Role of phyB
The phytochromes are photochromic plant photoreceptors (Bae & Choi 2008) encoded by a family of divergent genes. There are five phytochrome genes in Arabidopsis (PHYA-E) (Clack, Mathews & Sharrock 1994; Mathews & Sharrock 1997). The shade avoidance responses to low R : FR are primarily mediated by the photoreceptor phyB (for reviews, see Ballaré 1999; Franklin 2008). The active (Pfr) form of phyB, which dominates the photoequilibrium when the plant is exposed to light of high R : FR ratio, inhibits the expression of the shade avoidance phenotype (Smith 1982, 2000). When the plant experiences a drop in the R : FR ratio, for example because it receives FR reflected by other plants (Ballaréet al. 1987, 1990), the active form of phyB is depleted. Inactivation of phyB permits accumulation of a group of growth-promoting transcription factors, thereby unleashing the expression of the shade avoidance syndrome (see next section). That phyB is the principal photoreceptor involved in shade avoidance has been demonstrated for elongation, leaf angle and FR-induced phototropic responses by means of experiments carried out in real canopies under natural sunlight levels (for reviews, see Ballaré 1999; Franklin 2008). Phytochrome D (PhyD) and phytochrome E (phyE) have been shown in physiological experiments under laboratory conditions to act redundantly with phyB in the suppression of the shade avoidance under light of high R : FR (Franklin 2008), and presumably play important roles in fine-tuning the magnitude of the response to R : FR ratio in complex light environments.
PhyA is present at very low levels in plant tissues exposed to sunlight, because of rapid light-induced proteolytic degradation of the photoreceptor in the Pfr form (Sharrock & Clack 2002). PhyA has important functions in the control of seed germination, de-etiolation and day-length perception, but has a less prominent role in neighbour detection in plant canopies. In fact, because phyA signalling is enhanced at low R : FR ratios, which reduce the rate of phyA degradation (Hennig, Buche & Schäfer 2000), phyA, acting in the so called ‘HIR’ mode, might to some extent antagonize the expression of the shade avoidance syndrome (Salter, Franklin & Whitelam 2003). PhyA is likely to be responsible for the elongation responses to attenuation of total irradiance in the R + FR waveband mentioned earlier (Ballaréet al. 1991), because higher irradiances are predicted to preserve increased levels of phyA Pfr from degradation (Hennig et al. 2000), even under light qualities that establish relatively high Pfr % (Franklin, Allen & Whitelam 2007). Tobacco plants that over-express PHYA transgenes have been shown to be unable to mount normal shade avoidance responses to neighbour proximity, and stem elongation remained inhibited even in plants grown at high density (Ballaréet al. 1994; Schmitt, McCormac & Smith 1995). This aberrant behaviour is consistent with the idea that down-regulation of phyA is important for the proper expression of proximity responses controlled by phyB and phyB-like phytochromes (McCormac, Whitelam & Smith 1992).
Phytochrome targets and downstream processes
Although the functional significance of phyB in the mechanisms of neighbour perception and foraging for light has been established some time ago, the downstream processes that connect phyB inactivation with the elicitation of shade avoidance responses have only recently began to be uncovered (Fig. 1).
Figure 1. Simplified schematic of the functional connections between competition signals in the light environment (increased FR) and the expression of shade avoidance and defence responses. FR causes inactivation of phytochrome B (phyB) by photoconverting the active, Pfr form, into Pr. Removal of Pfr allows accumulation of phytochrome-interacting factors 4 and 5 (PIF4/5), which in turn activate several transcription factors such as ATHB2, ATHB4, etc (early SAS genes) and growth-promoting genes. These early responses are followed by activation of genes involved in the production of auxin (IAA) and bioactive gibberellins (GAs). Increased GA levels in turn facilitate degradation of DELLA proteins, which exert their biological function by inhibiting the transcriptional activity of PIFs. Phytochrome inactivation also leads to decreased sensitivity to jasmonates, and this phenomenon appears to be related to increased transcription of jasmonate signalling repressors (JAZ genes) and reduced expression of some positively acting transcription factors such as ERF1. Arrows indicate positive action (i.e. promotion, activation); blunt connectors indicate negative action (i.e. inhibition). Boxed arrows indicated protein destruction by the 26S proteasome. Dotted connectors indicate functional relationships where the molecular effectors are not firmly established.
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The initial critical event for elicitation of shade avoidance responses following phytochrome photoconversion to the inactive Pr form under low R : FR ratios is the increased stability of a group of growth-promoting transcription factors known as PIFs (Lorrain et al. 2008). PIFs are members of the bHLH family of transcriptional regulators that interact specifically with the active Pfr form of the phytochrome molecule (for reviews, see Castillon, Shen & Huq 2007; Monte et al. 2007). At least two of these PIFs (PIF4 and PIF5) interact predominatly with phyB (Huq & Quail 2002; Khanna et al. 2004; Shen et al. 2007). If phyB is in the active Pfr form, it is located in the nucleus, binding PIF4 and PIF5 proteins and targeting them for degradation in the 26S proteosome; when the plant is exposed to low R : FR ratios, PhyB Pfr is photoconverted to Pr, which no longer binds PIFs and exits the nucleus, thereby permitting greater stability of PIFs (Lorrain et al. 2008). PIF4 and PIF5 promote the expression of genes involved in cell elongation (such as β expansin) (de Lucas et al. 2008) and the elicitation of shade avoidance responses (Lorrain et al. 2008). Therefore, an increase in PIF stability following phyB inactivation by low R : FR ratios results in increased growth promotion. Simultaneous mutation of PIF4 and PIF5 in Arabidopsis (pif4 pif5) reduced the hypocotyl elongation response to low R : FR ratio, and partially reversed the elongated phenotype of the phyB mutant. In contrast, over-expression of PIF5 resulted in seedlings with elongated hypocotyls even under conditions of high R : FR ratio (Lorrain et al. 2008). These physiological data are consistent with the idea that PIF4 and PIF5 are necessary for the elicitation of shade avoidance responses induced by low R : FR or by the phyB mutation. However, the observation that low R : FR ratios have a growth-promoting effect even in pif4 pif5 double mutants indicates that additional, redundant molecular circuits connect R : FR perception with the activation of shade avoidance responses (Lorrain et al. 2008). These redundant signalling pathways still remain to be elucidated.
A few genes that are rapidly (within minutes) and reversibly activated following transfer of Arabidosis plants from high to low R : FR ratio have been described. These include ATHB2, ATHB4, PIF3-like 1 (PIL1), PHY RAPIDLY REGULATED 1 (PAR1) and HFR1 (reviewed in Jiao, Lau & Deng 2007). All of them (except PAR1) encode transcription factors. The up-regulation of these early genes by low R : FR or by phyB mutation is attenuated in pif4 pif5 double mutants, and their levels of expression are increased in PIF5 over-expressors (Lorrain et al. 2008). ATHB2 and ATHB4 are members of the homeodomain leucine zipper family of transcription factors (Carabelli et al. 1993; Ciarbelli et al. 2008); ATHB2 promotes shade avoidance responses by acting as a transcriptional repressor (Steindler et al. 1999). Evidence for a functional role of ATHB2 in the induction of shade avoidance responses was obtained by studying the phenotypes of transgenic plants expressing increased or reduced levels of this gene (Schena, Lloyd & Davis 1993; Steindler et al. 1999). The underlying mechanism has not been elucidated, but it likely involves changes in auxin homeostasis (Morelli & Ruberti 2002). In a further example of the operation of negative feedback controls in phytochrome-mediated behavioural responses, the product of one of the early shade avoidance genes, HFR, down-regulates the expression of others (such as ATHB2, ATHB4, PIL1 and PAR1), and eventually attenuates the expression of the shade avoidance response (Sessa et al. 2005). This down-regulation is supposed to prevent the expression of exaggerated elongation phenotypes under conditions of prolonged shade.
The involvement of hormones, particularly auxin and gibberellins in the orchestration of shade avoidance and light foraging responses, has long been suspected on the basis of the critical roles that these hormones play in controlling physiological processes that form the core of many foraging behaviours, such as tropisms, apical dominance, stem elongation, etc. However, only recently has a map of molecular interactions between photoreceptors and hormone homeostasis begun to take shape.
A recent study demonstrated that increased auxin production through a previously uncharacterized biosynthesis pathway is necessary for the expression of early shade avoidance responses in Arabidopsis, such as the increased petiole elongation and leaf hyponasty responses that plants present when exposed to reflected FR radiation (Tao et al. 2008). The sav3 mutant was originally isolated because the seedlings had a normal phenotype in darkness and in white light, but failed to induce normal elongation responses when placed under low R : FR ratios. The mutant has normal levels of induction of early shade avoidance genes (such as ATHB2, PAR1 and HFR1), but lacks TAA1, an aminotransferase that catalyses the formation of indole-3-pyruvic acid from l-tryptophan, the first step in a novel auxin biosynthesis pathway. This pathway appears to be essential for the production of the high levels of auxin that are required to initiate shade avoidance responses (Fig. 1).
The connection between R : FR responses and gibberellins was established long ago, even before the term phytochrome was widely accepted by the scientific community. Lockhart (1959) demonstrated that the inhibitory effects of low-intensity light on internode elongation in pea, which were presumably mediated by the ‘R : FR pigment system’, were reversible by gibberellic acid. However, the functional architecture of this relationship has been unclear until very recently. Among the genes that are up-regulated by low R : FR are genes that encode enzymes responsible for the production of bioactive gibberellins, such as the GA20 oxidases (Hisamatsu et al. 2005). This is consistent with the observation that low R : FR ratios, which result in increased PIF accumulation (Lorrain et al. 2008), promote the accumulation of growth-promoting bioactive GAs in some species (Garcia-Martinez & Gil 2001; Kurepin et al. 2007) (Fig. 1). Gibberellins also exert their biological action by means of the mechanism of targeted degradation of negative regulators (Hirano, Ueguchi-Tanaka & Matsuoka 2008). Following a functional chain that has obvious parallels with the mechanisms of auxin (Dharmasiri, Dharmasiri & Estelle 2005; Kepinski & Leyser 2005) and jasmonate signalling (Chini et al. 2007; Thines et al. 2007), bioactive gibberellins stimulate the physical interaction between GID1 (the GA receptor) and proteins of the DELLA family (which are repressors of GA responses), causing their degradation through the SCFGID2/26S proteasome pathway (Ueguchi-Tanaka et al. 2005; reviewed in Hirano et al. 2008). Therefore, increased synthesis of bioactive GAs under low R : FR ratios are predicted to cause increased DELLA degradation, thereby de-repressing growth responses associated with shade avoidance (Fig. 1). DELLA abundance in fact decreases in response to low R : FR ratios and in response to increases in planting density in canopies, and the onset of DELLA degradation precedes the appearance of measurable shade avoidance responses (Djakovic-Petrovic et al. 2007). Decreased DELLA abundance is also seen when seedlings are transferred from light to darkness, presumably in response to an increase in bioactive GA levels (Achard et al. 2007)
The mechanisms used by DELLAs to inhibit growth have been elusive, in part because studies using chromatin immunoprecipitation have failed to show specific binding of DELLAs to the promoters of genes known to be responsive to GA. In an interesting twist of the phytochrome–GA connection, two research groups discovered that the PIF proteins are a principal target of DELLAs. Thus, when present in the nucleus, DELLA proteins bind to and prevent PIFs from stimulating the transcription of growth-promoting genes (Feng et al. 2008; de Lucas et al. 2008). These observations suggest that low R : FR ratios stimulate growth via at least two related mechanisms: (1) PIF stabilization by removal of PhyB Pfr (the PIF-de-stabilizing form of phyB); and (2) GA-mediated degradation of DELLA proteins, which are inhibitors of PIF transcriptional function (Fig. 1).
Ethylene is another plant hormone that appears to play important roles in shade avoidance. Low R : FR ratios promote ethylene biosynthesis in various plant species (Finlayson, Lee & Morgan 1998; Pierik et al. 2004a), and ethylene is known to cause changes in plant morphology that are reminiscent of the shade avoidance response (such as increased elongation and leaf hyponasty) (Pierik, Sasidharan & Voesenek 2007). Transgenic tobacco plants impaired in ethylene perception showed reduced shade avoidance responses when grown in density-gradient experiments (Pierik et al. 2003), and reduced response to blue light attenuation (Pierik et al. 2004b). The mechanisms that mediate the effects of ethylene defining the shade avoidance phenotype remain to be elucidated, but it is well established that ethylene promotes apoplastic acidification and up-regulates expansin gene expression and protein activity during petiole elongation in Rumex palustris (Vreeburg et al. 2005). Because ethylene is also known to be involved in the regulation of plant defence, showing positive and negative interactions with jasmonate signalling (Winz & Baldwin 2001; Lorenzo & Solano 2005), it is likely to be an important player in the functional decisions that plants implement to solve the dilemma of competition versus defence allocation (Izaguirre et al. 2006; Pierik et al. 2007).
Light-controlled foraging for light is adaptive for the individual plant (Schmitt et al. 2003). For example, transgenic tobacco plants that do not show stem elongation responses to low R : FR ratio, because they over-express PHYA transgenes and display a persistent HIR phenotype, are easily outcompeted by their neighbours when placed in a crowded neighbourhood of photomorphogenically normal plants (Schmitt et al. 1995). Mutant phyB plants of cucumber, which are impaired in phototropic responses and other biomechanical processes controlled by phyB, are much less efficient than wild-type plants at locating and deploying new growth into canopy gaps (Ballaréet al. 1995). Shade avoidance responses to low R : FR are more intense in species or ecotypes of open habitats than in those that occur in chronically shaded environments, where these responses are unlikely to result in increased PAR capture (Morgan & Smith 1979; Dudley & Schmitt 1995). Selection for plasticity to R : FR has been explicitly demonstrated in natural populations (Donohue et al. 2000).
Because competition in crowded plant stands is essentially asymmetric [larger individuals obtain a disproportionate share of the light resource (for their relative size), and suppress the growth of smaller individuals] (Weiner & Thomas 1986), plant populations tend to develop increased size structuring in response to increased population density. This phenomenon has important implications for a number of ecological issues that range from the definition of effective population size in natural populations to the growth and yield of agricultural crops. Studies using photomorphogenic mutants (Ballaréet al. 1994; Ballaré & Scopel 1997) and detailed analysis of plant allometry in density-gradient experiments (Aphalo et al. 1999; Aphalo & Rikala 2006) demonstrate that phenotypic plasticity to R : FR contributes to buffer the population against the development of size structures at high density. Shade avoidance responses mounted by plants that are being suppressed by dominant individuals allow them to remain in the competition game and eventually produce seeds. In contrast, in canopies formed by plants that are unable to respond to low R : FR ratios, shaded plants become progressively more and more suppressed without reacting with adaptive changes in their allometry, providing a rather dramatic example of the importance of information in the definition of competitive interactions between neighbouring plants.