Diurnal regulation of plant growth*

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1 Growth regulation by light, clock and hormones K. Nozue & J. N. Maloof Plant, Cell and Environment (2006) 29, doi: /j x Diurnal regulation of plant growth* KAZUNARI NOZUE & JULIN N. MALOOF Section of Plant Biology, College of Biological Sciences, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA ABSTRACT Life occurs in an ever-changing environment. Some of the most striking and predictable changes are the daily rhythms of light and temperature. To cope with these rhythmic changes, plants use an endogenous circadian clock to adjust their growth and physiology to anticipate daily environmental changes. Most studies of circadian functions in plants have been performed under continuous conditions. However, in the natural environment, diurnal outputs result from complex interactions of endogenous circadian rhythms and external cues. Accumulated studies using the hypocotyl as a model for plant growth have shown that both light signalling and circadian clock mutants have growth defects, suggesting strong interactions between hypocotyl elongation, light signalling and the circadian clock. Here, we review evidence suggesting that light, plant hormones and the circadian clock all interact to control diurnal patterns of plant growth. Key-words: auxin; brassinosteroids; gibberellic acid; ethylene; phytochrome; cryptochrome; hypocotyl; diurnal; circadian clock; growth. HISTORY OF STUDIES ON DIURNAL AND CIRCADIAN-REGULATED RESPONSES Almost all organisms have evolved adaptations to diurnal (day/night) cycles. That these adaptations result in observable daily rhythms was first discovered in plants by Androsthenes, a Greek soldier, in the 4th century BC. He observed periodic daily movement of leaves ( sleep movement ) under natural diurnal cycles. This leaf movement has ecological significance. During the night, leaves are lowered, decreasing the area exposed to the sky and thus reducing radiant energy loss and chilling. During the day, leaves are raised to maximize exposure to light (More & Schopfer 1994). Not only were daily rhythms first discovered in plants, but plants also served as the first experimental organism for studying these rhythms (e.g. Darwin 1881). A significant early discovery was that sleep movements persist even in continuous darkness (for review, see Ward 1971; Sweeney 1987). By the early 20th century, plant biologists were Correspondence: Julin N. Maloof. Fax: ; jnmaloof@ucdavis.edu *This work is supported in part by NSF grant DBI to J. N. M and Cynthia Weinig. 396 searching for factor X an unknown environmental cue that synchronized movement under continuous darkness. For these experiments, a red safety light was used to allow plant watering in the dark. A plant biologist, Erwin Bünning, found that when plants were watered in the afternoon instead of in the morning, the maximal night leaf positions shifted from 3 4 AM to AM. Although shifts in periodicity had previously been observed, Bünning reasoned that the endogenous plant clock could be reset by light. (Salisbury & Ross 1978). Of particular relevance for this review, these experiments also illustrated that both the endogenous clock and external cues together control plant growth. Many other organisms, from cyanobacteria to humans, show persistent daily rhythms in continuous conditions. These endogenous rhythms have a period of about 24 h and thus are referred to as circadian rhythms (from the Latin circa diem, meaning approximately a day ). Although endogenous circadian rhythms may have periods more than 1 hour longer or shorter than 24 h in continuous conditions, in nature their periods are uniformly closer to 24 h because of the synchronizing effects of light at daybreak, referred to as entrainment (for review, see Salome & McClung 2005). From the mid-20th century, studies on the circadian clock in plants and other organisms have flourished, and these studies have led to the discovery of proteins essential for the function of the central oscillator (for review, see Salome & McClung. Stem elongation is one of the many plant processes that exhibit diurnal and circadian rhythms (Lecharny & Wagner 1984) and presents an attractive model because of the depth of accumulated knowledge on its regulation. Much of this knowledge has been learned from studying hypocotyls, the stems of young seedlings. The hypocotyl, like the adult shoot, is influenced by endogenous and environmental factors such as light, plant hormones, temperature and nutrition; furthermore, it exhibits rhythmic (circadian) growth (Dowson-Day & Millar 1999). Because light is a key factor during diurnal cycles, the focus of this review will be on how interactions between light, the circadian clock and plant hormones regulate hypocotyl growth; readers will be directed to the many excellent reviews describing individual pathways for additional details. LIGHT CONTROL OF HYPOCOTYL GROWTH Light is the primary regulator of hypocotyl growth. Seedlings have two developmental forms: dark-grown (etiolated) and light-grown. In nature, etiolated growth occurs Journal compilation 2006 Blackwell Publishing Ltd

2 Growth regulation by light, clock and hormones 397 as buried seedlings elongate their hypocotyls upwards, searching for the soil surface. Etiolated seedlings are tall and have yellow compressed cotyledons, undifferentiated chloroplasts and an apical hypocotyl hook that serves to protect the shoot apical meristem from damage. Light perception signifies soil emergence and causes de-etiolation: hypocotyl elongation ceases, the hook straightens, cotyledons unfold and green, photosynthetic growth begins. Light-grown seedlings are short because of the inhibition of hypocotyl elongation, and have open, green cotyledons. Genetic screens for mutants with defects in light perception have identified many genes involved in light signal transduction (for review, see Chen, Chory & Fankhauser. Light is perceived by at least three distinct families of photoreceptors: phytochromes (PHYA-PHYE in Arabidopsis) perceive red and far-red light, whereas cryptochromes (CRY1 and CRY2) and phototropins (PHOT1 and PHOT2) mediate distinct responses to blue light. Photoreceptor signals are integrated in part through the bzip transcription factors HY5 and its closely related homologue HYH (Oyama, Shimura & Okada 1997; Holm et al. 2002). Cloning of mutants with a constitutively photomorphogenic, de-etiolated phenotype (and hence known as cop or det) has revealed the importance of ubiquitin-proteoseome mediated protein degradation in light signalling. COP1 and DET1 likely function together as part of an E3 ubiquitin ligase (DCX DET1-COP1 ) to target positive light signalling transducers such as HY5 for proteasome degradation in the dark (for review, see Schwechheimer. A number of other COP genes are involved in this process: COP10 serves as an E2 ubiquitin conjugating enzyme, and the multiunit COP9 signalosome (CSN) is also required for degradation. In addition, a family of WD-repeat containing proteins, SPA1-4, function with COP1 to suppress photomorphogenesis (Laubinger, Fittinghoff & Hoecker. How does DCX DET1-COP1 mediate light signalling? Interaction between cryptochromes and COP1 is essential for cryptochrome-mediated blue-light signalling (Lin & Shalitin. Experiments suggest that a CRY:COP1:HY5 heterotrimeric complex exists and that blue light acts through CRY1 to attenuate the ubiquitin ligase properties associated with COP1. Thus, proteasome-mediated degradation of HY5 is inhibited in the light (Wang et al. 2001; Yang, Tang & Cashmore 2001). COP1 also binds to activated PHYA, facilitating PHYA protein degradation upon red or far-red illumination (Seo et al.. This binding is thought to cause removal of activated PHYA, thereby attenuating PHYA signalling. Interestingly, these mechanisms contrast with the COP1-CRY interaction where binding of CRY decreases COP1 activity, thereby increasing light-regulated signalling. In addition to the COP mutants, there are more than a dozen factors discovered to date that mediate phytochrome signalling (for review, see Chen et al.. Best characterized are a set of related bhlh transcription factors. The first to be characterized was PIF3, which binds phytochrome in a light-dependent manner and which binds the G-box regulatory element found in the promoters of many light-regulated genes. PIF3 and the related bhlhs function primarily as inhibitors of photomorphogenesis, although some positive effects have been reported (for review, see Duek & Fankhauser 2005). Activated phytochrome targets PIF3 for degradation, presumably allowing expression of light-activated genes. In contrast, a more distantly related bhlh, HFR1, acts as a positive transducer of PHYA-mediated signalling. Accordingly, HFR1 is degraded in the dark (by COP1) and stabilized by light. In etiolated seedlings, phytochrome is translocated from the cytoplasm to the nucleus in response to a red light pulse that photo-converts the red absorbing form (Pr) of phytochrome into the active far-red absorbing Pfr (for review, see Nagatani. The direct interaction of Pfr in the nucleus with transcription factors such as PIF3 is an important step in phytochrome regulation of hypocotyl elongation. In principle, interactions between hormones or the clock and light signalling could occur among the signalling components discussed here, or downstream at the promoters of the genes that they control. HORMONAL REGULATION OF HYPOCOTYL GROWTH Many aspects of plant development are regulated by both light and hormones (recent reviews include Nemhauser & Chory 2002; Halliday & Fankhauser 2003; Symons & Reid, suggesting interactions between the light and hormone signalling pathways. Figure 1a shows basic signal transduction pathways for the major growth control hormones (described below). Here, we present evidence supporting the idea that hypocotyl elongation is controlled differently by plant hormones in the light and dark, implying that light regulates the effectiveness or action mode of hormones. The plant hormone auxin (indole acetic acid, IAA) is important for the control of cell elongation. Experiments manipulating auxin transport, concentration and signalling suggest that there are different requirements for auxin in light versus dark conditions. Auxin transport is mediated by efflux (PIN) and influx (AUX/LAX) families, as well as by ABC transporters, and is essential for plant development (for review, see Woodward & Bartel 2005). The auxin transport inhibitor NPA and mutations in PIN3 reduce hypocotyl elongation in the light but not in the dark, suggesting that auxin or auxin transport is important for hypocotyl elongation of light-grown seedlings but not etiolated seedlings (Jensen, Hangarter & Estelle 1998; Friml et al. 2002). Consistent with this, PIN1 overexpression increases hypocotyl elongation in the light (De Grauwe et al. 2005). In contrast, mutations that disrupt PIN1 localization and polar auxin transport by knocking out specific ABC transporters or an interacting immunophilin cause shortened hypocotyls in both the light and the dark (Noh, Murphy & Spalding 2001, Noh et al. 2003; Geisler et al. 2003; Perez-Perez, Ponce & Micol 2004; Lin & Wang 2005). This apparent contradiction might be explained by these mutations having pleiotropic effects on both the auxin and brassinosteroid

3 398 K. Nozue & J. N. Maloof (a) PHYs, CRYs PIFs, PILs COP1 SPA1-4 ZTL ELF3 HYH, HY5 PRRs Cell wall Cytoskeleton GROWTH GAI RGA RGLs SCF SLY1 GA CCA1 LHY TOC1 EIN3, EIL1 ARFs BES1, BZR1 ELF4 GI EIN2 CTR1 IAAs SCF TIR1 BIN2 BRI1 ACS8 ACC ETR1 et al. Ethylene Auxin GH3s RED1 Conjugation/ Inactivation Brassinosteroids BAS1 SOB7 Inactivation (b) PHYs, CRYs PIFs, PILs COP1 SPA1-4 CCA1 LHY ZTL PRRs ELF4 GI ELF3 TOC1 ACS8 ACC HYH, HY5 Cell wall Cytoskeleton GROWTH EIN3, EIL1 ARF2 EIN2 CTR1 ETR1 et al. Ethylene HLS1 ARFs IAAs SCF TIR1 Auxin GH3s RED1 Conjugation/ Inactivation GAI RGA RGLs BES1, BZR1 SCF SLY1 BIN2 BRI1 GA Brassinosteroids BAS1 SOB7 Inactivation Figure 1. Multiple pathways interact to control hypocotyl elongation in diurnal conditions. (a) Core pathways controlling interaction. (b) Interactions among pathways. Each pathway is placed on a coloured background: peach (light signalling), yellow (clock), green (ethylene), blue (auxin), pink (brassinosteroid), magenta (GA). Black lines represent genetic interactions, blue lines show direct interactions, orange lines are for light and general light effects, and red lines show hypothesized interactions. Lines without arrowheads are for interactions where directionality is unknown. To maintain some clarity, not all relevant genes or interactions have been shown. pathways (Perez-Perez et al., by enhanced gravitropism (Noh et al. disrupting normal etiolated growth or by differential effects on polar versus non-polar auxin transport. Manipulation of auxin levels also affects hypocotyl elongation. Specific overexpression of auxin biosynthesis genes in the vasculature and apical meristem, or increasing endogenous auxin by raising temperature, causes elongated hypocotyls in the light but not in the dark (Romano et al. 1995; Gray et al. 1998; van der Graaff et al.. In other cases, changing auxin levels does affect dark growth. In mutants with increased auxin levels such as sur1/rty/hls3/ alf1 or yucca, or in the presence of exogenous auxin, hypocotyls are abnormally short in the dark (and still overly long in the light) (Boerjan et al. 1995; Celenza, Grisafi & Fink 1995; King et al. 1995; Lehman, Black & Ecker 1996; Thom-

4 Growth regulation by light, clock and hormones 399 ine et al. 1997; Barlier et al. 2000). These are essentially gain-of-function mutations; thus, it is unclear how they relate to etiolated growth in wild-type plants. However, a role for auxin in etiolated growth was also indicated by overexpressing iaalys, an IAA conjugating enzyme, which decreased hypocotyl elongation both under continuous light and in the dark (Gray et al. 1998). In summary, it seems that auxin may be important for both light and dark growth, but it is likely to play different roles or signal through different pathways in these two conditions. There is abundant evidence for cross-talk between the light and auxin signalling pathways. Auxin regulates gene expression by binding TIR1, which is part of the SCF TIR1 E3 ubiquitin ligase (Dharmasiri, Dharmasiri & Estelle 2005; Kepinski & Leyser 2005). Auxin activation of SCF TIR1 induces degradation of AUX/IAA proteins, which are short-lived nuclear proteins. AUX/IAA proteins in turn interact with and inactivate auxin response factors (ARFs), which function as transcription factors (for review, see Woodward & Bartel 2005). There are several links between specific AUX/IAA proteins and light signalling. A recent study found that HY5 promotes the expression of the negative auxin signalling factors AXR2/IAA7 and SLR/IAA14 and that overexpression of AXR2 can partially rescue the long-hypocotyl defect seen in hy5 mutants (Cluis, Mouchel & Hardtke. Another link was made when the shy2 mutant was found to be defective in IAA3 and IAA3 was found to interact with PHYB and TIR1 (Tian & Reed 1999). Additional gain-of function iaa mutants (iaa7/axr2 and iaa17/axr3) have constitutive photomorphogenic phenotypes (for review, see Woodward & Bartel 2005). Mutants with defects in AXR1, an ubiquitin activating E1 enzyme (Leyser et al. 1993), also are defective in COP1/ COP10/CSN-mediated repression of photomorphogenesis in the dark (Schwechheimer, Serino & Deng 2002). Finally, some recombinant IAAs are phosphorylated by phytochrome, suggesting another possible link between these signalling pathways (Colon-Carmona et al. 2000). Recent studies indicate regulation of auxin homeostasis as another area of light auxin cross-talk. The auxin-responsive gene family GH3 encodes IAA-aminosynthetases, which help maintain auxin homeostasis (Staswick et al. 2005). GH3 overexpression and antisense lines suggest that some of these genes affect hypocotyl elongation only in specific light conditions, whereas others affect growth both in the dark and in the light. Expression of several GH3 family members is light regulated, providing further support for their involvement in light auxin cross-talk (see References in Staswick et al. 2005). Seedlings of another mutant, red1, exhibit elongated hypocotyls and reduced cotyledon size specifically in continuous red light but not in continuous far-red light. RED1 was found to be allelic to SUR2/ATR4, which encodes a cytochrome P450 (CYP450) required for normal auxin homeostasis (Smolen & Bender 2002; Hoecker et al.. Transcripts of RED1/SUR2/ ATR4 are up-regulated by red light in a process that, surprisingly, does not require PHYB. In summary, there are multiple links between the auxin and light pathways. Determining when these links are relevant in wild-type plants and how they work mechanistically in many cases still needs to be investigated. Gibberellic acid (GA) is a terpenoid compound that, like auxin, regulates plant growth by influencing the rate and extent of cellular elongation (Cowling & Harberd 1999). GA influences hypocotyl elongation in light- and darkgrown plants. A GA biosynthesis deficient mutant, ga1, and a GA insensitive mutant, gai, are shorter than wild type under both continuous light and continuous dark. However, light- and dark-grown hypocotyls exhibit markedly different GA dose response relationships. The length of dark-grown hypocotyls is relatively unaffected by exogenous GA, while light-grown wild-type hypocotyl length is significantly increased by exogenous GA. The response of ga1 to exogenous GA in the dark is more pronounced than when grown in the light. Similarly, PHYB negatively regulates GA signalling or downstream responses because phyb ga1 double mutants show amplified responsiveness (but not sensitivity) to exogenous GA (Reed et al. 1996). Together, this suggests that light, sensed through PHYB, attenuates GA signalling or response and that the response is close to saturation in the dark. GA also participates in the repression of photomorphogenesis during etiolated growth. For example, the CAB2 gene is normally expressed only in the light; however, reduction of GA biosynthesis or signalling causes misexpression of CAB2 in etiolated seedlings (Alabadi et al.. Interactions between light, GA and auxin signalling are suggested by microarray experiments (Folta et al.. In cry1 mutants, expression of both GA biosynthesis genes and auxin response factors change. Consistent with these facts, inhibition of GA 4 biosynthesis restored wild-type growth kinetics in cry1 and suppressed its long-hypocotyl phenotype in blue light. A major mode of GA signal transduction is through regulation of ubiquitin-proteasome mediated degradation (for review, see Fleet & Sun 2005). In response to GA, GAI and four related DELLA proteins (likely transcriptional regulators) are degraded by the E3-ligase complex SCF SLY1. A GA receptor, GID1, has just been identified in rice. (Ueguchi-Tanaka et al. 2005). GID1 appears to regulate signal transduction by binding and promoting degradation of DELLA proteins in a GA-dependent manner. Limited information is available on the effects of these genes on hypocotyl elongation except for GAI (see above). Mutation of SLY1 reduces elongation in the dark, and rga hypocotyls are very short in the light (Silverstone et al. 1997; Steber & McCourt 2001). It will be important to determine if the light-regulated changes in GA response are mediated through changes in DELLA protein action. The brassinosteroids (BRs) are important regulators of cell elongation, and numerous experiments in Arabidopsis suggest that this hormone class plays a role in light signalling (for review, see Vert et al. 2005). BR biosynthesis mutants and plants treated with a BR biosynthesis inhibitor are shorter than wild-type seedlings in all light conditions and are short and de-etiolated in the dark (Li et al. 1996;

5 400 K. Nozue & J. N. Maloof Azpiroz et al. 1998; Asami et al. 2000). Therefore, a BR is required for growth in light-grown and etiolated seedlings. Analysis of BR signal transduction mutants (Vert et al. 2005) are consistent with this idea. In addition, two phytochrome A-mediated responses are altered in BR biosynthesis mutants, supporting the involvement of BR in light signalling (Luccioni et al. 2002). BR signal transduction starts from a leucine-rich repeat receptor kinase, BRI (Vert et al. 2005). The signal from BRI is transduced by a GSK3-like kinase, BIN2/UCU1. In the absence of BR, BIN2 phosphorylates and inactivates two transcription factors, BES1 and BZR1. BR binding leads to the inhibition of BIN2 activity, allowing the accumulation of unphosphorylated BES1 and BZR1 and subsequent changes in transcription. How does BR signalling mediate the de-etiolation process? So far there is no evidence that BR signalling components are modified by light. However, light may modulate BR synthesis and degradation. Pra2, a small G protein found in pea, has been shown to stimulate activity of DDWF1, a CYP450 hydroxylase involved in BR synthesis (Kang et al. 2001). Transcription of Pra2 is dark-induced, suggesting a mechanism for regulating BR levels by light. Arabidopsis has no obvious Pra2 homologue, so this may not be a highly conserved mechanism (or the Pra2 sequence has diverged while retaining its function). In contrast, two CYP450 monooxygenases, BAS1 and SOB7, are involved in BR inactivation. BAS1 and SOB7 are partially redundant, and single and double mutants are hyporesponsive to white, blue, red and far-red light (Turk et al. 2005). There is some evidence of light-regulated transcription of BAS1 and SOB7, but the details of how light regulates their activity need further investigation. A framework for signal transduction downstream from the gaseous hormone ethylene has begun to emerge (for review, see Guo & Ecker. Briefly, ethylene is perceived by a family of two-component receptors (ETR1, ETR2, ERS1, ERS2 and EIN4). Ethylene inhibits the action of the receptors, which, in the absence of ethylene, activate an MAPKKK-type kinase, CTR1. In the absence of ethylene, CTR1 negatively regulates EIN2, a membrane protein of unknown biochemical function. Activation of EIN2 by ethylene perception allows the accumulation of two transcription factors (EIN3 and EIL), which in turn initiate a transcriptional cascade activating many ethyleneresponsive genes. Ethylene has been shown to inhibit hypocotyl elongation in the dark (Bleecker et al. 1988). However, in long days (16 h light and 8 h dark), ethylene induces hypocotyl elongation (Smalle et al. 1997). ctr1-1 mutants have longer hypocotyls than wild type in long days (Smalle et al. 1997). Interestingly, seedling ethylene sensitivity has also been shown to change during illumination (Knee, Hangarter & Knee 2000). Exogenous ethylene causes enhanced curvature of the hypocotyl hook in darkness; however, light-grown plants have open hooks even at high ethylene concentrations, indicating that light attenuates ethylene sensitivity. How might this occur? Ethylene-regulated hook development requires HLS1, a protein whose expression is negatively regulated by light. In the presence of light, HLS1 is not expressed, thereby preventing ethylene-mediated hook curvature (Li et al.. INTERACTION BETWEEN HORMONES Hormones interact with one another at many steps of their signalling pathways (for review, see Halliday 2004; Swain & Singh 2005; Woodward & Bartel 2005). One example of possible ethylene auxin cross-talk comes from further studies on HLS1. The synthetic auxin-responsive reporter DR5::GUS is expressed asymmetrically in the hook and this expression is abolished in hls1 mutants. Mutations in ARF2 restore hooks and differential DR5::GUS staining in hls1 mutant hypocotyls. Furthermore, HLS1 regulates ARF2 degradation (Li et al.. However, microarray studies failed to find a role for ARF2 in auxin-mediated gene expression (Okushima et al. 2005), suggesting that ARF2 and HLS may influence auxin signalling indirectly. Auxinregulated root elongation is mediated by DELLA proteins, which are GA signalling components (Fu & Harberd. For hypocotyl elongation, interactions among auxin, GA and ethylene have been reported by some groups (Smalle et al. 1997; Saibo et al., whereas others have failed to find interactions (Collett, Harberd & Leyser 2000). The reasons for such discrepancies are not known but might involve differences in growth conditions. A recent study showed that auxin and BR effects on hypocotyl elongation are interdependent: auxin- and BR-mediated growth promotion by exogenous hormone required both pathways to be intact (Nemhauser, Mockler & Chory. In addition, auxin- and BR-responsive genes overlap; this, with further studies, suggests that the auxin and BR signalling pathways converge at gene promoters (Nemhauser et al.. Finally, hormones can regulate each others biosynthesis; for example, auxin stimulates ethylene synthesis (Tsuchisaka & Theologis. Whether these hormone interactions are regulated by light or the circadian clock remains to be determined. CLOCK REGULATION OF HYPOCOTYL GROWTH In most organisms studied, transcriptional translational negative feedback loops are central to the circadian oscillator [however, in Synechococcus, rhythms can be generated by a protein phosphorylation cycle (Nakajima et al. 2005)]. Three major clock genes have been identified in Arabidopsis: CCA1, LHY and TOC1/PRR1. CCA1 and LHY are homologous DNA-binding proteins that recognize a sequence in the promoter of TOC1, a pseudoresponse regulator gene (Strayer et al. 2000; Alabadi et al. 2001). CCA1 and LHY are expressed in the early subjective day (Schaffer et al. 1998; Wang & Tobin 1998) and are thought to repress TOC1/PRR1 expression (Alabadi et al. 2001). TOC1/PRR1 expression in the evening has been proposed to activate CCA1 and LHY transcription, completing the loop (Alabadi et al. 2001). The activation could be indi-

6 Growth regulation by light, clock and hormones 401 rect, because it takes 8 h from peak TOC1/PRR1 expression to CCA1 and LHY activation and may require at least two other genes, GI (Fowler et al. 1999) and ELF4 (Doyle et al. 2002). Four Arabidopsis PRR genes homologous to TOC1/PRR1 are expressed rhythmically, in sequence every 2 3 h from dawn to dusk (for review, see Mizuno & Nakamichi 2005). Mutation of TOC1/PRR1 has a greater effect on the circadian clock than do other PRR single mutants. These PRRs appear to modulate phytochrome signalling, because all PRR mutants examined (except toc1-1) have long hypocotyls under red light. Protein degradation, as well as light signalling, is important for normal clock function. The ztl mutant confers a long period phenotype; ZTL encodes an F-box protein, a component of a SKP-type E3 ligase 2000; Han et al.. ZTL binds and degrades TOC1/PRR1, regulating circadian period (Mas et al. 2003b). Interestingly, ZTL contains a photosensitive light, oxygen, voltage (LOV) domain similar to the light-sensing domains in PHOT1 and PHOT2, suggesting that light may directly regulate the ability of ZTL to degrade TOC1 (Imaizumi et al.. There is abundant evidence that the clock regulates hypocotyl elongation. Hypocotyl growth is rhythmic even under continuous light (Dowson-Day & Millar 1999), and there is a strong correlation between circadian and hypocotyl mutant phenotypes (Table 1). Arrhythmic clock mutants, such as elf3 and CCA1-overexpressing plants, show continuous growth (Hicks et al. 1996; Dowson-Day & Millar 1999; Thain et al.. Strikingly, elf3 arrhythmia is only found in the light (Hicks et al. 1996), consistent with its proposed role as a modulator of light input to the clock (Covington et al. 2001). The co-occurrence of circadian and hypocotyl phenotypes might suggest that the clock is intrinsically linked with hypocotyl elongation. However, an exception is an allele of toc1 (toc1-1) that exhibits a short period both in CAB2 gene expression and in hypocotyl growth, but that does not have a hypocotyl length phenotype, showing that these phenotypes are separable (Somers et al. 1998; Dowson-Day & Millar 1999; Mas et al. 2003a). Although the current data clearly show that the circadian clock regulates hypocotyl elongation, much has yet to be learned about the underlying mechanisms. GATING MODEL OF HYPOCOTYL ELONGATION BY THE CIRCADIAN CLOCK Light inhibits hypocotyl elongation and hypocotyl elongation is regulated by the circadian clock. How do light and the clock coordinately regulate hypocotyl elongation? One model is that there is a linear connection between light signalling, the circadian oscillator and the hypocotyl elongation machinery. In this model, light inhibits hypocotyl elongation through the circadian clock. An alternative model is that the circadian clock and the hypocotyl elongation machinery are separately regulated by light and that the clock further modulates hypocotyl elongation (Fig. 1b). Recent experiments, showing that the circadian clock can modulate the effectiveness of simulated plant shade on hypocotyl elongation, help to distinguish these models. A TOC1/PRR1 interacting protein, PIL1, was found to be rapidly induced by low red/far-red (simulated shade) illumination (Salter, Franklin & Whitelam. The authors found that under constant conditions, a pulse of low red/ far-red light was only able to induce hypocotyl elongation at subjective dusk, meaning that responsiveness to light quality is gated by the clock. This gated response is absent or reduced in toc1-2, pil1 and phyb mutants, supporting the idea that phyb-mediated shade avoidance is regulated by the circadian clock. The fact that shade avoidance is gated strongly favours the second model discussed above: light regulates both clock function and hypocotyl regulation, with further modulation of hypocotyl elongation by the clock. How do clock mutants alter growth? As noted above, arrhythmic mutants seem to abolish the daily arrest in hypocotyl growth, at least in continuous conditions. Short and longer period mutants with hypocotyl phenotypes could alter either the duration or the amplitude of the growth phase. Time-lapse photography could help distinguish these possibilities. It will be interesting to compare plant growth in both constant and diurnal lighting to examine the interplay of the clock and photoperception. POSSIBLE MECHANISMS OF CLOCK- REGULATED GROWTH Mechanistically, how might the clock alter growth? Broadly, the clock could affect light signalling, hormone production/action or cell architecture components (Fig. 1b). The clock could also act by regulating light/hormone or light/cell architecture interactions. One way to investigate clock-regulated growth is by expression analysis. Microarray data have implicated the circadian clock in the regulation of genes involved in light signalling, auxin transport and responses, ethylene responses and cell-wall modification (Staiger, Apel & Trepp 1999; Harmer et al. 2000; Schaffer et al. 2001; Sharrock & Clack 2002). Although intriguing, this transcriptional regulation needs to be further investigated to determine if it is important for circadian or diurnal patterns of growth. In addition to transcriptional regulation, there are many ways in which light signalling components are affected by the circadian clock. Because light is used to help set the clock, this implies a clock light feedback loop that may be important for entrainment (Salome & McClung 2005). Both phytochromes and cryptochromes participate in clock input. PhyB interacts with ZTL (Jarillo et al. 2001) and ELF3 (Liu et al. 2001), and CRY1 interacts with ZTL (Jarillo et al. 2001), implying direct coupling of photoreceptors with circadian clock-associated proteins. In addition, TOC1/PRR1 interacts with PIF4 and PIL6, members of the PIF3 bhlh transcription factor family. The transcription of both PIF4 and PIL6 is clock regulated (Yamashino et al., but the pil6 mutant does not affect the expression of CCA1 (pif4 has not yet been examined). Both pil6 and pif4

7 402 K. Nozue & J. N. Maloof Table 1. Hypocotyl phenotype of circadian clock mutants hypocotyl phenotype clock phenotype dark L/D W B R FR LHY OX AR (Schaffer et al. 1998) (Schaffer et al. 1998) CCA1 OX AR (Wang &Tobin1998), LA (Harmer & Kay 2005) cca1 lhy AR, SP (Alabadi et al. 2002; Mizoguchi et al.2002) (Mas et al. 2003a) (Mas et al. 2003a) toc1-1 SP (Millaretal.1995) (Mas et al. 2003a) toc1-2 or RNAi TOC1 /PRR1 OX SP, LA, AR (Alabadi et al. 2002) (Mas et al. 2003a) LP, AR (Makino et al. 2002) (Mas et al. 2003a) elf3 AR(LL), N(DD) (Hicks et al. 1996) LA(DD) (Covington et al. 2001) (Wang & Tobin 1998) (Mas et al. 2003a) 1998) 1998) (Liu et al. 2001) (Zagotta et al. 1996) (Liu et al. 2001) (Zagotta et al. 1996) (Mas et al. 2003a) 1998) 1998) (Mas et al. 2003a) (Mas et al. 2003a) (Mas et al.2003a) (Mas et al. 2003a) (Mas et al. 2003a) (Mas et al.2003a) (Zagotta et al. 1996) ELF3 OX LP(LL) (Covington et al. 2001) (Liuet al. 2001) elf4 VP, AR (Doyle et al. 2002) (Khanna et al. (Doyle et al. 2002) (Doyle et al. 2002) (Khanna et al. ztl LP (Somers etal.2000) 2000) ZTL OX SP (Somers etal. (Kiyosue & Wada, 2000; Nelson et al. 2000) LKP2 OX AR (Schultz et al. 2001) (Schultz et al. 2001) gi SP, LA (Fowler et al. 1999; Park et al. 1999),LP(Park et al. 1999) srr1 SP (Staiger et al. (Staiger et al. spy LP (Tseng et al. (Swain et al. 2001) spy gi LP, LA (Tseng et al. (Tseng et al. SPY OX SP, HA (Tseng et al. (Swain et al. 2001) (Huq et al.2000) (Araki & Komeda,1993) (Staiger et al. (Staiger et al. phyb oop1 PS (Salome et al. 2002) (Salome et al. 2002) (Schultz et al. 2001) (Staiger et al. (Salome et al. 2002) (Zagotta et al. 1996) (Khanna et al. 2000) (Schultz et al. 2001) (Huq et al.2000) (Huq et al.2000) (Staiger et al. (Tseng et al. (Tseng et al. (Salome et al. 2002) (Staiger et al. (Tseng et al. (Tseng et al. (Salome et al. 2002) Abbreviations: AR, arrhythmic; SP, short period; LP, long period; VP, variable period; PS, phase shift; HA, high amplitude; LA, low amplitude; N, normal. LL, continuous light; LD, light/dark cycle; DD, continuous dark; W, white light; B, blue light; R, red light; FR, far-red light. Yellow (short), green (normal/wild-type) and blue (tall) colors indicate hypocotyl length. Only the first reference for each reported phenotype is cited to conserve space. See Mizuno and Nakamichi (2005) for summary of pseudo-response regulators.

8 Growth regulation by light, clock and hormones 403 are hypersensitive to red light, suggesting that PIL6 (and perhaps PIF4) function downstream of the circadian clock to repress phytochrome signalling (Huq & Quail 2002; Fujimori et al.. There is evidence for circadian regulation of hormone production and signalling, increasing the complexity of circadian growth-control networks. A GA biosynthetic enzyme has been shown to be regulated both by the clock and by phytochrome (Hisamatsu et al. 2005). In some species, ethylene production is clock regulated (for review, see McClung 2000). Rhythmic ethylene production is controlled by TOC1/PRR1 and CCA1 (Thain et al.. However, rhythmic ethylene synthesis is not required for circadian leaf movement, hypocotyl elongation or CAB2 gene expression (Thain et al.. In contrast, decapitation of the shoot meristem induces a strong inhibition of circadian-controlled floral stem elongation and IAA restores rhythmic growth (Jouve et al. 1999). Furthermore, an auxin transport inhibitor reduces elongation and abolishes rhythmicity. Both free and conjugated auxin levels oscillate, suggesting that auxin fluctuation contributes to rhythmic growth. Are other hormones involved in circadian clock regulation of gene expression, leaf movement and hypocotyl elongation? Much work remains to be done. In addition to the factors described so far, there might be circadian regulation of cell architecture components such as those of the cytoskeleton (for review, see Wasteneys & Yang and cell wall (for review, see Somerville et al.. One target could be cellulose synthases such as PRC1 (Fagard et al. 2000). prc1 mutants have hypocotyl elongation defects in the dark but appear wild type in the light (Desnos et al. 1996). Mutations in the KOR gene, which encodes endo-1,4-β-d-glucanase, result in the same phenotype (Nicol et al. 1998). This suggests that different mechanisms may govern growth in dark- and light-grown seedlings. Consistent with this notion, transcription of many genes encoding cell-wall enzymes is induced by light (Tepperman et al. 2001) and controlled by the circadian clock (Harmer et al. 2000). DIURNAL REGULATION OF CLOCK GENE EXPRESSION Plant growth is normally governed not just by the clock, but also by the interaction of endogenous rhythms and external cues. To better understand the relationship between circadian and diurnal rhythms, it is helpful to examine the relationship between gene expression in these two environments. While 11% of Arabidopsis mrnas were found to be diurnally regulated, only a subset of these show circadian rhythms in continuous conditions (Schaffer et al. 2001). In part this represents the difference between acute light (or dark) induction and circadian regulation. However, clock mutants cause altered gene expression under diurnal cycles, indicating that some diurnal changes are regulated in part by the circadian clock (Green et al. 2002; Mizoguchi et al. 2002). Similarly, the clock is involved in the regulation of hypocotyl elongation under diurnal cycles because some clock mutants have a hypocotyl phenotype under both diurnal and continuous conditions (Table 1). To understand the role of the circadian system in diurnal rhythms, it would be helpful to know the expression patterns of clock genes under diurnal cycles. Unfortunately, such data are limited. As summarized in Fig. 2, the pattern of CCA1 and LHY in light/dark cycles is similar to that in continuous light (Daniel, Sugano & Tobin. The amount of TOC1/PRR1 and ELF3 proteins is reduced in the dark period (Liu et al. 2001); in the case of TOC1/PRR1 this is due to the action of ZTL (Mas et al. 2003b). As a result of this diurnal regulation, ELF3 protein level reaches a maximum level at dusk, well before the peak of mrna expression (Hicks, Albertson & Wagner 2001; Liu et al. 2001). This is in contrast to expression in continuous light, where ELF3 protein levels peak at subjective dawn (Liu et al. 2001). These results highlight the complex interactions between the circadian clock and light signalling during diurnal cycles. To obtain a better understanding of the role of clock genes in diurnal cycles, protein expression patterns of the remaining clock components in wild type and clock mutants should be determined. DIURNAL REGULATION OF PROTEIN LOCALIZATION AND ABUNDANCE Light-dependent translocation of signalling components between the cytosol and the nucleus is likely to play an important role in diurnal regulation of hypocotyl elongation. As discussed above, phytochrome is regulated in this manner, being cytoplasmic in the light and nuclear in the dark (Kircher et al. 2002). In contrast, light induces translocation of COP1 from the nucleus to the cytosol, allowing accumulation of HY5 and HFR1 and transduction of the light signal to downstream components (Osterlund et al. 2000; Duek et al. 2004; Jang et al. 2005; Yang et al. 2005). Therefore, the diurnal cycle of COP1 localization could be a critical aspect of light signal transduction (Stacey, Hicks & von Arnim 1999, Stacey et al. 2000; Subramanian et al.. PIF3 protein is also regulated by light. In etiolated seedlings, PIF3 is localized in nuclear speckles, but upon illumination, PIF3 is degraded rapidly, suggesting that PIF3 CCA1/LHY TOC1/PPR1 ELF3 ZTL Figure 2. Diurnal protein expression patterns of circadian clockregulated genes.schematic drawing of protein amount of CCA1 (Wang & Tobin 1998), LHY (Kim, Song et al., TOC1/PRR1 (Mas et al. 2003b), ELF3 (Liu et al. 2001) and ZTL (Kim, Geng & Somers. Top bar indicates diurnal periods (white is light and black is dark period). Darker shading indicates higher protein amounts.

9 404 K. Nozue & J. N. Maloof functions at dark/light transitions (Bauer et al.. Indeed, during diurnal conditions PIF3 protein level is high immediately before dawn, decreases rapidly upon light irradiation, remains low during the day, and gradually increases during the dark (Monte et al.. It remains to be seen whether the circadian system regulates subcellular localization of any of these components. TIME-LAPSE OBSERVATIONS OF HYPOCOTYL ELONGATION One useful method for better understanding diurnal and circadian regulation of plant growth is time-lapse photography. As mentioned previously, this technique was used to establish that there is circadian regulation of hypocotyl growth (Dowson-Day & Millar 1999). High-resolution time-lapse photography has proven especially helpful for determining the contributions of specific photoreceptors and effectors to the early effects of illumination on hypocotyl growth (Parks, Folta & Spalding 2001). One example of how this technique could be employed to help understand diurnal growth control is the study of diurnal changes in hormone action. As noted above, the roles of hormones in hypocotyl elongation during diurnal cycles are not simple. Low levels of exogenous auxin promote hypocotyl elongation, while high concentrations of auxin inhibit it (Collett et al. 2000). This could be due to a combination of differential auxin effects on hypocotyl elongation during day and night. Time-lapse monitoring of hypocotyl length could test this hypothesis by identifying periods of rapid and slow elongation during diurnal cycles and examining the effects of applying exogenous hormone at different hours of the day. We have begun to use this technique to test other aspects of hypocotyl growth. The gating model assumes that light inhibition of hypocotyl elongation is affected by the circadian clock but is not dependent upon it. This can be tested by determining whether hypocotyl elongation of arrhythmic clock mutants is light regulated. In short days, CCA1 overexpressing and elf3 mutant plants showed simple growth curves as if inhibition of hypocotyl elongation is solely regulated by light (K. N and J. N. M., unpublished results). In contrast, wild-type plants show a delayed response to lights off, suggesting that the circadian clock is modulating plant growth responses to light. A likely candidate for such gating of the light-induced inhibitory pathway is ELF3, because the elf3 mutant lacks gating of lightinduced CAB2 gene expression (McWatters et al. 2000). Time-lapse monitoring of mutants of all classes (light signalling, clock, hormone, etc.) should be helpful to understand when and how hypocotyls elongate. PHYSIOLOGICAL RELEVANCE OF CIRCADIAN CLOCK REGULATION OF GROWTH Why do plants bother to regulate their growth according to circadian or diurnal cycles? Recently, it has been shown that proper synchronization of the clock to external cycles increases photosynthetic activity, biomass, survival and competitive advantages (Dodd et al. 2005). This is also true of non-plant organisms; arrythmic strains of cyanobacteria are outcompeted by wild-type counterparts under diurnal cycles but not in constant conditions, confirming the adaptive value of circadian clocks in rhythmic environments (Woelfle et al.. Compared to the delayed response of hypocotyl growth to diurnal light changes in wild-type seedlings, arrhythmic clock mutants are immediately responsive to light signals (see previous paragraph). This observation supports the idea that the circadian clock has buffering effects against sudden unexpected changes in the environment such as alteration in light or temperature (Covington et al. 2001). The buffering action might serve to prevent rapid changes in photosynthetic or growth activity in response to transient changes in light intensity or quality, just as a surge protector protects a computer in the case of sudden changes in electrical signal. The circadian clock may also serve to anticipate regular changes in plant physiology and the environment; growth would be timed to occur when growth material is most likely to abundant. CONCLUSION It is well known that light, the circadian clock and plant hormones regulate hypocotyl elongation under continuous light or darkness. Although diurnal cycles are known to affect hypocotyl elongation, little is known about the molecular mechanisms underlying growth patterns under more natural diurnal cycles. To understand the interaction of endogenous and external factors, we need to accumulate knowledge on diurnal expression patterns including temporospatial expression changes and post-transcriptional and post-translational control of clock, light signalling, hormone, and cell architecture genes and proteins. Further information on diurnal changes in hormone levels and sensitivity are also required. An integrated model of diurnal growth control will only be developed once we have measured the factors described above while perturbing each pathway. These studies will help untangle the multiple ways in which hormones, light signalling and the circadian clock interact to control plant development in diurnal cycles. ACKNOWLEDGEMENTS We would like to thank Stacey Harmer, Michael Covington, Jennifer Nemhauser and several reviewers for critical reading and helpful comments on the manuscript. REFERENCES Alabadi D., Oyama T., Yanovsky M.J., Harmon F.G., Mas P. & Kay S.A. 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