JXB Advance Access originally published online on June 23, 2006
Journal of Experimental Botany 2006 57(10):2379-2390; doi:10.1093/jxb/erj210
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Published by Oxford University Press [2006] on behalf of the Society for Experimental Biology.
RESEARCH PAPER |
Quantitative contributions of blue light and PAR to the photocontrol of plant morphogenesis in Trifolium repens (L.)
Unité d'Ecophysiologie des Plantes Fourragères, INRA, Lusignan, F-86600, France
*To whom correspondence should be addressed. E-mail: christop{at}ensam.inra.fr
Received 17 October 2005; Accepted 22 March 2006
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Abstract |
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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 [photosynthetically 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 µmol m–2 s–1) at constant PAR (300 µmol m–2 s–1) and three PAR levels (338, 705, and 163 µmol m–2 s–1) at constant BL (36 µmol m–2 s–1). Effects on morphogenetic processes were analysed as quantitative modulations of ontogenic trends and response curves were produced. White clover (Trifolium repens L.) was used, as it is a typical shade-avoider displaying 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 mechanisms, such as a trophic effect and a signal. Both PAR and BL actions involved organ-specific differences, 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
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Introduction |
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Plants have evolved a high level of plasticity in response to light conditions. In many species, light-grown plants react to canopy shade by a suite of morphogenetic responses, involving (i) an enhancement of stem and petiole elongation and an associated reduction in lamina expansion, (ii) an increase in main axis development compared with that of existing branches, and (iii) a reduction in the number of branches (Smith and Whitelam, 1997). This suite of photomorphogenetic responses has been called the shade-avoidance syndrome (Grime, 1981) and is interpreted as an adaptive light-foraging behaviour (for reviews see de Kroon and Hutchings, 1995; Ballaré et al., 1997). However, the way that these responses are controlled by shading is not yet fully understood.
Many questions remain unanswered, such as, which spectral components of light are involved and to what extent? Generally, studies of the shade-avoidance syndrome are primarily concerned with the perception of the changes in the red far:red light ratio (R:FR) through phytochromes. This has now been well characterized (for reviews see Smith and Whitelam, 1997; Smith, 2000; Franklin and Whitelam, 2005). However, it is known that approximately half of the total shade-avoiding responses can also be observed under neutral shading at a constant R:FR ratio, thus involving other photocontrolling mechanisms ( Lötscher and Nösberger, 1997; Stuefer and Huber, 1998). More recently, manipulation of light conditions during plant development and the use of various photoreceptor mutants have indeed revealed the specific role of the blue light (BL) fluence rate in the shade-avoidance syndrome, acting through cryptochromes (Ballaré et al., 1991; Kozuka et al., 2005). However, despite significant advances in the understanding of the molecular mechanisms involved in BL perception (Lin and Shalitin, 2003) and in the BL control of seedling morphogenesis (Ahmad et al., 2002), there is still little information about the effects of BL on light-grown plants. And even fewer studies have been conducted on light-grown plants to separate BL fluence rate from changes in phytochrome photoequibrium and in photosynthetically active radiation (PAR) (Ballaré et al., 1991; Gautier et al., 1997, 1998; Dougher and Bugbee, 2001a).
Additionally, there is evidence indicating that PAR is also implicated in the control of plant morphogenesis. For example, light-grown plants displayed different responses in stem elongation or growth under two levels of PAR with no reduction in BL and in the R:FR ratio (Ballaré 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 on Arabidopsis using sugar-insensitive mutants (Kozuka et al., 2005). However, the effects of PAR are still rarely documented and no quantitative insights have been given on the relative importance of BL and PAR responses in a given amount of shading. Indeed, most of studies on the effects of PAR on plant morphogenesis are focused upon the so-called ‘neutral shading’, which reduces light homogeneously in the 400–700 nm waveband with a constant phytochrome photoequilibrium but inducing a proportional reduction of BL. Interpretation of the effects of PAR and BL on plant morphogenesis in these studies is complicated because the light sources used and the BL:PAR ratios vary considerably. Plants may respond simultaneously, but in different ways, to changes in BL and in PAR so these studies can only yield circumstantial conclusions about the photocontrol of shade-avoidance responses.
Another central question concerns the photocontrol of the different, and possibly antagonistic responses, of the plant parts involved in the adaptive shade-avoidance syndrome, for example, stimulation of stem elongation and inhibition of lamina expansion (Kozuka et al., 2005). Several hypotheses can be proposed to explain such differences. The simplest one is based on the fact that plant growth during ontogenesis is generally allometric rather than isometric. So an effect of light on the rate of plant development would change the absolute growth rates of organs, whereas the allometric relationships would be kept constant (Wright and Mc Connaughay, 2002). This potential confusion between plasticity in growth rates and in allometries could be overcome by taking plant ontogeny into account (through an index of developmental stages) when studying adaptive responses of plant morphology to shading (Huber and Stuefer, 1997). Alternatively, growth allometries could be directly controlled by light-inducing contrasted plastic responses of the organs (Wright and McConnaughay, 2002). In that case, the contrasted plastic responses of the different organs could be explained by two hypotheses: (i) the photoperception mechanisms are the same, but the selection of morphogenesis responses is organ specific; or (ii) the photoperception sensitivity is organ-dependent (the different organs being more or less sensitive to the different spectral components of light). An approach to test these hypotheses is to consider light fluence-rate response curves. Indeed, if the hypothesis of organ-specific selection holds true, then all the responses that are selected in all the organs should display fluence-rate response curves with similar shapes (as they depend on the same process of photoperception). By contrast, if the photoperception itself is organ-dependent, then different fluence-rate response curves should be produced. However, until now, few BL response curves have been characterized (Wheeler et al., 1991; Dougher and Bugbee, 2001a), and possible changes in developmental rates caused by shading were not considered. Moreover, no PAR response curves independent of BL have ever been produced, as far as is known.
The aim of this study was to identify and quantify the relative contributions of BL and PAR in the photocontrol of aerial morphogenesis of a typical shade-avoiding species (Trifolium repens L.). This species presented the advantage that the analysis of its shade-avoidance syndrome is not complicated by flowering time. Although this work required controlled environmental conditions, light conditions were selected to mimic those found in natural shade, in the range in which PAR and BL responses are likely to become significant (Ballaré et al., 1991). Developmental ontogenic trends were quantified to investigate how morphogenetic processes are plastically affected by shading, separating responses of the rate of ontogenic development from specific plastic responses of organ growth. Additional light conditions were also studied to produce PAR and BL fluence-rate response curves to determine how each spectral component of light controls the different morphogenetic responses for each organ.
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Materials and methods |
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Defining ecologically relevant artificial light treatments
Each artificial light treatment was controlled to simulate the PAR and BL irradiances experienced by a plant that is shaded by a white clover canopy of a given leaf area index (LAI) in natural conditions. The PAR incident in the growth chambers was fixed to produce a typical natural incident light. The levels of PAR and BL irradiances to be applied in the artificial treatments were determined using the relationship between the fraction of transmitted light in the visible 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 for details on the shade conditions retained). The fraction of light transmitted in the BL waveband was assumed to be similar to that of incident light, as the BL:PAR ratio has been shown to be independent of the amount of shading (Holmes, 1981; Messier and Bellefleur, 1988).
As the aim was to vary independently BL and PAR fluence rates, each light treatment was designated as BLx_PARy where x is the fraction of light transmitted in the BL range and y that for PAR. When x and y are equal, the treatment corresponds to a neutral shade treatment that could be achieved under a natural canopy.
Experimental design and artificial light treatments
In the growth chambers, the incident light was fixed to simulate a 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 daily incident 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, 0.07°E, 46.3°N, meteorological data).
Two neutral shade treatments, BL45_PAR45 and BL20_PAR20, were applied to study the effects of neutral shading. These fractions of transmitted light in BL and PAR correspond to ranges of natural shading (LAIs from 1.4 to 2.8) in which (i) PAR and BL responses are likely to become significant (Ballaré 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 to measure quantitatively the separated contributions of PAR and BL reductions in this neutral shading (Fig. 1). The contribution of BL reduction at a constant PAR was assessed by comparing BL45_PAR45 and BL20_PAR45 treatments. That of PAR reduction at a constant BL was assessed by comparing BL20_PAR45 and BL20_PAR20.
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Two additional light treatments, BL20_PAR100 and BL2_PAR45 (Fig. 1), were defined to establish PAR and BL fluence-rate response curves. BL20_PAR100, with no PAR reduction (i.e. with the same PAR level as that of the incident light) was preferred to a deep PAR reduction, to prevent the plants from being under the point of compensation of photosynthesis. BL2_PAR45 treatment, corresponding to 4 µmol m–2 s–1 BL in the present conditions, was retained as the effects of BL were sometimes found maximum for photon fluxes lower than 40 µmol m–2 s–1 BL (Wheeler et al., 1991; Dougher and Bugbee, 2001a). Moreover it is similar to the BL (–) treatment used by Gautier et al. (1997) in white clover. Fluence-rate response curves to BL at constant PAR could then be established considering BL45_PAR45, BL20_PAR45, and BL2_PAR45, and that of PAR at constant BL considering BL20_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 of metallic halide lamps (HQI, 400 W; Osram, France). In the BL45_PAR45 treatment no filter was used. In the other light treatments, PAR and BL irradiances required were obtained from a similar ceiling but using one layer of a filter (Lee Filters, ATOHAAS France SA Argenteuil, France; transmission curves of the filters are shown in Fig. 2A) on top of the plants. BL2_PAR45 was 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), and BL20_PAR20 by adding a perforated reflector (Lee Filter 270). The light spectrum at plant level (Fig. 2B) was measured for each treatment using an LI-1800 spectroradiometer (Li-Cor Inc., Lincoln, NE, USA) and relevant actinic light fluxes were then computed for all the treatments (Table 1). For PAR, the standard definition of the PPFD (photosynthetic photon flux density at 400–700 nm) was used. For BL, fluence rate was measured as the photon fluence rate in the 350–500 nm waveband (Ahmad et al., 2002; Lin and Shalitin, 2003). In order to eliminate the confusion between BL and photosynthetic effects, the photosynthetic efficiency (light spectrum times the action spectrum of the leaf photosynthetic yield; Sager et al., 1988) was computed for all the light treatments and controlled to be decorrelated with BL (Table 1).
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In all treatments, the phytochrome photoequilibrium was maintained at its maximal ‘unshaded’ values (i.e. slightly higher than that of incident natural solar radiation), the UV fluence rate was negligible (Fig. 2) and the photoperiod was 10 h.
Note that, from the commercially available filters, it was not possible to produce the levels of BL and PAR fluence rates matching exactly the targeted percentage of light transmitted. The greatest deviation 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 (20x12x8 cm) filled with sterilized sand (Gautier et al., 1997). During the pre-experiment period, all the cuttings were grown together and subjected to the BL45_PAR45 treatment (Table 1). After 2 weeks, plants were selected for uniformity of (i) internode and petiole lengths and (ii) unfolding foliar stage using the decimal scale proposed for white clover by Carlson (1966) (10 stages from 0.1 when the leaf first becomes visible to 1 when the leaf is fully unfolded). Eight plants were then randomly assigned to each light treatment. Pots were watered liberally by excess solution eight times a day, using a complete nutrient solution containing nitrogen (Gastal and Saugier, 1986) to avoid any water or nutrient limitation. Relative air humidity was maintained constant at 80%. Air temperature was measured at plant height using six thermocouples per growth chamber and maintained at 23 °C day/20 °C night. A more complete description of the growth conditions is presented in Table 1. Note that with a 10 h photoperiod all the plants in all the treatments remained vegetative.
Plant measurements
Plant morphology:
White clover (Trifolium repens L.) is a clonal stoloniferous plant with plagiotropic axes (main axis and lateral branches) and orthotropic petioles bearing leaves (a leaf consists of three leaflets). An axis can be viewed as a succession of phytomers comprising a node, an internode, an axillary bud, a leaf, and two nodal root primordia (Bell, 1984).
Number of leaves:
The number of leaves and their unfolding stage (using the Carlson decimal scale; Carlson, 1966) were recorded every 3 d, on each axis of the cutting, for eight plants per treatment. These data were used to calculate the usual indices of development representing the number of leaves of a given axis (cumulated Carlson stage; Gautier et al., 1997) and of the whole plant (plant cumulated Carlson stage; Christophe et al., 2003), in decimal units. Note that the development of the whole plant depends on both (i) the rate of leaf appearance on the axes and (ii) the rate of branch production.
Rate of leaf appearance:
In each treatment, the rates of leaf appearance (RLA; leaf °C–1 d–1) on the whole plant and on each axis were estimated by fitting the number of leaves (in Carlson decimal units) that were visible on the whole plant and on each axis against thermal time. Thermal time was calculated as the cumulative degree-days from emergence, assuming a base temperature of 0 °C (Simon et al., 1989; Gautier et al., 1997).
The balance between the rate of leaf appearance on the lateral branches and that on the main axis was estimated by the slope of the relationship between the number of leaves of each branch (in Carlson decimal units) and the number of leaves of the main axis (in Carlson decimal units) produced at the same time.
Rate of branch production per bud:
The rate at which newly formed axillary buds were produced on new lateral branches was estimated by the slope of the relationship between the number of branches and the number of leaves of the whole plant produced over the same time interval (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 every 3 d using callipers. For each treatment, the individual leaf area was estimated from an allometric relationship between the leaflet area and the midrib length determined from a sample of leaves using an Li-Cor planimeter (Li-3100, Li-Cor Inc.). In order to study plastic growth responses, the plant axis length and the plant leaf area were compared between treatments at the same plant developmental stage (Christophe et al., 2003). Thus the rates of the plant axis elongation and of the plant leaf expansion were estimated by fitting the plant axis length and the plant leaf area, respectively, against the number of leaves of the whole plant (in Carlson decimal units). Lengths of petioles were compared between treatments at their final stage of leaf development (when maximal length is achieved). Only the petiole lengths of leaves 3 and 4 of the main axis are given, as they were the only petioles that completed the entire visible growth during the light treatments.
Statistical analysis
Final plant measurements were compared using a one-way analysis of 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 linear model, with the mixed procedure (Proc Mixed) in SAS version 6.12 (Littell et al., 1996). The effects of individual plants (nested within the treatment) were added as random effects in the error of the model, using the repeated option in Proc Mixed. The variance–covariance matrix of the error was specified by a ‘spatial’ power structure [SP (Pow)]. SP (Pow) takes into account uneven intervals in the data sets. For the present data sets, it displayed the highest Akaike's information criterion (AIC) and Schwarz' Bayesian criterion (SBC) (as defined in SAS version 6.12; Littell et al., 1996). For the study of developmental kinetics, kinetics were fitted using a linear or polynomial regression model. The light treatments were first tested as fixed effects on the heterogeneity of the parameters of this model (slopes, higher order coefficients). If this test was significant, the parameters were compared using the estimate statement; otherwise a covariance analysis was performed. When the common slope was not different from zero, the model was reduced to a (mixed) analysis of variance, with the light treatment effects as fixed effects.
Whenever a statistically significant effect was found for at least one light treatment, fluence-rate response curves were constructed using means values (and standard error).
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Morphology of shaded plants
At the end of experiment, the effects of neutral shading on plant morphology were tested by comparing plants grown under the two neutral shade treatments (Table 2). Shaded plants had produced about half the number of leaves and branches than plants grown under high-light conditions. Shaded plants also presented a reduction in the number of leaves on the branches and in the total leaf area. By contrast, the shaded plants produced petioles much longer than plants under high-light conditions. The number of leaves on the main axes and the total stem length were not affected by shading (Table 2).
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Neutral shading induced changes in both plant development and growth, leading to important differences in plant morphology. Morphogenetic processes were thus studied during ontogeny in order to separate responses of the rate of the ontogenic development and their possible indirect consequences on organ growth from direct plastic responses of organs. Then, for each morphogenetic process, the effects of the neutral shading and the contribution of the effects of PAR and BL shading were analysed by quantifying modulations of these ontogenic trends.
Ontogenic development trends
In all the treatments, the plants had a similar initial developmental stage and their number of leaves increased exponentially during development. As a consequence, the rate of the ontogenic development can be easily quantified by estimating the slope of the changes in the number of leaves 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 over time for the period considered (23 d). The balance between the rate of leaf appearance on the primary branches and that on the main axis (i.e. the slope of the curve in Fig. 3C) was also constant over the time. Additionally, as there was no significant difference in this ratio between the first four branches, the data were pooled. In all the treatments, this ratio was below 1, indicating that the production of leaves of the main axis was always superior to that of the primary branches. The relationship between the total number of leaves and the total number of branches was complex and exhibited a cubic pattern when the plant had <10 branches (Fig. 3D). This suggests that the rate of branch production (i.e. the inverse of the slope of this curve) was not constant over the developmental range investigated (up to six branches). This rate increased rapidly during the early stage of plant development, reaching a peak when the plant had two branches, and then declined continuously (data not shown). Considering growth of the whole plant, the total leaf area increased proportionally with the number of leaves of the whole plant (Fig. 4A), whereas the plant axis length increased as a three-quarters power law function of the total number of leaves (Fig. 4B). Thus a three-quarter power allometry between total internode length and total leaf area per plant was maintained during development.
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Despite significant differences in the final petiole length of leaves 3 and 4 (Fig. 4C) there was no significant interaction between shade treatments and leaf rank (P=0.3944) and all the petioles on all the branches were similarly affected, even when correcting for total leaf number (data not shown).
Effects of neutral shading and PAR and BL reductions on development of the whole plant
The neutral shading significantly slowed down the relative rate of the ontogenic development of the plant (by –0.0013, –24%, Fig. 3A; Table 3), and this was shown to be caused entirely by the reduction in PAR (Table 3). In the present conditions, the rate of branch production was not significantly affected by either neutral shading or by BL and PAR reductions (Fig. 3D). Only the rate of leaf appearance (RLA) on the different axes was differentially affected by the treatments (Fig. 3B, C; Table 3).
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Under the neutral shading, the RLA on the main axis was not significantly modified (Table 3) and its mean value was 0.0122 leaf °C–1 d–1. However, individually, both BL and PAR reductions affected the RLA on the main axis (Table 3). BL reduction significantly increased RLA by +0.002 leaf °C–1 d–1 whereas, on the contrary, PAR reduction decreased RLA by –0.002 leaf °C–1 d–1. This explains the absence of neutral shading effects.
The neutral shading significantly reduced the ratio of the rates of leaf appearance on the main axis and primary branches by 21% (Table 3). This was the consequence of a reduction of the RLA on the primary branches (–0.002 leaf °C–1 d–1, –20%). BL reduction decreased this ratio by 13% (Table 3), which was due to a greater increase of the RLA on the main axis (three times more) than that on the branches (+0.0004 leaf °C–1 d–1, +4%). PAR reduction also decreased this ratio by 10.5% (Table 3) but, in this case, it was due to a greater reduction of the RLA on the branches (–0.003 leaf °C–1 d–1, –23%) than that on the main axis.
Effects of neutral shading and PAR and BL reductions on plant growth
No significant modification in the rate of the plant leaf expansion and of the plant axis elongation was observed in response to neutral shading, BL and PAR reductions (Fig. 4; Table 3). By contrast, the final petiole length of the mature petioles was significantly increased by the neutral shading (Fig. 4C; Table 3). Neutral shading induced a significant increase of 28 mm of the petiole length of leaves 3 and 4 of the main axis (corresponding to an increment of 35%). BL reduction increased the petiole length of leaves 3 and 4 by 12.5 mm (corresponding to an increment of 16%). PAR reduction increased the petiole length of leaves 3 and 4 by 15.5 mm (corresponding to an increase of 17%).
PAR and BL fluence-rate response curves
The morphogenetic parameters shown to be significantly affected by at least one light treatment were studied further by the analysis of the fluence-rate response curves, including the two additional light treatments BL20_PAR100 and BL2_PAR45. Plant axis length and branching production were not affected under the present treatments (P=0.2691 and P=0.074, respectively).
The shape of the fluence-rate response curves varied between responses (Fig. 5A, B). Two response curves to PAR (the rate of leaf appearance on the main axis and the petiole length; Fig. 5A) displayed no significant changes between 300 and 750 µmol m–2 s–1 despite a 2.5-fold difference in irradiance, whereas they displayed clear and opposite responses under 300 µmol m–2 s–1. These two responses thus probably tend to saturate for irradiance over 300 µmol m–2 s–1 or, at least, reach a maximum between 300 and 750 µmol m–2 s–1. Surprisingly, the ratio between the rates of leaf appearance on the main axis and on the branches did not show such a saturating pattern, although it seemed to display damped increments between 300 and 750 µmol m–2 s–1. Lateral axes probably displayed a different range and shape of PPFD sensitivity than the main axis. Also surprising, the rate of leaf expansion was never significantly affected by the levels of PAR in the conditions (P=0.2395), contrary to the petiole.
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The rate of leaf appearance on the main axis increased for moderate BL reductions (Fig. 5B) and then levelled off with BL fluence rates lower than 38 µmol m–2 s–1, or at least reach some maximum for a deep BL reduction. The response of the ratio between the rates of leaf appearance on the main axis and on the branches was distinct from that of the rate of leaf appearance on the main axis, as noted for PAR response, but to a higher extent. Indeed a deep BL reduction decreased very significantly the ratio on the leaf appearance rates. Contrary to the PAR responses, BL reduction increased both the rate of leaf expansion and the petiole final length to similar extents of, respectively, +35% and +40% (between extreme treatments).
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Varying BL and PAR independently within ecologically relevant ranges
The action spectra of phytochromes, cryptochromes, and photosynthetic efficiency largely overlap (Schäfer et al., 1983). Therefore, uncoupling experimentally blue light (BL) and photosynthetically active radiation (PAR) within a realistic broad-waveband light without affecting phytochrome photoequilibirum (
c) is not simple. The experimental design reported here is the first as far as is known that was constructed (i) to quantify the relative contributions of BL and PAR in the photocontrol of plant morphogenesis, and (ii) to control the level of artificial light irradiance in BL and PAR ranges to correspond to that of natural shading (as defined with a relationship between the fraction of transmitted light and the leaf area index (LAI; see Materials and methods). It is a generalization of the method of ‘light equivalence’ designed to analyse the BL effects at constant c using two monochromatic lights (Schäfer et al., 1983). Dougher and Bugbee (2001a) produced BL variations for two levels of PAR by combining different light sources and filters in a way similar to the one used in the present study. But, in their experimental design, the relative contribution of the effects of BL and of PAR in shading could not be fully analysed. Note also that the present approach can be conducted over the whole range of plant development (the light treatments can be applied at different times during plant development) and the whole range of possible shade. Indeed, it would be interesting to extend the experimental design (with additional light treatments) to study the effects of c, and the possible interactions between the different spectral components in the control of shade-avoidance in natural ‘green shading’. Moreover, such experiments could be repeated using other species where photoreceptor mutants are available (for example, Arabidopsis thaliana; Kozuka et al., 2005). Such a combination with genetic analysis could be very useful to analyse the mechanisms of photocontrol as the two approaches are complementary. Note, however, that the use of the LAI reference for defining the range of natural shading might not be ecologically meaningful for a ruderal plant such as Arabidopsis thaliana that may never live in canopies.
Neutral shading produced typical shade-avoidance responses
Neutral shading primarily slowed down ontogenic plant development. At the end of the experiment (i.e. at the same chronological age), shaded plants were smaller than plants under high-light conditions with a total number of leaves reduced by half. Additionally, shaded plants showed a reduction in the final number of branches, a reduction in the development of existing branches than that of the main axis, and an enhancement of the petiole elongation associated with a reduction in total leaf area per plant. All these responses are consistent with previous work on the effects of neutral shading in a range of species (Lötscher and Nösberger, 1997; Stuefer and Huber, 1998; Tsukaya et al., 2002) and are typical of what is termed the shade-avoidance syndrome (Smith and Whitelam, 1997).
BL and PAR control the shade-avoidance syndrome through antagonist effects on leaf production and additive effects on petiole elongation
PAR and BL reductions were differently involved in producing the shade-avoidance responses of the plants grown under neutral shading. PAR reduction induced a decrease in the leaf production on the main axis, probably through the reduction of photosynthesis. This inhibitory effect of PAR was more pronounced on the branches. By contrast, a BL reduction protected the leaf production on the main axis, but its promoting effects on the rate of leaf appearance declined steeply on the branches. These responses of the rate of leaf appearance to BL, in an axis-dependent way is in accordance with previous reports in white clover (Gautier et al., 1998). As a result, in neutral shading, the combined contributions of the antagonistic PAR and BL effects maintained the leaf production on the main axis, and decreased it in the branches, as described in the shade-avoidance syndrome. Increasing shade might not change this trend as BL stimulation of the leaf production on the main axis levelled off in deeper BL reduction. Thus, differential PAR and BL controls of leaf production appear to be important in the establishment of the linear growth pattern involved in light-foraging behaviour.
By contrast to the antagonistic effects of PAR and BL reductions on leaf production, BL and PAR reductions displayed additive promoting effects on petiole elongation. Both contributed to the increase in petiole length by the neutral shading studied, with an almost equal share. BL reduction accounted for 45% and PAR reduction for 55% of the neutral shading effects. The effect of BL reduction on petiole elongation confirms previous reports (Gautier et al., 1997; Kozuka et al., 2005). But, the quantification of a PAR control in this shade-avoidance response is a novel and major finding. Increased shade might not change the additive behaviour of the BL and PAR effects as the response curves for petiole length tended to be parallel with increasing shade (Fig. 5).
Effects of neutral shading on branching and on leaf area versus stem growth were only the consequence of the effects of PAR on leaf appearance
The strong decrease of the leaf appearance rates due to PAR reduction, despite its mitigation by BL effects, clearly produced indirect effects on branching. No direct effect of PAR and BL reductions was found on the relative rate of bud outgrowth. These results indicated that the effects of shade on branching usually reported in the literature for dicots (for example, for white clover; Lötscher and Nösberger, 1997), are probably an indirect consequence of the effects on the leaf appearance rate reducing the amount of axillary buds.
By the same token, a clear allometry between total stem length and leaf area per plant was found during development (Fig. 4). Except in the deep BL reduction, no direct effect of BL or PAR was found on total stem length or leaf area developmental trend (Fig. 4). The effects of neutral shading on leaf area versus stem growth were only an allometric consequence of the reduction of the leaf appearance rate by decreased PAR. In the literature, there is controversy concerning the effects of neutral shading on internode elongation (for a review see de Kroon and Hutchings, 1995) and on lamina expansion (Lötscher and Nösberger 1997; Tardieu et al., 1999). This could be due to the fact that the ranges of neutral shading were different and that, generally, possible changes in the rates of leaf appearance were not monitored. The present results demonstrated that an analysis of the growth responses during ontogenesis is crucial to identify plastic responses directly affected by shading (see also Huber and Stuefer, 1997; Wright and McConnaughay, 2002) and to get response curves informative about the underlying mechanisms of perception.
PAR and BL actions also involved organ-specific differences in sensitivity
The responses of five types of organs were studied: main apices, lateral apices, internodes, petioles, and laminas. Fluence-rate response curves differed both qualitatively and quantitatively between organs and between processes. For example, the response of lateral apices differed from that of primary apices for both PAR and BL. This difference in the shape of the response curves might track possible mechanistic differences. A diversity of shapes was also described for other responses to BL reduction by Dougher and Bugbee (2001a). Thus, in light-grown plants, the control of the shade-avoidance responses might not be explained by similar mechanisms of photoperception and signalling pathways 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 the PAR waveband?
BL reduction had a global enhancing effect on the production of leaves and on petiole growth. By contrast, PAR reduction had an opposite effect on the production of leaves, whereas it also enhanced petiole growth (even when corrected for changes in developmental rates). This suggests two distinct mechanisms for the action of PAR. The negative effect on the rate of leaf appearance is likely to correspond to the expected reduction of growth and development due to decreased photosynthesis, i.e. a trophic control (Ryle et al., 1992). Indeed the response curve for the rate of leaf appearance to PAR displays a downward concavity and an upper asymptote, like the response curve of the net CO2 assimilation. From the literature, there are two putative candidates for PAR control of petiole elongation. The first one is a photon-counting effect mediated by phytochromes (i.e. the high irradiance responses mode of phytochrome action; Casal et al., 1998; Nagy and Schäfer, 2002). Indeed, in the present conditions, both R and FR fluence rates are highly correlated with PAR (although the R:FR ratio and c were constant). This would be consistent with previous inferences suggesting that spectral wavebands distinct from BL (350–500 nm) may be directly implicated in the photomorphogenetical control of growth (Ballaré et al., 1991; Dougher and Bugbee, 2001b). The alternative candidate for PAR control is sugar signalling. Indeed, Kozuka et al. (2005), using ABA and ethylene pathway mutants in Arabidopsis, argued that sugar signalling (which is likely to be PAR-dependent) could be involved in petiole and leaf blade responses and interacted with BL perception in an organ-specific manner.
Concerning the effects of BL reduction, they are unlikely to be neither trophic-mediated responses, nor phytochrome-mediated high irradiance responses, as no correlation exists in this work between fluence rates in BL and in any other spectral waveband. This is in accordance with the already well-substantiated evidence that BL can trigger light signalling pathways for plant morphogenetic processes acting through specific photoreceptors like cryptochromes or phototropins. Moreover, although interactions between cryptochromes and the low fluence-rate mechanism of phytochrome perception have been documented genetically and molecularly (Nagy and Schäfer, 2002; Lin and Shalitin, 2003), they are probably not involved here as the phytochrome photoequilibrium (c) was kept constant in this experiment. Control by BL was thus likely to involve the BL signalling pathway on its own.
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Conclusion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferences |
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The experimental design presented here is suited to quantify the relative contribution of each spectral component of natural shading on plant morphogenesis, as illustrated here for BL and PAR. Separating and quantifying the morphogenetic effects of BL and PAR reductions revealed that both are involved in the control of the contrasted responses that produce the shade-avoidance syndrome. However, only two responses were directly controlled by both BL and PAR reductions: leaf appearance and petiole extension. All the other typical responses to shading were indirect consequences of changes in the leaf appearance rates. BL reduction was promotive on both responses, whereas PAR reduction was inhibitory for leaf appearance and promotive on petiole expansion. However, the sensitivity to BL or PAR varied between the different apices. These differences are very important in producing the shade-avoidance syndrome. This demonstrates that the quantitative aspects of BL and PAR sensitivity have to be considered to understand the control of the shade-avoidance responses, in addition to phytochrome-mediated responses to the R:FR ratio.
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Acknowledgements |
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We thank Dr F Tardieu, Dr S Cookson, the two anonymous referees for fruitful comments on the manuscript, and S Cookson for revising the English text.
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Present address: Laboratoire d'Ecophysiologie des Plantes sous Stress Environnementaux, UMR INRA – ENSAM, place Viala, F-34060 Montpellier cedex 1, France. 
Present address: UMR PIAF, INRA, Site de Crouël, 234 avenue du Brézet, F-63039 Clermont-Ferrand cedex 02, France.
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