Light signals, phytochromes and cross-talk with other environmental cues

Advance Access published December 12, 2003 Journal of Experimental Botany, Vol. 55, No. 395, Cross-talk in Plant Signal Transduction Special Issue, Page 1 of 6, January 2004 DOI: 10.1093/jxb/erh026 Light signals, phytochromes and cross-talk with other environmental cues Keara A. Franklin and Garry C. Whitelam* Department of Biology, University of Leicester, Leicester LE1 7RH, UK Received 29 April 2003; Accepted 8 October 2003 Abstract Plants have evolved highly complex sensory mechanisms to monitor their surroundings and adapt their growth and development to the prevailing environmental conditions. The integration of information from multiple environmental cues enables the coordination of development with favourable seasonal conditions and, ultimately, determines plant form. Light signals, perceived via the phytochrome, cryptochrome and phototropin photoreceptor families, are especially important environmental signals. Redundancy of function among phytochromes and their interaction with blue light photoreceptors enhance sensitivity to light signals, facilitating the accurate detection of, and response to, environmental uctuations. In this review, current understanding of Arabidopsis phytochrome functions will be summarized, in particular, the interactions among the phytochromes and the integration of light signals with directional and temperature sensing mechanisms. Key words: Arabidopsis, environmental cues, light signals, photoreceptors, temperature sensing mechanisms. Introduction As sessile organisms that cannot choose their surroundings, plants need to modify their growth and development to suit their ambient environment. Such developmental plasticity involves the integration of multiple environmental signals, enabling plants to synchronize their growth with seasonal changes and compete effectively with neighbours for essential resources. Light signals are amongst the most important environmental cues regulating plant development. In addition to light quantity, plants monitor the quality, periodicity and direction of light and use the information to modulate multiple physiological responses, from seed germination and seedling establishment through to mature plant architecture and the onset of reproductive development. In higher plants there are three principal families of signal-transducing photoreceptors; the red/far-red (R/FR) light-absorbing phytochromes and the UV-A/blue light-absorbing cryptochromes and phototropins (reviewed in Quail, 2002). The phytochromes are reversibly photochromic biliproteins that absorb maximally in the red (R) and far-red (FR) regions of the spectrum. In Arabidopsis thaliana, ve discrete apophytochrome-encoding genes, PHYA±PHYE, have been isolated and sequenced. These can be clustered by protein similarity into three subfamilies: A/C, B/D and E (Clack et al., 1994; Mathews and Sharrock, 1997). Phytochrome is synthesized in its inactive R-absorbing (Pr) form and activity is acquired upon phototransformation to the FR-absorbing (Pfr) isomer (Kendrick and Kronenberg, 1994). The photoconversion of phytochrome from its Pr to Pfr form has been demonstrated, at least for phya and phyb, to trigger translocation of a proportion of the photoreceptor from the cytoplasm to the nucleus (Sakamoto and Nagatani, 1996; Kircher et al., 1999; Yamaguchi et al., 1999). In the nucleus, for phyb at least, Pfr can interact with PIF3, a transcriptional regulator to control expression of a number of target genes (Ni et al., 1999; Martinez-Garcia et al., 2000). Elucidation of the roles of individual phytochromes in mediating plant growth is often confounded by their redundant and overlapping mechanisms of action. The isolation of mutants de cient in individual phytochromes and the subsequent creation of multiple mutant combinations have, therefore, been essential in the determination of individual phytochrome functions and the dissection of * To whom correspondence should be addressed. Fax: +44 (0)116 252 2791. E-mail: gcw1@le.ac.uk Journal of Experimental Botany, Vol. 55, No. 395, ã Society for Experimental Biology 2004; all rights reserved

Page 2 of 6 Franklin and Whitelam functional interactions between family members. In addition to providing insights into individual phytochrome functions, the use of multiple mutant combinations has also enabled progress in the understanding of how light signals integrate with other environmental cues. The focus here is on the use of Arabidopsis mutants, de cient in multiple phytochromes, in order to elucidate the complex overlapping roles of individual family members during photomorphogenesis. The role of these mutants in dissecting cross-talk between light-, gravity- and temperaturesensing mechanisms is also discussed. Photoreceptor interactions Seedling establishment The light environment in which a seedling develops can in uence not only the timing of seed germination but the ensuing developmental strategy of a plant. Induction of Arabidopsis seed germination by R involves both phya and phyb (Shinomura et al., 1994, 1996). Germination responses displaying R/FR reversibility are characteristic of the low uence response (LFR) mode of phytochrome action and enables buried seeds to detect proximity to the soil surface and exposed seeds to identify canopy gaps. The retention of R/FR reversible germination responses in phyaphyb double mutants implicated the participation of another phytochrome in this response, a role subsequently assigned to phye (Hennig et al., 2002). Many seeds that have been imbibed in darkness acquire extreme sensitivity to light that is typical of the phyamediated very low uence response (VLFR) mode of phytochrome action. It is estimated that these sensitized seeds would be induced to germinate following exposure to only a few milliseconds of daylight, thus enabling the opportunistic exploitation of very brief soil disturbances (Smith, 1982). Inhibition of germination following prolonged exposure to FR, most likely represents the phyamediated high irradiance response (FR-HIR) mode of phytochrome action and may be ecologically relevant as a means of delaying the germination of seeds situated under chlorophyllous vegetation or leaf litter (Smith and Whitelam, 1990). Following the induction of germination, light signals act to constrain hypocotyl extension while initiating the expansion of cotyledons and the concomitant synthesis of chlorophyll. Despite showing no obvious mutant phenotype following growth under white light or R, mutants de cient in phya have revealed a unique role for this photoreceptor in mediating the inhibition of hypocotyl elongation growth under FR and FR-enriched light environments (Nagatani et al., 1993; Parks and Quail, 1993; Whitelam et al., 1993). By contrast, phyb-de ciency confers no aberrant phenotype under FR, but leads to a marked loss of seedling sensitivity to R for a wide range of de-etiolation responses (Koornneef et al., 1980; Somers et al., 1991; Reed et al., 1993). Seedlings de cient in both phya and phyb display a greater insensitivity to R than monogenic phyb seedlings (Reed et al., 1994). Thus, although phyb plays the major role in inhibition of hypocotyl elongation in red light, phya can also contribute to this response. An additional minor role is performed by phyd (Aukerman et al., 1997) whereas the contribution of phye to seedling de-etiolation appears negligible (Devlin et al., 1998). The recent identi cation of mutants at the PHYC locus has revealed a role for this phytochrome in the R-mediated inhibition of hypocotyl elongation (Franklin et al., 2003a; Monte et al., 2003). The combined loss of phya and phyc in the Ws ecotype (phyc-1) resulted in a signi cant increase in hypocotyl length, an effect greater than that observed in phyc-1 plants. Since loss of phya alone has no effect on sensitivity to R, the possibility exists that phya and phyc act redundantly to regulate the R-control of hypocotyl growth (Franklin et al., 2003a). The role of phyc in this response was most pronounced at low uence rates and not observable in the phyb mutant background, suggesting a possible role for phyc in modulating phyb function (Franklin et al., 2003a). No role for phyc was identi ed in the inhibition of hypocotyl elongation in FR (Franklin et al., 2003a; Monte et al., 2003). The isolation and characterization of mutants de cient in cryptochromes 1 and 2 (cry1 and cry2) have de ned roles for these photoreceptors throughout seedling development (Lin et al., 1996, 1998). Despite uncertainty over the exact nature of co-action, it is accepted that B-mediated de-etiolation involves the interaction of both phytochrome and cryptochrome signalling (Yanovsky et al., 1995; Ahmad and Cashmore, 1997; Casal and Mazzella, 1998). A physical interaction between CRY1 and PHYA proteins has been demonstrated (Ahmad et al., 1998; Ahmad, 1999) in addition to a functional interaction between cry2 and phyb (MaÂs et al., 2000). Mutant combinations de cient in phyc displayed elongated hypocotyls in B, an effect most evident at low uence rates (Franklin et al., 2003a). Under these conditions, it has been shown that the cry2 function predominates in the regulation of hypocotyl elongation (Lin et al., 1998). The hyposensitivity of phyc mutants to low uence rate of B may therefore indicate a possible functional interaction between phyc and cry2. There is also evidence of functional redundancy between phytochromes and cryptochromes. For example, the inhibition of hypocotyl growth by a R pulse in phyb seedlings that have been pretreated with white light, requires the presence of either phyd or cry1 (Hennig et al., 1999). Mature plant development In Arabidopsis and many other plant species, de ciency of phyb has a marked effect on the architecture of the mature light-grown plant. Phytochrome B-de cient plants display

an elongated growth habit, retarded leaf development, increased apical dominance, and early owering (Robson et al., 1993; Halliday et al., 1994; Devlin et al., 1996). This pleiotropic phenotype resembles the shade avoidance syndrome shown by wild-type plants following the perception of low R:FR ratio and suggests a predominant role for phyb in suppressing this response under natural conditions (Whitelam and Devlin, 1997). The ability to respond to the perceived threat of shading, and therefore to execute architectural changes before canopy closure, provides a crucial competitive strategy to plants growing in dense stands (Ballare et al., 1990). The retention of shade avoidance responses in phyb null mutants indicated the involvement of additional phytochromes (Whitelam and Smith, 1991; Robson et al., 1993; Halliday et al., 1994). Multiple mutant analyses have since revealed that the perception of low R:FR in Arabidopsis is mediated solely by phyb, D and E, acting in a functionally redundant manner (Devlin et al., 1996, 1998, 1999; Franklin et al., 2003b). These represent the most recently evolved members of the phytochrome family and form a distinct subgroup (Mathews and Sharrock, 1997). It is therefore possible that competition for light may have provided the selective pressure for their evolution (Devlin et al., 1998). Adult Arabidopsis plants structure their leaves in a compact rosette phenotype. The elongated internodes observed in phyaphybphye-triple mutant plants was the basis on which the phye mutation was isolated and led to the proposal that maintenance of the rosette phenotype is regulated, redundantly, by phya, B and E (Devlin et al., 1998). The elongated appearance of phyaphybphydphyequadruple mutants grown under white light, a phenotype not displayed in phybphydphye-triple mutants has supported such a proposal (Franklin et al., 2003b). Physiological comparison of these genotypes also revealed a signi cant role for phya in the modulation of rosette leaf expansion and petiole elongation in high R:FR (Franklin et al., 2003b). Analysis of mutants de cient in phyc revealed this phytochrome to play a similar role to phya in regulating rosette leaf elongation in high R:FR (Franklin et al., 2003b; Monte et al., 2003). Interaction of light and directional sensing systems Gravity provides plants with a continuous and unidirectional signal, enabling emerging seedlings to orientate themselves within the soil. Correct spatial orientation ensures that primary roots grow downwards to nd water and essential minerals (positive gravitropism) while primary shoots grow upwards towards light at the soil surface (negative gravitropism). Multiple plant organs are also subject to reorientation and can change their growth angle in response to gravitational signals (Hangarter, Sensory mechanisms of environmental monitoring Page 3 of 6 1997). The angle from the vertical at which an organ shows no gravity-induced differential growth is termed the gravitational set-point angle (GSA) (Digby and Firn, 1995). Gravitropism responses are, however, not independent from other environmental stimuli and are often modulated by light signals perceived through phytochromes. Exposure of dark-grown maize roots to R can alter the GSA from perpendicular to the gravity vector to a positively gravitropic orientation (Lu et al., 1996). The gravitropism of stems is also modulated by light. In etiolated Arabidopsis seedlings, R and FR, acting through either phya or phyb, lead to agravitropism of the hypocotyl (Liscum and Hangarter, 1993; Poppe et al., 1996; Robson and Smith, 1996). It is possible that this phenomenon is of ecological signi cance, effectively leading to `enhancement' of phototropic curvature via elimination of gravitropic compensation which can occur when phototropically-stimulated plant organs bend in relation to the gravitational vector. In the lazy-2 mutant of tomato, R was shown to reverse the GSA of hypocotyls, resulting in positive gravitropism (Gaiser and Lomax, 1993). In addition to effects on primary roots and shoots, light can also regulate gravitropism in apical hooks and secondary stems (Myers et al., 1994). The phytochromes are also known to interact more directly with phototropism. For example, R, acting predominantly through phya is known to lead to enhancement of subsequent phototropic curvature (Parks et al., 1996; Janoudi et al., 1997a). This appears to be a discrete response, not related to phytochrome-mediated agravitropism, mediated by both phya and phyb. It has also been established that manifestation of rst positive phototropic curvature is abolished in Arabidopsis seedlings which are doubly null for phya and phyb, indicating a speci c requirement for phytochrome action for the display of this archetypal blue light response (Janoudi et al., 1997b). Interaction of light and temperature sensing Alterations in the ambient growth temperature can dramatically affect plant physiology. In many species, exposure to a period of cold treatment provides seasonal information, enabling plants to germinate (strati cation) or initiate reproduction (vernalization) under more favourable conditions. The ability to anticipate and, consequently, prevent the adverse effects of a particular seasonal environment is selectively advantageous to plants through reducing competition for resources and increasing the chances of outbreeding and genetic recombination. Such information is often integrated with other environmental stimuli, such as daylength. Extended periods of cold temperature and reduced daylength provide plants with a reliable indication of seasonal progression and are amongst the most important environmental factors governing the

Page 4 of 6 Franklin and Whitelam timing of oral transition (Samach and Coupland, 2000; Simpson and Dean, 2002). Sensitivity to the timing of light and darkness, termed photoperiodism, involves the integration of temporal information, provided by the circadian oscillator, with light/dark discrimination provided by speci c photoreceptors. In the long-day plant (LDP) Arabidopsis, owering is accelerated under photoperiods exceeding a critical daylength. Here, the presence of light is perceived through the action of either cry2 or phya (Yanovsky and Kay, 2002). Genetic and physiological analyses have grouped mutations that delay owering into independent promotory pathways. These include the long-day pathway, the autonomous pathway and the gibberellic acid (GA)- dependent pathway (Koornneef et al., 1998). The vernalization response acts separately, but similarly, to the autonomous pathway to repress transcript levels of the oral repressor FLC (Michaels and Amasino, 1999). The stable repression of FLC levels requires a nuclear localized protein, VRN2, although this is not required for the initial reduction in transcript levels (Gendall et al., 2001). The requirement of vernalization is conferred by dominant alleles of the FRI gene, the product of which promotes FLC accumulation (Johanson et al., 2000). Downstream targets of FLC include the oral integrators FT and SOC1/ AGL20 (Lee et al., 2000; Rouse et al., 2002). In addition to photoperiod and vernalization, ambient growth temperature can have profound effects on owering (BlaÂzquez et al., 2003; Halliday et al., 2003). Growth of Arabidopsis plants at reduced temperatures has revealed a novel thermosensory pathway controlling oral initiation (BlaÂzquez et al., 2003). Delayed owering was observed in wild-type plants grown at 16 C, a response absent in the autonomous pathway mutants, fca-1 and fve-1 (BlaÂzquez et al., 2003). The authors propose a dual role for these proteins in owering control: a temperature-dependent mechanism, where they act jointly and a temperatureindependent mechanism where they act redundantly. Both FCA and FVE are believed to down-regulate the oral repressor FLC (Sheldon et al., 2000; Michaels and Amasino, 2001). The modest increases in FLC levels observed at lower temperatures suggest that the temperature-dependent control of owering time by FCA and FVE operates through an FLC-independent pathway (BlaÂzquez et al., 2003). This information is believed to be integrated with other environmental signals, such as daylength, through the oral promoter FT (Kardailsky et al., 1999; Kobayashi et al., 1999). Observations revealing temperature-dependency in the owering responses of photoreceptor mutants have provided insight into the interaction between light and temperature signalling (BlaÂzquez et al., 2003; Halliday et al., 2003). Mutants de cient in the B-photoreceptor cry2 (fha) displayed an exaggerated owering response to change in ambient temperature, with a signi cant delay at 16 C. The combined de ciency of cry2 and phya revealed synergism between these photoreceptors, with the double mutant owering as late at 23 C as the fha/cry2 mutant at 16 C (BlaÂzquez et al., 2003). Growth at 16 C also abolished the early owering phenotypes of phyb and phyaphybphyd-triple mutants, despite plants retaining elongation responses characteristic of the shade avoidance syndrome (Halliday et al., 2003). The early owering response of plants at 23 C was shown to correlate with elevated levels of the oral promoter FT and provides evidence of discrete pathways controlling the owering and elongation components of low R:FR perception (Halliday et al., 2003). Repression of owering in the phyaphybphyd-triple mutant at 16 C was relieved by the additional loss of phye, suggesting a novel and important role for this phytochrome in repressing oral induction at lower temperatures. Conclusions Light signals are amongst the most important environmental factors that regulate the growth and development of plants. The phenotypic plasticity awarded by effective monitoring of the ambient light environment confers considerable advantage to plants growing in natural communities. The capacity to respond to the perceived threat of shading promotes survival by enabling plants to overtop competing vegetation and precociously initiate reproduction. The integration of gravitropic and phototropic signalling are of adaptive signi cance in orientating plant organs within their environment. Such adaptations enhance photosynthetic ef ciency by enabling plants to grow towards sunlight and position their lateral organs for optimum light capture and gas exchange. Synchronization of owering time to environmental cues, such as photoperiod and temperature, enhance survival through promoting seed set during optimal seasonal conditions and increasing the chance of out-breeding and genetic recombination. The multiplicity of responses available to plants in response to environmental light signals results from functional divergence within the phytochrome family of photoreceptors. Redundancy between family members and co-actions with blue light-sensing mechanisms increase sensitivity to environmental uctuations and permit an array of developmental responses. Phylogenetic studies in Arabidopsis have revealed common evolutionary ancestry between phytochromes B, D and E. These form a distinct subgroup that act redundantly to control responses to perceived vegetational shade in addition to their own, individual, functions. Phytochrome A shares a common ancestry with phyc and performs regulatory roles in plant architecture, owering and FR sensing. Until recently, the absence of a phyc mutant has precluded functional analysis of this phytochrome. Mutant analyses have since

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