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Keywords:

  • DELLA;
  • phytochrome;
  • PIF (PHYTOCHROME INTERACTING FACTOR);
  • red to far-red ratio (R : FR);
  • shade avoidance

Abstract

  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. Shade avoidance responses
  5. III. Photoreceptor regulation of shade avoidance
  6. IV. Molecular mechanisms in shade avoidance signalling
  7. V. Crosstalk in shade avoidance signalling
  8. VI. Future perspectives
  9. Acknowledgements
  10. References

Contents

 Summary930V.Crosstalk in shade avoidance signalling939
I.Introduction931VI.Future perspectives940
II.Shade avoidance responses932 Acknowledgements940
III.Photoreceptor regulation of shade avoidance932 References940
IV.Molecular mechanisms in shade avoidance signalling934   

Summary

The threat to plant survival presented by light limitation has driven the evolution of highly plastic adaptive strategies to either tolerate or avoid shading by neighbouring vegetation. When subject to vegetational shading, plants are exposed to a variety of informational signals, which include altered light quality and a reduction in light quantity. The former includes a decrease in the ratio of red to far-red wavelengths (low R : FR) and is detected by the phytochrome family of plant photoreceptors. Monitoring of R : FR ratio can provide an early and unambiguous warning of the presence of competing vegetation, thereby evoking escape responses before plants are actually shaded. The molecular mechanisms underlying physiological responses to alterations in light quality have now started to emerge, with major roles suggested for the PIF (PHYTOCHROME INTERACTING FACTOR) and DELLA families of transcriptional regulators. Such studies suggest a complex interplay between endogenous and exogenous signals, mediated by multiple photoreceptors. The phenotypic similarities between physiological responses habitually referred to as ‘the shade avoidance syndrome’ and other abiotic stress responses suggest plants may integrate common signalling mechanisms to respond to multiple perturbations in their natural environment.


I. Introduction

  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. Shade avoidance responses
  5. III. Photoreceptor regulation of shade avoidance
  6. IV. Molecular mechanisms in shade avoidance signalling
  7. V. Crosstalk in shade avoidance signalling
  8. VI. Future perspectives
  9. Acknowledgements
  10. References

As sessile photoautotrophs, plants must continuously adjust their growth and development to optimize photosynthetic activity in fluctuating conditions. This developmental plasticity is achieved through the perception, transduction and integration of multiple environmental signals. In addition to providing a key energy resource for photosynthesis, light signals provide plants with important spatial and temporal information about their surrounding environment. Light signals are perceived by specialized information-transducing photoreceptors which include the red (R) and far-red (FR) light-absorbing phytochromes and the blue/UV-A light-absorbing cryptochromes and phototropins (see Section III). Phytochromes are reversibly photochromic biliproteins which exist in two photo-convertible isomers, a biologically inactive red light-absorbing Pr form, and a biologically active far-red light-absorbing Pfr form. Phytochromes are synthesized in the Pr form and acquire biological activity following phototransformation to the Pfr form (reviewed in Schäfer & Nagy, 2006). In natural light environments, phytochrome exists in a dynamic equilibrium of Pr and Pfr forms, the relative proportions of each being largely determined by ambient light quality. The parameter commonly used to describe the light quality of natural environments is the ratio of photon irradiance in the red region of the spectrum to that in the FR region (termed R : FR ratio). This relates directly to the properties of phytochrome and is often more precisely defined as the following:

  • image

Eqn 1

The R : FR ratio of daylight has been shown to vary little with weather conditions or season (Holmes & Smith, 1977), but undergoes daily fluctuations at dawn and dusk, periods commonly referred to as twilight. During twilight periods, solar elevation drops below 10°, increasing the path length of the solar beam through the earth's atmosphere. This results in enhanced absorption and scattering of shorter wavelengths in the outer atmosphere, enhancing the proportion of longer wavelengths at the earth's surface. Twilight periods are therefore often associated with a significant drop in R : FR, particularly at northern latitudes where twilight duration is also enhanced. The magnitude and duration of twilight reductions in R : FR can provide important seasonal information to boreal species, regulating processes such as bud development in some trees (Linkosalo & Lechowicz, 2006).

The most significant alteration in R : FR occurs when daylight is reflected from (or transmitted through) living vegetation. Preferential absorption of red and blue wavebands by chlorophyll and carotenoid pigments results in the selective enrichment of lesser absorbed regions of the spectrum in reflected/transmitted light. The latter include green and FR wavebands, making plants appear green to the human eye. Although not detectable by human vision, the greatest enrichment occurs in FR wavelengths, thereby reducing the R : FR ratio. The low R : FR ratio signal of reflected light can provide early warning of the presence and proximity of neighbouring vegetation, enabling the initiation of adaptive developmental strategies to either tolerate or avoid vegetational shade before canopy closure. When shaded by neighbouring vegetation, plants additionally experience a reduction in light quantity (in particular photosynthetically active radiation (PAR), 400–700 nm) and elevated concentrations of the gaseous hormone ethylene (see Section V). The integration of multiple environmental stimuli therefore enables plants to distinguish between the threat of vegetational shading (proximity perception) and actual shading (shade perception), ensuring initiation of the appropriate developmental response. The spectral photon distribution of daylight and daylight filtered through Arabidopsis leaves is shown in Fig. 1. In this experiment, a single Arabidopsis leaf filter covering the spectroradiometer sensor reduced the PAR of daylight from 1500 to 120 µmol m−2 s−1 and the R : FR ratio from 1.2 to 0.2. Further reductions of PAR and R : FR to 40 µmol m−2 s−1 and 0.1, respectively, were recorded following the addition of a second leaf. These data highlight the dramatic alteration in light quality and significant reduction in PAR experienced by plants developing under a vegetational canopy. The ability of individual plants to effectively tolerate or avoid shading by neighbouring vegetation thereby significantly enhances competitiveness, and ultimately the probability of reproductive success, in rapidly growing populations.

image

Figure 1. Spectral photon distribution of daylight and daylight filtered through Arabidopsis leaves. Spectra were recorded at noon during October in Leicester, UK.

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II. Shade avoidance responses

  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. Shade avoidance responses
  5. III. Photoreceptor regulation of shade avoidance
  6. IV. Molecular mechanisms in shade avoidance signalling
  7. V. Crosstalk in shade avoidance signalling
  8. VI. Future perspectives
  9. Acknowledgements
  10. References

Considerable intra- and interspecies variation occurs in the response of plants to vegetative shade and is thought to confer selective advantage in different ecological habitats. In shade-tolerant species such as Alocasia macrorrhiza, leaves display adaptations in photosynthetic structures to optimize efficiency at low light intensities. These include thinner leaves, higher chlorophyll content and lens-shaped epidermal cells to focus light within mesophyll tissue (Boardman, 1977; Middleton, 2001). In shade-avoiding species (e.g. Sinapis alba, Chenopodium album), perception of low R : FR results in a suite of developmental responses, collectively referred to as the ‘shade avoidance syndrome’ (Smith & Whitelam, 1997). The most striking phenotypes observed in dicotyledonous plants subject to low R : FR are a rapid elongation of stems and leaves (Morgan & Smith, 1976, 1978, 1981) and an upward reorientation of leaves (leaf hyponasty) (Whitelam & Johnson, 1982). These adaptations serve to elevate leaves within the canopy, a response that is likely to enhance light-foraging capacity in dense stands and enable plants to overtop competing vegetation. Indeed, horizontally propagated, reflected FR within the lower vegetational strata of canopies has been shown to be a major regulatory signal controlling stem elongation in shade-avoiding species (Ballaréet al., 1987, 1990). The rate of stem elongation responses to targeted supplementary FR was measured in Sinapis alba seedlings by Harry Smith and colleagues, using linear voltage displacement transducers and a fibreoptic light source (Morgan et al., 1980). An increase in stem elongation rate was recorded following a lag phase of just 10–15 min, demonstrating the rapidity of this response.

Additional responses to low R : FR include reduced leaf chlorophyll content and increased apical dominance (Smith & Whitelam, 1997). If the reduced R : FR ratio signal persists and the plant is unable to overtop competing vegetation, flowering is accelerated, thereby promoting seed set and enhancing the probability of reproductive success (Halliday et al., 1994; Dudley & Schmitt, 1995; Donohue et al., 2001). These adaptations are often accompanied by reductions in leaf area, shoot biomass and the size of harvestable organs, a likely consequence of the reallocation of resources towards reproductive structures (Keiller & Smith, 1989; Robson et al., 1993; Devlin et al., 1996, 1999). An Arabidopsis thaliana plant grown in high and low R : FR light is shown in Fig. 2. Elongation of petioles, reduced leaf area and decreased leaf chlorophyll are clearly visible. In monocotyledonous plants, low R : FR-induced increased apical dominance results in significantly reduced tiller formation, reduced harvest yield and increased risk of lodging, making shade avoidance a major determinant in crop planting density (reviewed in Kebrom & Brutnell, 2007).

image

Figure 2. Arabidopsis thaliana grown in 16 h photoperiods of high red to far red (R : FR) (a) and low R : FR light (b).

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III. Photoreceptor regulation of shade avoidance

  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. Shade avoidance responses
  5. III. Photoreceptor regulation of shade avoidance
  6. IV. Molecular mechanisms in shade avoidance signalling
  7. V. Crosstalk in shade avoidance signalling
  8. VI. Future perspectives
  9. Acknowledgements
  10. References

1. Phytochromes

Angiosperms contain three major types of phytochromes, phyA, phyB and phyC, the apoproteins of which are encoded by the genes PHYA, PHYB and PHYC, respectively. In the model species A. thaliana, five phytochrome genes (PHYA-PHYE) have been sequenced and characterized (Sharrock & Quail, 1989; Clack et al., 1994). These can be divided into two subgroups based on sequence homology: PHYA/PHYC and PHYB/PHYD/PHYE (Goosey et al., 1997). Responses to low R : FR ratio are primarily mediated by phyB, which acts to restrain shade avoidance responses under high R : FR conditions. Mutants deficient in phyB mimic growth of wild-type plants in low R : FR ratios and display phenotypes referred to as ‘constitutive shade avoidance’ (López-Juez et al., 1990; Nagatani et al., 1991; Somers et al., 1991). The involvement of additional phytochromes was suggested, following observations of residual shade avoidance responses in phyB mutants of multiple species (Whitelam & Smith, 1991; Smith et al., 1992; Robson et al., 1993). The isolation of a naturally occurring phyD mutation in the Wassilewskija (Ws) accession of Arabidopsis enabled the role of this phytochrome in shade avoidance to be examined. Similar elongation responses to end of day (EOD-FR) treatments were observed in phyD mutants and control plants containing an introgressed functional PHYD allele (Aukerman et al., 1997). In plants grown in light : dark cycles, EOD-FR treatment mimics growth in low R : FR by decreasing Pfr amounts before the onset of darkness, thereby maintaining a low Pfr throughout the dark period. Functional roles for phyD were, however, observed following the creation and analyses of multiple mutant combinations. Double mutants, deficient in phyB and phyD, displayed longer hypocotyls, longer petioles and earlier flowering than either monogenic parent, suggesting that phyD acts redundantly with phyB in mediating the suppression of shade avoidance (Aukerman et al., 1997; Devlin et al., 1999). An additional regulatory role for phyE was proposed following the isolation of mutants deficient in this photoreceptor (Devlin et al., 1998). In an elegantly devised screen, the authors exploited observations that phyAphyB double mutants treated with EOD-FR displayed visibly elongated internodes between rosette leaves and flowered early when compared with untreated controls. Mutagenized phyAphyB mutant seed was grown in light : dark cycles and plants displaying phenotypes consistent with EOD-FR treatment were identified. Sequencing of resulting mutants revealed a single base-pair deletion at the PHYE locus, implicating a role for this photoreceptor in the suppression of these responses (Devlin et al., 1998). Similar to phyD mutants, monogenic phyE mutants displayed no obvious phenotype, suggesting that phyE acts redundantly with phyB and phyD in the suppression of shade avoidance (Devlin et al., 1998). A role for phyC was dismissed following observations that phyBphyDphyE triple mutants treated with low R : FR displayed no further elongation or promotion of flowering (Franklin et al., 2003).

In contrast to other phytochromes, phyA displays light lability in the Pfr form and accumulates to relatively high amounts in etiolated seedlings. Accumulated phyA can be envisaged to function as a highly sensitive antenna, triggering the germination and subsequent photomorphogenesis of buried seeds following a brief exposure to light (Smith & Whitelam, 1990). The unique properties of phyA enable this photoreceptor to operate as an effective FR sensor in the high irradiance response (HIR) mode (Hennig et al., 2000). Mutants deficient in phyA are unable to de-etiolate in continuous FR, a property which formed the basis of screens for mutants null at the PHYA locus (Nagatani et al., 1993; Parks & Quail, 1993; Whitelam et al., 1993). The ability of phyA to function as a FR sensor has implications for R : FR ratio signalling. In low R : FR conditions, the photoequilibrium of phyB, D and E is shifted to the inactive Pfr form, whilst phyA signalling is enhanced through the FR-HIR response mode. Mutants deficient in phyA have been shown to display enhanced responses to low R : FR, suggesting that phyA may perform an important role in natural light environments in ‘antagonizing’ shade avoidance (Johnson et al., 1994; Salter et al., 2003). This suggestion is supported, in part, by observations showing phyA mutants grown in dense vegetational shade to display excessive elongation, with many seedlings failing to become established and dying prematurely (Yanovsky et al., 1995). Given the extreme nature of the shade treatment in these experiments, it could be argued that in most natural light environments, the adaptive significance of this phyA-mediated ‘antagonism’ is limited and may simply represent an unavoidable consequence of the physical properties of phyA, the evolutionary selection of which was driven by the importance of this photoreceptor in seedling de-etiolation.

Monocotyledonous plants contain only three phytochromes, phyA, phyB and phyC (Mathews & Sharrock, 1996, 1997). In maize (Zea mays), duplications have arisen in all three family members (Sheehan et al., 2004). Mutants deficient in phyB have been isolated in multiple species, including rice (Oryza sativa), barley (Hordeum vulgare), maize and sorghum (Sorghum bicolour). The roles of different phytochromes in grasses are less clear than in Arabidopsis and it is likely that all may play a role in the suppression of shade avoidance. In maize, both phyB alleles (phyB1 and phyB2) display functional redundancy in the suppression of leaf elongation and apical dominance. Partial redundancy was, however, observed between these alleles in the regulation of mesocotyl elongation and flowering time, where phyB1 and phyB2 were observed to predominate respectively (Sheehan et al., 2007). Sorghum mutants deficient in phyB1 (inline image) have been reported to constitutively display some phenotypes consistent with shade avoidance (Pao & Morgan, 1986; Childs et al., 1997), whilst analyses of rice mutants, deficient in individual and multiple combinations of phytochromes, have revealed a role for all three family members in the repression of flowering (Takano et al., 2005).

2. Cryptochromes

When subject to vegetational shading, plants experience a significant reduction in light quantity, in particular red and blue wavebands, which are absorbed by the canopy and used to drive photosynthesis (Fig. 1). In higher plants, sensitivity to blue wavebands is conferred primarily by the flavoproteins, cryptochromes and phototropins (Cashmore et al., 1999; Briggs & Huala, 1999) with suggested blue light-sensing roles for the proteins FKF1, ZTL/ADO and LKP2 (Somers et al., 2000; Schultz et al., 2001; Imaizumi et al., 2003). When growing in dense stands, reductions in blue light quantity are perceived by stems well before leaves are shaded (Ballaréet al., 1987, 1991b). Glasshouse experiments using selective spectral filters have shown the removal of blue wavelengths to result in pronounced stem elongation in multiple species (Ballaréet al., 1991a,b). Similar elongation responses were not observed following the removal of green wavelengths, confirming the blue light specificity of this response. Increased leaf hyponasty has also been observed in tobacco plants subject to reduced photon irradiances of blue light (Pierik et al., 2004b). Reductions in blue light quantity can therefore elicit some physiological responses characteristic of low R : FR ratio perception.

Cryptochromes show structural similarity to DNA photolyases and regulate an array of developmental responses throughout plant photomorphogenesis (Briggs & Huala, 1999). In Arabidopsis, the blue light-mediated inhibition of hypocotyl growth is regulated by two cryptochromes (cry1 and cry2), which differ in light lability and fluence rate specificity (Ahmad et al., 1995; Lin et al., 1998). An additional cryptochrome (cry3) has been identified in Arabidopsis and shown to function as a single-stranded DNA photolyase (Selby & Sancar, 2006). At higher photon irradiances of blue light, cry1 function predominates in the inhibition of hypocotyl growth. Under these conditions, cry2 displays considerable light lability (Lin et al., 1998). At lower photon irradiances (< 1 µmol m−2 s−1), cry2 displays greater stability and performs a significant role in the inhibition of hypocotyl growth, thereby enhancing blue light sensitivity in light-limiting conditions (Lin et al., 1998). When grown in high-photon-irradiance white light, elongated hypocotyls were observed in cry1 but not cry2 mutants (Mazzella et al., 2001). Double mutants deficient in both photoreceptors displayed greater elongation than cry1 monogenic mutants, suggesting a redundant role for cry2 in suppressing this response (Mazzella et al., 2001). It is therefore likely that cry1 and cry2 are the primary photoreceptors mediating elongation responses to reductions in blue light quantity. Reversal of Arabidopsis cry1 function by green light (500–600 nm) was suggested by Lin et al. (1995) following the identification of a green light-absorbing, flavin semiquinone state of this photoreceptor. More recently, a similar semi-reduced state has been identified for Arabidopsis cry2 (Banjeree et al., 2007). Observations showing increased hypocotyl length following green light supplementation of red and blue light mixtures suggest that, under certain conditions, green light may reverse cryptochrome-mediated growth inhibition (Folta, 2004, Bouly et al., 2007). The possibility therefore exists that in light reflected from/transmitted through living vegetation, the effects of reduced blue light quantity are further exacerbated by green light-mediated inactivation of cryptochrome signalling.

3. Phototropins

Two phototropins (phot1 and phot2) have been identified in Arabidopsis (Christie et al., 1998; Jarillo et al., 1998) and display overlapping regulatory roles in plant development. These include the control of directional growth in response to directional light (phototropism), intracellular chloroplast movement in response to light quantity and leaf expansion (Jarillo et al., 2001; Sakai et al., 2001; Sakamoto & Briggs, 2002). A recent study has additionally shown that phot1 (and to a lesser extent, phot2) performs a significant role in promoting plant growth in low intensities of blue light (Takemiya et al., 2005). In these experiments, the addition of low-photon-irradiance blue light (0.1 µmol m−2 s−1) to a background of red light (25 µmol m−2 s−1) resulted in a marked increase in leaf expansion and plant biomass. This response was severely attenuated in phot1 mutants and abolished in phot1phot2 double mutants. The authors propose that the blue light-mediated increases in plant biomass may result, in part, from phototropin-mediated increases in chloroplast accumulation and stomatal opening. Furthermore, growth of phot1phot2 double mutants in low-photon-irradiance white light (25 µmol m−2 s−1) resulted in a striking reduction in plant biomass when compared with wild-type controls and monogenic parents. This phenotype was not observed at a higher photon irradiance (70 µmol m−2 s−1), suggesting a major role for phototropins in mediating plant growth and development in conditions of low light quantity (Takemiya et al., 2005).

IV. Molecular mechanisms in shade avoidance signalling

  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. Shade avoidance responses
  5. III. Photoreceptor regulation of shade avoidance
  6. IV. Molecular mechanisms in shade avoidance signalling
  7. V. Crosstalk in shade avoidance signalling
  8. VI. Future perspectives
  9. Acknowledgements
  10. References

The most significant developments in shade avoidance research over the past decade have undoubtedly resulted from the identification of multiple R : FR ratio-regulated genes and elucidation of molecular mechanisms by which phytochromes control their expression. Early observations reporting stem elongation to occur within 10–15 min of FR supplementation appeared to preclude the involvement of gene expression changes in the initiation of these responses (Morgan et al., 1980). Quantitative kinetic analyses of low R : FR-mediated gene expression later revealed similar rapidity, presenting an exciting opportunity to investigate the molecular mechanisms controlling these responses (Salter et al., 2003). The combined research efforts of both the photomorphogenesis and gibberellin signalling fields have recently converged, providing a more holistic understanding of how plants regulate elongation growth at the molecular level. In this review, I present a (simplified) hypothetical model of how plants may initiate elongation growth following perception of low R : FR (Fig. 3). The mechanisms presented are based on evidence from published literature and are described in detail throughout this section.

image

Figure 3. Hypothetical model depicting molecular mechanisms controlling red to far red ratio (R : FR)-mediated elongation growth. (a) In daylight, phyB exists predominantly in the Pfr form (Pfr B). Phytochrome A Pfr (Pfr A) is degraded in both the cytosol and the nucleus. Following import into the nucleus, Pfr B binds PIF4 (and PIF5 proteins), resulting in their degradation via the 26S proteosome. The binding of DELLA and PIF proteins simultaneously results in PIF inactivation. An enhancement of DELLA activity relative to PIF4 activity results in the suppression of genes involved in elongation growth. (b) In vegetational shade, a reduction in R : FR results in conversion of phyB Pfr to the inactive Pr form, which dissociates from PIFs and exits the nucleus, increasing PIF stability. Antagonism of shade avoidance is provided by phyA operating in the high irradiance response (HIR) mode. In low R : FR, a small pool of cycled phyA is protected from degradation and binds some PIF4 protein, targeting it for degradation. The overall increase in PIF4 activity results in increased expression of genes involved in elongation growth. These include genes involved in the biosynthesis of gibberellin (GA). (c) In prolonged vegetational shade, GA concentrations may increase, thus enhancing DELLA degradation. Increased DELLA degradation would further enhance PIF stability, resulting in prolonged elongation growth.

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1. R : FR ratio-regulated genes

Research efforts to identify R : FR ratio-regulated genes progressed considerably following the development of microarray technology (Devlin et al., 2003; Salter et al., 2003; Sessa et al., 2005). Before this, the only genes reported to be reversibly regulated by changes in R : FR were the transcription factors ATHB2 (formerly HAT4) and ATHB4 (Carabelli et al., 1993, 1996). Both are members of the homeodomain leucine zipper (HDZip) family of transcription factors and interact with the DNA binding site CAATNATTG (Henriksson et al., 2005). Transcript abundance of ATHB2 was shown to increase rapidly following low R : FR and EOD-FR treatments (Carabelli et al., 1996). This response was partially retained in phyB mutants, suggesting the involvement of additional phytochromes (Carabelli et al., 1996). Subsequent analysis of multiple phytochrome-deficient mutants revealed phyE to act redundantly with phyB to suppress ATHB2 expression (Franklin et al., 2003). A role for ATHB2 in the regulation of shade avoidance was proposed following analyses of transgenic plants, expressing altered abundances of ATHB2 transcript (Schena et al., 1993; Steindler et al., 1999). Plants expressing elevated abundances of ATHB2 displayed reduced apical dominance and earlier flowering, phenotypes reminiscent of the shade avoidance syndrome (Schena et al., 1993). A reciprocal phenotype was observed in plants expressing an antisense ATHB2 construct, which displayed shorter stature and a reduced developmental rate (Schena et al., 1993). Plants expressing elevated abundances of ATHB2 were subsequently observed to display reduced growth of secondary tissues and decreased lateral root formation, phenotypes associated with altered auxin transport (Steindler et al., 1999). Such observations suggested the involvement of auxin in shade avoidance signalling, a notion which has gained increased credence in recent years (see Section V).

Microarray-based investigations have enabled additional genes displaying robust R : FR reversibility to be identified. In a study by the Whitelam laboratory, transcripts eroding the bHLH transcription factors PIL1 (PIF3 LIKE1) and a close homologue, PIL2, were shown to display rapid up-regulation by low R : FR treatment (Salter et al., 2003). These proteins were named based on their close sequence similarity to a group of phytochrome-binding bHLH transcription factors termed PHYTOCHROME INTERACTING FACTORs (PIFs) (see later). Differences in the responsivity of PIL1 and PIL2 to low R : FR were recorded at different times of day, suggesting the possible involvement of the circadian clock in their regulation. In a series of free-running experiments, the authors established circadian gating of both gene expression and hypocotyl elongation responses to low R : FR. Intriguingly, plants expressing altered abundances of PIL1 displayed a phase shift in the timing of maximal hypocotyl elongation response. Further connections between PIL1 and the circadian clock are provided by the reported physical interaction of PIL1 with the central oscillator component TOC1 (Makino et al., 2002) and observations that pil1 null mutants display reduced light-regulated expression of the putative clock component PSEUDO RESPONSE REGULATOR 9 (PPR9) (Khanna et al., 2006). A confusing observation from the Salter et al. study involved the relative timings of different low R : FR ratio-mediated responses. Gene expression responses to low R : FR were observed to occur maximally post-subjective dawn, whereas hypocotyl elongation displayed maximum responsivity pre-subjective dusk (Salter et al., 2003). Elongation responses to R : FR therefore mapped on to the period of maximum seedling elongation observed in free-running experiments (Dowson-Day & Millar, 1999), yet were at variance with the timing of gene expression responses. This apparent temporal paradox has recently been addressed in a seminal study by Nozue et al. (2007) and is discussed later in this section.

Global analysis of low R : FR ratio-mediated gene expression has additionally revealed a number of genes associated with cell wall loosening and auxin signalling to be up-regulated in low R : FR, including a number of the indole-3-acetic acid (IAA) family of auxin-regulated transcription factors (Devlin et al., 2003). In a later study, the bHLH transcription factor HFR1, the auxin responsive gene IAA29 and the xyloglucan endotransglycosylase XTH15 were shown to display significant up-regulation following simulated shade treatment (Sessa et al., 2005). Together, these results present further support for the involvement of auxin in low R : FR signalling and provide potential candidates for the regulation of cell wall metabolism during elongation responses. The latter study additionally reports enhanced shade avoidance responses in hfr1 null mutants. Based on these observations, the authors suggest the existence of a HFR1-mediated negative feedback loop controlling the magnitude of shade avoidance responses (Sessa et al., 2005). Given the well documented role of HFR1 in phyA signalling (Fairchild et al., 2000; Fankhauser & Chory, 2000; Soh et al., 2000), the possibility exists that reported phenotypes may represent a defect in the phyA-mediated ‘antagonism’ of shade avoidance (see Section III).

2. Phytochrome interacting factors (PIFs)

Following photoconversion to the active Pfr form, phytochromes undergo translocation to the nucleus (Sakamoto & Nagatani, 1996; Kircher et al., 1999, 2002). Nuclear-localized Pfr binds directly to a family of bHLH transcription factors termed PIFs (reviewed in Duek & Fankhauser, 2005; Monte et al., 2007). The first PIF protein (PIF3) was identified by yeast-two-hybrid screening and shown to display photoreversible binding to phyA and phyB Pfr in vivo (Ni et al., 1998, 1999). A number of closely related PIFs have since been identified which, together with PIF3, perform varied and overlapping regulatory roles in seedling de-etiolation (Halliday et al., 1999; Huq & Quail, 2002; Kim et al., 2003; Huq et al., 2004; Khanna et al., 2004, 2007; Monte et al., 2004; Leivar et al., 2008). Binding of phyB and PIFs has been shown to occur via a conserved APB (Active Phytochrome Binding) motif present in PIF proteins (Khanna et al., 2004). In a series of de-etiolation studies, binding of photoactivated phytochromes to PIFs has been shown to result in rapid PIF phosphorylation and degradation via the 26S proteosome (Bauer et al., 2004; Park et al., 2004; Shen et al., 2005, 2007; Al-sady et al., 2006; Lorrain et al., 2008).

The importance of PIF stability in shade avoidance has been highlighted in a pivotal recent study by the Fankhauser and Whitelam labs (Lorrain et al., 2008). In this work, the authors demonstrate rapidly increased stability of PIF4 and PIF5 proteins following transfer of de-etiolated plants to low R : FR. Immunoprecipitation assays have shown PIF4 to display strong binding to phyB with weaker binding to phyA (Huq & Quail, 2002). By contrast, PIF5 has only been demonstrated to bind phyB (Shen et al., 2007). This observation does not accord with mutant analyses, showing a clear requirement for phyA in PIF5 degradation (Shen et al., 2007). The authors speculate that either a weak phyA/PIF5 interaction exists in planta, which is unstable in the assay conditions used, or phyA may mediate PIF5 turnover through one or more intermediary factors (Shen et al., 2007). The analyses of null mutants and transgenic plants displaying altered abundances of PIF4 and PIF5 have revealed significant roles for these proteins in mediating shade avoidance responses (Lorrain et al., 2008). Monogenic pif4 mutants displayed short hypocotyls in both high and low R : FR, conditions in which pif5 monogenic mutants resembled WT plants. Double mutants, deficient in PIF4 and PIF5, however, displayed reduced elongation of both hypocotyls and petioles in high and low R : FR conditions. By contrast, plants overexpressing PIF5 were considerably elongated and displayed a ‘constitutive shade avoidance’ phenotype in high R : FR. Similar phenotypes were observed in plants overexpressing PIF4, but difficulties in line maintenance led the authors to focus on lines overexpressing PIF5 (Lorrain et al., 2008). Parallel analyses of gene expression responses revealed a similar pattern. All mutants (pif4, pif5, pif4pif5) displayed reduced expression of shade avoidance marker genes in low R : FR, whilst plants overexpressing PIF5 displayed constitutively elevated amounts of these genes in high R : FR. Transgenic plants expressing PIF4 and PIF5 constructs with a deleted APB motif were additionally investigated in this study. The deletion of this motif prevents phy/PIF interaction (Khanna et al., 2004) and resulted in exacerbation of the ‘constitutive shade avoidance’ phenotype (Lorrain et al., 2008). The authors conclude that PIF4 and PIF5 promote the expression of genes mediating elongation growth. In high R : FR, PIF proteins are degraded, following binding of photoactivated phytochrome. In low R : FR, the photoconversion and subsequent nuclear export of phyB Pfr results in enhanced stability of PIF4 and PIF5, thereby increasing the expression of genes mediating elongation growth (Lorrain et al., 2008). This model suggests that R : FR ratio signalling operates, in part, through the targeted degradation of transcriptional regulators, an emerging regulatory mechanism already established in auxin and gibberellin (GA) signalling (reviewed in Huq, 2006). Further support for this model is provided by observations that pif4 and pif5 mutations can partially restore the constitutively elongated phenotype of a phyB mutant (Lorrain et al., 2008).

Transfer of etiolated seedlings to red light has recently been shown to result in some degradation of the phyB photoreceptor (Khanna et al., 2007). This effect is marginally reduced in pif5 mutants and considerably exacerbated in PIF5 overexpressing lines. The authors suggest that PIF5 may display functional duality, acting as both transcriptional regulator and mediator of photoreceptor abundance. Observations of pronounced elongation phenotypes in plants expressing elevated amounts of PIF4 and PIF5 proteins with a deleted phytochrome-binding APB motif suggests these PIFs can promote shade avoidance independently of phytochrome interaction (Lorrain et al., 2008). Such observations are at variance with the suggestion that PIF5 promotes elongation growth through reducing phyB abundance, and support the notion that phyB acts to target PIF5 for degradation. An alternative explanation for the observed results may be that the light-regulated turnover of PIF5 results in some unavoidable loss of phyB photoreceptor, as a result of incomplete disassociation of the complex, before degradation. As both proteins display light-mediated proteolysis (Khanna et al., 2007; Lorrain et al., 2008), a circular argument could be initiated to explain the functional significance of this interaction in the regulation of elongation growth. Comparative analysis of PIF5 and PHYB protein amounts in different R : FR ratios would therefore be of interest in assessing the functional significance of this interaction in shade avoidance.

The importance of PIF4 and PIF5 in elongation growth is additionally highlighted in recent work from the Maloof and Fankhauser labs (Nozue et al., 2007). The authors demonstrate that in light : dark cycles, the hypocotyl growth of Arabidopsis seedlings is regulated through an external coincidence model, integrating both the circadian regulation of PIF4 and PIF5 transcript abundance and light-mediated proteolysis of PIF4 and PIF5 proteins. Under these conditions, transcript abundance of PIF4 and PIF5 is high from the end of the dark period and peaks in the middle of the day. In agreement with other studies (Shen et al., 2007; Lorrain et al., 2008), the authors show rapid light-mediated degradation of both proteins. The coincidence of high PIF4 and PIF5 transcript abundances and enhanced protein stability at the end of the dark period promotes maximum elongation growth during this time. This observation is in contrast to studies in continuous light, whereby maximum hypocotyl growth occurred at subjective dusk (Dowson-Day & Millar, 1999). Double pif4pif5 mutants displayed loss of the dawn growth peak, confirming their importance in the regulation of this response (Nozue et al., 2007). Furthermore, an advancement of the growth peak was observed in 16 h photoperiods when compared with experiments in 8 h photoperiods (Nozue et al., 2007). In these conditions, maximum hypocotyl growth was observed at approx. 4 h post-dawn, thereby coinciding with maximum low R : FR ratio-induced gene expression (Lorrain et al., 2008). It would therefore appear that previous temporal inconsistencies between low R : FR ratio-mediated gene expression and elongation growth have arisen as a result of the analysis of plants in continuous light (Salter et al., 2003). It is likely that in light : dark cycles, maximum elongation growth in response to transient low R : FR occurs in the early morning, thereby coinciding with maximum gene expression responses. Moreover, the circadian regulation of PIF4 and PIF5 transcript abundances may ultimately provide a molecular mechanism for the gating of rapid shade avoidance.

3. DELLA proteins

The retention of shade avoidance responses in pif4pif5 double mutants provides evidence for the existence of multiple regulatory mechanisms (Lorrain et al., 2008). Recent studies suggest that GA and phytochrome-mediated signalling pathways may converge to regulate elongation growth. The DELLA family of growth repressing proteins are mediators of the GA signalling pathway (Peng et al., 1997; Silverstone et al., 1998; Dill & Sun, 2001). In Arabidopsis, five DELLA homologues exist: GAI, RGA, RGL1, RGL2 and RGL3 (reviewed in Hussain & Peng, 2003). The relief-of-restraint model proposed by Harberd (2003) suggests that DELLAs act to restrain plant growth whilst GAs promote growth through overcoming DELLA-mediated growth restraint. It is now known that GAs regulate turnover of DELLAs via binding of a receptor, GID1 (Ueguchi-Tanaka et al., 2005; Griffiths et al., 2006; Nakajima et al., 2006; Willige et al., 2007). Interaction of DELLAs with GA-bound GID1 promotes interaction with the F-box protein SLEEPY1 (SLY1). This interaction results in polyubiquitination and degradation of DELLAs via the 26S proteosome, thereby relieving inhibition of growth (McGinnis et al., 2003; Dill et al., 2004).

Numerous reports exist of interactions between light and GA signalling (López-Juez et al., 1995; Reed et al., 1996; Pierik et al., 2004a). More recently, Pierik and colleagues have provided evidence supporting a central role for DELLA proteins in mediating shade avoidance responses (Djakovic-Petrovic et al., 2007). Confocal imaging of transgenic Arabidopsis plants expressing a pRGA:GFP-RGA construct revealed increased DELLA degradation in canopy-grown plants. As expected, these plants displayed greater hypocotyl and petiole elongation than isolated control plants. The identity of the canopy signal regulating DELLA stability was investigated through analyses of plants subject to low R : FR and blue light depletion. Increased DELLA degradation was observed in low R : FR-treated hypocotyls and petioles, confirming the involvement of phytochrome in these responses. Similar degradation was also observed in hypocotyls subject to blue light depletion, suggesting interplay between multiple photoreceptors. The requirement for GA in hypocotyl and petiole growth responses was confirmed by analyses of GA-deficient mutants. These plants displayed short hypocotyls which lacked responsivity to both low R : FR and blue light depletion. All phenotypes were rescued by GA application. A similar result was also observed in petioles treated with low R : FR. Quadruple mutants deficient in four DELLAs (gai/rga/rgl1/rgl2) did not, however, display elongated petioles, when compared with wild-type controls. Hypocotyls of these mutants were elongated, but retained responsivity to both low R : FR and blue light depletion. These data suggest that low R : FR-mediated DELLA degradation is not sufficient to induce shade avoidance responses in petioles, but, together with other signalling mechanisms, performs a more central role in hypocotyl growth (Djakovic-Petrovic et al., 2007).

The importance of light in the regulation of DELLA stability is supported by a parallel study from the Harberd lab (Achard et al., 2007). In this work, the authors show increased GFP-RGA stability in etiolated hypocotyls transferred to light. Comparison of hypocotyl length ratios (wild-type/quadruple DELLA mutant) revealed an increased contribution of DELLAs to growth restraint with increasing photoperiods. Conversely, GA-deficient plants displayed short hypocotyls in the dark, but wild-type hypocotyl lengths at longer photoperiods. Interestingly, transcript abundance of enzymes involved in GA metabolism showed rapid up-regulation upon transfer to light (Achard et al., 2007). The authors conclude that light-mediated growth restraint during photomorphogenesis involves the stabilization of DELLA proteins. Further stabilization may be achieved through light-regulated increases in GA metabolism. This notion is supported by observations in pea, showing decreased bioactive GA1 concentrations upon transfer of etiolated seedlings to light (Gil & García-Martínez, 2000; O’Neill et al., 2000).

4. PIF/DELLA interaction – a molecular mechanism for R : FR ratio signalling?

A molecular mechanism integrating light and GA signalling has recently been provided by parallel studies from the Deng and Prát labs (De Lucas et al., 2008; Feng et al., 2008). Both report the physical interaction of PIF and DELLA proteins through yeast-two hybrid and immunoprecipitation assays. The former study focuses on PIF3 and the latter, PIF4. Deletion studies confirmed the PIF4/RGA interaction to require the bHLH DNA recognition domain in PIF4 and a conserved leucine repeat motif in the RGA protein (De Lucas et al., 2008). The physiological significance of PIF/DELLA binding is elegantly demonstrated in both studies through chromatin immunoprecipitation (ChIP) experiments. Here, the physical interaction between PIFs and DELLAs was shown to block transcriptional activity, thereby inhibiting PIF function. These studies highlight PIFs as an integration node for both light and GA signalling. The authors propose that GA acts to target DELLAs for degradation, thereby relieving the inhibition of PIF activity and promoting elongation growth (De Lucas et al., 2008; Feng et al., 2008).

A hypothetical model for the R : FR ratio-mediated regulation of elongation growth is presented in Fig. 3. In daylight (Fig. 3a), photoactivation of phyB (and likely phyD and phyE) results in nuclear import of Pfr and binding to PIF4. A similar role for PIF5 can also be speculated. This physical interaction results in targeted PIF4 phosphorylation and degradation (Lorrain et al., 2008). In high R : FR, DELLA stability would also be enhanced (Djakovic-Petrovic et al., 2007), resulting in PIF4 binding and inactivation (De Lucas et al., 2008). Phytochrome A would be degraded in both the cytosol and the nucleus. Overall, the increased relative enhancement of DELLA to PIF4 activity would restrain elongation growth.

When subject to reduced R : FR (Fig. 3b), phyB is converted to the inactive Pr form. This dissociates from PIF proteins and exits the nucleus. The reduction in phyB Pfr would therefore result in increased abundance of nuclear PIF4. Increased PIF4 activity would result in enhanced expression of genes involved in elongation growth (Nozue et al., 2007; Lorrain et al., 2008). Under these conditions, phyA activity in the FR-HIR response mode would provide some antagonism of elongation growth, possibly through the binding and targeted degradation of PIF4 (Huq & Quail, 2002). Overall, the relative enhancement of PIF4 to DELLA activity would promote rapid elongation growth.

In prolonged vegetational shade (Fig. 3c), an additional role for GA can be proposed. Increased expression of transcripts encoding GA 20-oxidase have been reported following low R : FR, EOD-FR and simulated shade treatment in Arabidopsis (Devlin et al., 2003; Salter et al., 2003; Hisamatsu et al., 2005; Sessa et al., 2005). Such observations suggest GA concentrations may increase in vegetational shade. Increased concentrations of GA would enhance DELLA degradation, thereby relieving DELLA-mediated inhibition of PIF function (Djakovic-Petrovic et al., 2007; De Lucas et al., 2008). It can therefore be speculated that in prolonged low R : FR, increased GA synthesis would increase DELLA degradation, further enhancing PIF4 function and maintaining elongation growth.

5. Flowering

Flowering time in Arabidopsis is regulated through the complex integration of multiple environmental signals which act together to regulate the expression of floral meristem identity genes (for review, see Simpson & Dean, 2002). The light-quality pathway mediating acceleration of flowering in low R : FR has been shown to operate through the floral integrator FLOWERING TIME (FT), independently of the transcriptional regulator CONSTANS (CO) (Cerdán & Chory, 2003; Halliday et al., 2003). Indeed, expression of both FT and the meristem identity gene LEAFY (LFY) have been shown to display constitutively elevated expression in early-flowering phyB mutants (Blázquez & Weigel, 1999; Cerdán & Chory, 2003; Halliday et al., 2003). A potential signalling component linking phyB to FT expression was identified in a mutagenesis screen by Cerdán & Chory (2003). A plant displaying late flowering behaviour in both long and short days was isolated. Sequencing of the mutation revealed a nuclear-localized protein with similarity to some transcriptional activators, termed PHYTOCHROME AND FLOWERING TIME 1 (PFT1). Mutants deficient in PFT1 displayed impaired flowering responses to EOD-FR treatments. Double mutants, deficient in phyB and PFT1, displayed petiole lengths similar to phyB controls, but flowered at the same time as wild-type plants. Together, these observations suggest separate signalling mechanisms operate downstream of phytochromes to regulate elongation and flowering responses to low R : FR. This notion is supported by reports of natural Arabidopsis accessions displaying low R : FR-mediated elongation growth but no acceleration of flowering (Botto & Smith, 2002).

V. Crosstalk in shade avoidance signalling

  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. Shade avoidance responses
  5. III. Photoreceptor regulation of shade avoidance
  6. IV. Molecular mechanisms in shade avoidance signalling
  7. V. Crosstalk in shade avoidance signalling
  8. VI. Future perspectives
  9. Acknowledgements
  10. References

The interaction of phytochrome signalling with other environmental cues facilitates the accurate detection of, and response to, environmental fluctuations. An emerging body of evidence suggests that plants integrate R : FR information with multiple signalling pathways to elicit developmental responses. These include blue light signals, the circadian clock and gibberellin status (as discussed earlier). Additional interactions with auxin, ethylene and temperature signalling have been established and will be discussed briefly.

1. Auxin

The phytohormone auxin regulates multiple aspects of plant growth and development. These include elongation growth and apical dominance, processes commonly associated with shade avoidance (Chatfield et al., 2000; Sawa et al., 2002). Low R : FR, EOD-FR and simulated shade treatments have all been demonstrated to enhance expression of genes associated with auxin signalling (Tanaka et al., 2002; Vandenbussche et al., 2003; Sessa et al., 2005; Roig-Villanova et al., 2007). Such observations strongly suggest the involvement of auxin in shade avoidance. Circumstantial evidence is provided by experiments using auxin transport inhibitors and analyses of mutants deficient in auxin responsivity and signalling (Steindler et al., 1999; Vandenbussche et al., 2003). Such experiments are, however, confounded by pleiotropic morphological phenotypes, requiring careful interpretation.

2. Ethylene

The role of ethylene as a proximity perception signal within canopies was investigated by Pierik and colleagues, following observed similarities between physiological adaptations to flooding stress in the wetland species Rumex palustris and responses commonly termed shade avoidance (Pierik et al., 2003). Such adaptations include increased stem elongation and rapid leaf hyponasty, responses induced during flooding by ethylene accumulation in the shoot (reviewed in Pierik et al., 2005). Exposure of tobacco plants to low concentrations of ethylene resulted in similar phenotypes (Pierik et al., 2003). Furthermore, analysis of transgenic tobacco plants displaying ethylene insensitivity revealed delayed shade avoidance responses in crowded canopies. Interestingly, the delayed shade avoidance phenotypes observed were demonstrated to result from insensitivity of transgenic plants to low quantities of blue light, suggesting crosstalk between multiple signalling pathways (Pierik et al., 2004a). Ethylene increases have been recorded following treatment of multiple plant species with low R : FR (Finlayson et al., 1998, 1999; Pierik et al., 2004b) and reduced light quantity (Vandenbussche et al., 2003). Studies in tobacco have additionally reported that the growth-promoting effects of ethylene require GA and suggest that ethylene functions, in part, through altering GA responsiveness (Pierik et al., 2004b). Intriguingly, ethylene has been demonstrated to regulate DELLA stability in Arabidopsis (Achard et al., 2003). More recently, seedlings overexpressing PIF5 have been shown to display elevated ethylene concentrations (Khanna et al., 2007), suggesting PIF and DELLA proteins may function as integrators of multiple hormone signalling pathways. Together, these data suggest that elevated ethylene concentrations in crowded canopies may augment low R : FR ratio and reduced blue light signalling to maximize shade avoidance responses.

3. Temperature

The integration of phytochrome and temperature signalling pathways has been reported in the regulation of multiple developmental processes, including germination (Penfield et al., 2005; Heschel et al., 2007; Donohue et al., 2008) and flowering (Halliday et al., 2003; Halliday & Whitelam, 2003). More recently, low R : FR and temperature signalling have been shown to interact to regulate freezing tolerance in Arabidopsis (Franklin & Whitelam, 2007). Transcriptomic analysis of plants treated with low R : FR at 16 and 22°C revealed ambient temperature-dependent, light-quality regulation of the CBF regulon, a suite of genes involved in cold acclimation and the acquisition of freezing tolerance. During cold acclimation, the perception of low temperature (usually < 4°C) results in increased expression of the CBF family of transcriptional activators. These lead to the up-regulation of downstream COLD-REGULATED (COR) genes and metabolic changes which enhance tolerance to subzero temperatures (Thomashow, 1999). Low R : FR treatment of Arabidopsis plants at both 16 and 22°C resulted in increased CBF transcript abundance in a circadian gated manner. Maximum responsivity was observed between ZT4 and ZT8, times at which maximum responsivity to low-temperature treatment has also been observed (Fowler et al., 2005). Intriguingly, low R : FR treatment at 16°C but not 22°C led to significant up-regulation of COR genes and enhanced freezing tolerance (Franklin & Whitelam, 2007). The authors speculate that the reduced ambient temperature, shorter day-length and longer twilight periods experienced during autumn months at northern latitudes may provide the ideal environmental conditions to initiate COR gene expression. The use of light quality signals to seasonally enhance freezing tolerance would confer some protection to plants subject to sudden decreases in temperature. The uncoupling of this response at 22°C may serve to prevent the unnecessary accumulation of COR gene products at higher temperatures.

VI. Future perspectives

  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. Shade avoidance responses
  5. III. Photoreceptor regulation of shade avoidance
  6. IV. Molecular mechanisms in shade avoidance signalling
  7. V. Crosstalk in shade avoidance signalling
  8. VI. Future perspectives
  9. Acknowledgements
  10. References

The identification of multiple genes robustly and reversibly regulated by R : FR has enabled considerable advancement in our understanding of shade avoidance signalling. Observations of PIF/DELLA interactions provide an exciting insight into how R : FR ratio and GA signalling integrate to regulate elongation growth (Djakovic-Petrovic et al., 2007; De Lucas et al., 2008; Feng et al., 2008; Lorrain et al., 2008). The increased stability of PIF4 and PIF5 in low R : FR implicate significant roles for these transcription factors in shade avoidance signalling and suggest a considerably wider role for PIF proteins beyond de-etiolation and seedling establishment. The existence of residual shade avoidance responses in pif4/pif5 and quadruple DELLA-deficient mutants clearly demonstrates the involvement of additional, as yet unidentified, molecular mechanisms. Observations that plants subject to other environmental perturbations display phenotypes consistent with low R : FR treatment suggests that ‘the shade avoidance syndrome’ may in fact, represent a more generic stress response. Examples include the leaf hyponasty and petiole extension responses observed during flooding (Pierik et al., 2005) and the auxin-mediated extension growth and precocious flowering observed in response to high-temperature treatment (Gray et al., 1998; Balasubramanian et al., 2006). Indeed, phenotypes similar to shade avoidance have even been reported in plants defective in plant pathogen signalling (Faigón-Soverna et al., 2006). These plants also displayed enhanced ATHB2 and HFR1 expression, raising a pertinent question: do all stress responses resulting in ‘shade avoidance’-like phenotypes target the same downstream genes? Recent observations that PIFs act as nodes to integrate light and GA signalling tempt speculation that similar mechanistic interactions may operate in other light signal crosstalk interactions (De Lucas et al., 2008; Feng et al., 2008). The emerging picture of shade avoidance is of a complex signalling network, regulated by multiple light signals, mediated by multiple photoreceptors and modulated by multiple endogenous and exogenous cues. The molecular dissection of such a complex network presents photobiologists with a daunting future challenge but should hopefully result in a more complete understanding of this important biological phenomenon.

References

  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. Shade avoidance responses
  5. III. Photoreceptor regulation of shade avoidance
  6. IV. Molecular mechanisms in shade avoidance signalling
  7. V. Crosstalk in shade avoidance signalling
  8. VI. Future perspectives
  9. Acknowledgements
  10. References