would beinteresting to extend the experimental design (with addi-tional light treatments) to study the effects of /c, and thepossible interactions between the different spectral compon-ents in the control of shade-avoidance in natural ‘greenshading’. Moreover, such experiments could be repeatedusing other species where photoreceptor mutants are avail-able (for example, Arabidopsis thaliana; Kozuka et al.,2005). Such a combination with genetic analysis could bevery useful to analyse the mechanisms of photocontrol asthe two approaches are complementary. Note, however,that the use of the LAI reference for defining the range ofnatural shading might not be ecologically meaningful fora ruderal plant such as Arabidopsis thaliana that may neverlive in canopies.

Neutral shading produced typical shade-avoidanceresponses

Neutral shading primarily slowed down ontogenic plantdevelopment. At the end of the experiment (i.e. at the samechronological age), shaded plants were smaller than plantsunder high-light conditions with a total number of leavesreduced by half. Additionally, shaded plants showed areduction in the final number of branches, a reduction inthe development of existing branches than that of the mainaxis, and an enhancement of the petiole elongationassociated with a reduction in total leaf area per plant. Allthese responses are consistent with previous work on theeffects of neutral shading in a range of species (Lotscherand Nosberger, 1997; Stuefer and Huber, 1998; Tsukayaet al., 2002) and are typical of what is termed the shade-avoidance syndrome (Smith and Whitelam, 1997).

BL and PAR control the shade-avoidance syndromethrough antagonist effects on leaf production andadditive effects on petiole elongation

PAR and BL reductions were differently involved inproducing the shade-avoidance responses of the plantsgrown under neutral shading. PAR reduction induceda decrease in the leaf production on the main axis, probablythrough the reduction of photosynthesis. This inhibitory

effect of PAR was more pronounced on the branches. Bycontrast, a BL reduction protected the leaf production onthe main axis, but its promoting effects on the rate of leafappearance declined steeply on the branches. Theseresponses of the rate of leaf appearance to BL, in an axis-dependent way is in accordance with previous reports inwhite clover (Gautier et al., 1998). As a result, in neutralshading, the combined contributions of the antagonisticPAR and BL effects maintained the leaf production on themain axis, and decreased it in the branches, as described inthe shade-avoidance syndrome. Increasing shade might notchange this trend as BL stimulation of the leaf productionon the main axis levelled off in deeper BL reduction. Thus,differential PAR and BL controls of leaf production appearto be important in the establishment of the linear growthpattern involved in light-foraging behaviour.

By contrast to the antagonistic effects of PAR and BLreductions on leaf production, BL and PAR reductionsdisplayed additive promoting effects on petiole elongation.Both contributed to the increase in petiole length by theneutral shading studied, with an almost equal share. BLreduction accounted for 45% and PAR reduction for 55%of the neutral shading effects. The effect of BL reductionon petiole elongation confirms previous reports (Gautieret al., 1997; Kozuka et al., 2005). But, the quantificationof a PAR control in this shade-avoidance response isa novel and major finding. Increased shade might notchange the additive behaviour of the BL and PAR effectsas the response curves for petiole length tended to be par-allel with increasing shade (Fig. 5).

Effects of neutral shading on branching and on leafarea versus stem growth were only the consequenceof the effects of PAR on leaf appearance

The strong decrease of the leaf appearance rates due to PARreduction, despite its mitigation by BL effects, clearlyproduced indirect effects on branching. No direct effect ofPAR and BL reductions was found on the relative rateof bud outgrowth. These results indicated that the effectsof shade on branching usually reported in the literaturefor dicots (for example, for white clover; Lotscher andNosberger, 1997), are probably an indirect consequence ofthe effects on the leaf appearance rate reducing the amountof axillary buds.

By the same token, a clear allometry between total stemlength and leaf area per plant was found during develop-ment (Fig. 4). Except in the deep BL reduction, no directeffect of BL or PAR was found on total stem length or leafarea developmental trend (Fig. 4). The effects of neutralshading on leaf area versus stem growth were only anallometric consequence of the reduction of the leaf ap-pearance rate by decreased PAR. In the literature, thereis controversy concerning the effects of neutral shadingon internode elongation (for a review see de Kroon and




Hutchings, 1995) and on lamina expansion (Lotscherand Nosberger 1997; Tardieu et al., 1999). This couldbe due to the fact that the ranges of neutral shading weredifferent and that, generally, possible changes in the ratesof leaf appearance were not monitored. The present re-sults demonstrated that an analysis of the growth responsesduring ontogenesis is crucial to identify plastic responsesdirectly affected by shading (see also Huber and Stuefer,1997; Wright and McConnaughay, 2002) and to getresponse curves informative about the underlying mechan-isms of perception.

PAR and BL actions also involved organ-specificdifferences in sensitivity

The responses of five types of organs were studied: mainapices, lateral apices, internodes, petioles, and laminas.Fluence-rate response curves differed both qualitatively andquantitatively between organs and between processes. Forexample, the response of lateral apices differed from that ofprimary apices for both PAR and BL. This difference in theshape of the response curves might track possible mech-anistic differences. A diversity of shapes was also describedfor other responses to BL reduction by Dougher andBugbee (2001a). Thus, in light-grown plants, the controlof the shade-avoidance responses might not be explainedby similar mechanisms of photoperception and signallingpathways which are just on or off depending on the organ.In both BL and PAR responses, the control involves organ-and process-specific quantitative differences in sensitivity.

Two distinct mechanisms for light in thePAR waveband?

BL reduction had a global enhancing effect on the pro-duction of leaves and on petiole growth. By contrast, PARreduction had an opposite effect on the production ofleaves, whereas it also enhanced petiole growth (even whencorrected for changes in developmental rates). This sug-gests two distinct mechanisms for the action of PAR. Thenegative effect on the rate of leaf appearance is likely tocorrespond to the expected reduction of growth anddevelopment due to decreased photosynthesis, i.e. a trophiccontrol (Ryle et al., 1992). Indeed the response curve forthe rate of leaf appearance to PAR displays a downwardconcavity and an upper asymptote, like the response curveof the net CO2 assimilation. From the literature, there aretwo putative candidates for PAR control of petiole elonga-tion. The first one is a photon-counting effect mediated byphytochromes (i.e. the high irradiance responses mode ofphytochrome action; Casal et al., 1998; Nagy and Schafer,2002). Indeed, in the present conditions, both R and FRfluence rates are highly correlated with PAR (althoughthe R:FR ratio and /c were constant). This would be con-sistent with previous inferences suggesting that spectral

wavebands distinct from BL (350–500 nm) may be directlyimplicated in the photomorphogenetical control of growth(Ballare et al., 1991; Dougher and Bugbee, 2001b). Thealternative candidate for PAR control is sugar signalling.Indeed, Kozuka et al. (2005), using ABA and ethylenepathway mutants in Arabidopsis, argued that sugar signal-ling (which is likely to be PAR-dependent) could beinvolved in petiole and leaf blade responses and interactedwith BL perception in an organ-specific manner.

Concerning the effects of BL reduction, they are unlikelyto be neither trophic-mediated responses, nor phytochrome-mediated high irradiance responses, as no correlation existsin this work between fluence rates in BL and in any otherspectral waveband. This is in accordance with the alreadywell-substantiated evidence that BL can trigger light sig-nalling pathways for plant morphogenetic processes actingthrough specific photoreceptors like cryptochromes orphototropins. Moreover, although interactions betweencryptochromes and the low fluence-rate mechanism ofphytochrome perception have been documented geneticallyand molecularly (Nagy and Schafer, 2002; Lin and Shalitin,2003), they are probably not involved here as the phyto-chrome photoequilibrium (/c) was kept constant in thisexperiment. Control by BL was thus likely to involve theBL signalling pathway on its own.

Conclusion

The experimental design presented here is suited to quantifythe relative contribution of each spectral component ofnatural shading on plant morphogenesis, as illustrated herefor BL and PAR. Separating and quantifying the morpho-genetic effects of BL and PAR reductions revealed that bothare involved in the control of the contrasted responses thatproduce the shade-avoidance syndrome. However, onlytwo responses were directly controlled by both BL and PARreductions: leaf appearance and petiole extension. All theother typical responses to shading were indirect consequen-ces of changes in the leaf appearance rates. BL reductionwas promotive on both responses, whereas PAR reductionwas inhibitory for leaf appearance and promotive on petioleexpansion. However, the sensitivity to BL or PAR variedbetween the different apices. These differences are veryimportant in producing the shade-avoidance syndrome.This demonstrates that the quantitative aspects of BL andPAR sensitivity have to be considered to understand thecontrol of the shade-avoidance responses, in addition tophytochrome-mediated responses to the R:FR ratio.

Acknowledgements

We thank Dr F Tardieu, Dr S Cookson, the two anonymousreferees for fruitful comments on the manuscript, and S Cooksonfor revising the English text.




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