Genetic and molecular analysis of light-regulated plant development

Light regulates many physiological and developmental events in plants through the action of multiple sensory pigment systems. Although our understanding of the regulatory photoreceptors, including phytochromes (that principally absorb red and far-red energy) and blue light receptors, has advanced considerably in the recent past, the mechanisms of light signal transduction in higher plants are poorly understood. To unravel the molecular events associated with light-regulated plant development, a large number of photomorphogenic mutants have been isolated in several different plant species, including Arabidopsis, cucumber, tomato, pea, Brassica and Sorghum, which are either impaired in normal perception of light signal (photoreceptor mutants) or are affected in some specific or a sub-set of phenotypic traits (signal transduction mutants). Their physiological and molecular analysis is proving to be valuable in (1) assigning specific function to discrete phytochrome species, (2) elucidation of elements that constitute the transduction pathway downstream of signal perception, and (3) determining how different photosensory systems regulate many diverse responses. The progress made in the analysis of photomorphogenic mutants, as reviewed in this article, clearly indicates that multiple photoreceptors, either of the same or different class, interact through an intricate network of signal transduction pathways to finally determine the light-dependent phenotype of both monocots and dicots.     

Photomorphogenic mutants of Arabidopsis thaliana reveal activities of multiple photosensory systems during light-stimulated apical-hook opening

Fluence rate-response curves were generated for red-, far-red-, and blue-light-stimulated apical-hook opening in seedlings of several photomorphogenic mutants of Arabidopsis thaliana (L.) Heynh. Compared to wild-type plants, hook opening was reduced in the phytochrome-deficient hy1, hy2, and hy6 mutants in red and far-red light at all fluence rates tested, and in low-fluence blue light, but was normal under high-irradiance blue light. In contrast, the blue-light-response mutants (blu1, blu2, and blu3) lacked the high-irradiance-dependent hook-opening response in blue light while hook opening was normal in low-fluence blue light and in red and farred light at all fluence rates tested. Hook opening in the phytochrome-B-deficient hy3 mutant was similar to wild type in all light conditions tested. The effects of the different mutations on light-induced hook opening indicate that a phytochrome(s) other than phytochrome B mediates hook opening stimulated by red, far-red and lowfluence blue light, while a blue-light-absorbing photoreceptor mediates the blue-light-sensitive high-irradiance response. Although the phytochrome and blue-light photosensory systems appear to work independently for the most part, some of their signal-transduction components may interact since the hy4, and hy5 mutants showed reduced hook-opening responses under conditions dependent on the phytochrome and blue-light-photosensory systems.We thank Jeff Young and Brian Parks for their many helpful suggestions during the progress of this research. This work was supported by National Science Foundation Grant No. DCB-9106697.     

Blue light sensory systems in plants

Plant development is influenced by many environmental stimuli, including light, temperature and gravity. Of these stimuli, light is of particular importance because plants depend on it for energy and, thus, for survival. Moreover, virtually all stages of plant development are regulated in part by light through the action of various photosensory systems. Examples of light-regulated processes include germination, stem growth, leaf and root development, tropic responses and flower induction. This review provides an analysis of recent investigations of blue light sensory systems in plants. Current results suggest that plants respond to blue light through a complex photosensory network that incorporates the action of multiple blue light perception systems.     

Photosensory Perception and Signal Transduction in Higher Plants — Molecular Genetic Analysis

Light plays a crucial role throughout the life cycle of higher plants modulating various aspects of their growth and development, such as seed germination, leaf differentiation, flowering, and senescence. Plants have thus evolved extremely sensitive mechanisms to continually detect the changing ambient light conditions and transduce the information to the gene expression machinery. The elucidation of this complex information sensing and transduction machinery is fundamental to our understanding of the molecular mechanisms involved in light-regulated plant development. The last decade has witnessed an immense upsurge in information in this regard and the mechanism of photosensory perception and phototransduction is turning out to be quite intricate, involving an array of cellular effectors and biochemical messengers. The analysis of photomorphogenic mutants, predominantly of Arabidopsis, has revealed interesting facts, not only about the intricacies of light signaling circuitry, but also about the multiplicity of the photoreceptors and their specialized or overlapping photosensory functions. In addition, these studies have also highlighted, and in some cases even redefined, the role of conventional plant growth regulators in modulating photomorphogenic development. Employing standard recombinant DNA techniques, substantial information has also become available about the regulatory cis-acting DNA sequences that make a gene amenable to light control and the trans-acting protein factors that can potentially interact with these cis-acting sequences on receiving the signal from the upstream transduction components. The information available to date on these emerging trends in photomorphogenesis research has been summarized and critically evaluated in this review.     

The C-terminal kinase fragment of Arabidopsis phototropin 2 triggers constitutive phototropin responses

Phototropins mediate various blue-light responses such as phototropism, chloroplast relocation, stomatal opening and leaf flattening in plants. Phototropins are hydrophilic chromoproteins that are mainly bound to the plasma membrane. One of two phototropins in Arabidopsis thaliana, phot2, associates with the Golgi apparatus in a light-dependent manner. In this study, we analyzed the biological activities of the N-terminal photosensory and C-terminal kinase domains of phot2. For this purpose, these domains were fused to green fluorescent protein (GFP) and ectopically expressed in the wild-type and a phot1 phot2 double mutant of Arabidopsis. The kinase domain fused to GFP (P2CG) was localized to the plasma membrane and the Golgi apparatus, whereas the photosensory domain fused to GFP (P2NG) was uniformly localized in the cytosol. Hence, the kinase domain rather than the photosensory domain is responsible for the membrane association. Interestingly, the P2CG plants exhibited constitutive blue-light responses even in dark conditions, i.e. stomata were open and chloroplasts were in the avoidance position. By contrast, P2CG with a mutation that abolishes the kinase activity (P2C[D720/N]G) failed to exhibit these responses. phot2 kinase is therefore suggested to be correctly localized to functional sites in the cell and to trigger light signal transduction through its kinase activity. In contrast to P2CG, P2NG did not affect the phot2 responses, except for partial inhibition of the phototropic response caused by the endogenous phototropins.     

Arabidopsis Mutants Lacking Blue Light-Dependent Inhibition of Hypocotyl Elongation

We have isolated a new class of photomorphogenic mutants in Arabidopsis. Hypocotyl elongation is not inhibited in the mutant seedlings by continuous blue light but is inhibited by far red light, indicating that these mutations are phenotypically different from the previously isolated long hypocotyl (hy) mutants. Complementation analysis indicated that recessive nuclear mutations at three genetic loci, designated blu1, blu2, and blu3, can result in the blu mutant phenotype and that these mutants are genetically distinct from other long hypocotyl mutants. The BLU genes appear to be important only during seedling development because the blu mutations have little effect on mature plants, whereas hypocotyl elongation and cotyledon expansion are altered in seedlings. The genetic separation of the blue and far red sensitivities of light-induced hypocotyl inhibition in the blu and hy mutants demonstrates that two photosensory systems function in this response.     

植物蓝光反应突变体分子生物学研究

植物具备一套复杂的由3种蓝光受体和多种信号转导下游组分组成的蓝光感应系统,通过感受光照强度、光的方向和光周期,调节自身对蓝光的应答。本文综述了植物蓝光反应突变体分子生物学研究进展,探讨蓝光受体及信号转导下游组分在植物发育中的作用及蓝光诱发植物作出反应的分子机制。     

Interactions of light and ethylene in hypocotyl hook maintenance in Arabidopsis thaliana seedlings

Etiolated seedlings frequently display a hypocotyl or epicotyl hook which opens on exposure to light. Ethylene has been shown to be necessary for maintenance of the hook in a number of plants in darkness. We investigated the interaction of ethylene and light in the regulation of hypocotyl hook opening in Arabidopsis thaliana . We found that hooks of Arabidopsis open in response to continuous red, far-red or blue light in the presence of up to 100 μl l⁻¹ ethylene. Thus a change in sensitivity to ethylene is likely to be responsible for hook opening in Arabidopsis, rather than a decrease in ethylene production in hook tissues. We used photomorphogenic mutants of Arabidopsis to demonstrate the involvement of both blue light and phytochrome photosensory systems in light-induced hook opening in the presence of ethylene. In addition we used ethylene mutants and inhibitors of ethylene action to investigate the role of ethylene in hook maintenance in seedlings grown in light and darkness.     

Functional and signaling mechanism analysis of rice CRYPTOCHROME 1

Cryptochromes (CRY) are blue-light photoreceptors that mediate various light responses, such as inhibition of hypocotyl elongation, enhancement of cotyledon expansion, anthocyanin accumulation and stomatal opening in Arabidopsis. The signaling mechanism of Arabidopsis CRY is mediated through direct interaction with COP1, a negative regulator of photomorphogenesis. CRY has now been characterized in tomato, pea, moss and fern, but its function in monocots is largely unknown. Here we report the function and basic signaling mechanism of rice cryptochrome 1 (OsCRY1). Overexpresion of OsCRY1b resulted in a blue light-dependent short hypcotyl phenotype in Arabidopsis, and a short coleoptile, leaf sheath and leaf blade phenotype in rice (Oryza sativa). On fusion with beta-glucuronidase (GUS), the C-terminal domain of either OsCRY1a (OsCCT1a) or OsCRY1b (OsCCT1b) mediated a constitutive photomorphogenic (COP) phenotype in both Arabidopsis and rice, whereas OsCCT1b mutants corresponding to missense mutations in previously described Arabidopsis cry1 alleles failed to confer a COP phenotype. Yeast two-hybrid and subcellular co-localization studies demonstrated that OsCRY1b interacted physically with rice COP1 (OsCOP1). From these results, we conclude that OsCRY1 is implicated in blue-light inhibition of coleoptile and leaf elongation during early seedling development in rice, and that the signaling mechanism of OsCRY1 involves direct interaction with OsCOP1.     

Distinct white collar-1 genes control specific light responses in Mucor circinelloides

Light regulates many developmental and physiological processes in a large number of organisms. The best-known light response in the fungus Mucor circinelloides is the biosynthesis of beta-carotene. Here, we show that M. circinelloides sporangiophores also respond to light, exhibiting a positive phototropism. Analysis of both responses to different light wavelengths within the visible spectrum demonstrated that phototropism is induced by green and blue light, whereas carotenogenesis is only induced by blue light. The blue regulation of both responses suggests the existence of blue-light photoreceptors in M. circinelloides. Three white collar-1 genes (mcwc-1a, mcwc-1b and mcwc-1c) coding for proteins showing similarity with the WC-1 photoreceptor of Neurospora crassa have been identified. All three contain a LOV (light, oxygen or voltage) domain, similar to that present in fungal and plant blue-light receptors. When knockout mutants for each mcwc-1 gene were generated to characterize gene functions, only mcwc-1c mutants were defective in light induction of carotene biosynthesis, indicating that mcwc-1c is involved in the light transduction pathway that control carotenogenesis. We have also shown that positive phototropism is controlled by the mcwc-1a gene. It seems therefore that mcwc-1a and mcwc-1c genes control different light transduction pathways, although cross-talk between both pathways probably exists because mcwc-1a is involved in the light regulation of mcwc-1c expression.     

Light-induced absorbance changes in Phycomyces: evidence for cryptochrome-associated flavosemiquinones

Light-induced absorbance changes (LIACs), which are associated with early photochemical events of blue-light transduction, were detected in growing zones of Phycomyces sporangiophores. The novel LIACs meet all the essential requirements for a spectrophotometric photoreceptor assay which was previously unavailaible for blue-light receptors (cryptochromes). In-vivo absorption spectra of growing zones were derived from reflection spectra which were measured with a novel rapid-scan spectrophotometer. To detect photoreceptor-associated absorbance changes white mutants were employed which lack the interfering bulk pigment β-carotene. Blue and white light, not however red light, induced in these strains absorbance changes near 460–490 and 600–620 nm. The LIACs were absent in light-insensitive mutants with defects in the genes madA, madB and madC. Because these genes affect photosensory adaptation and the blue-light receptor itself, the novel in-vivo LIACs must be associated with photochemical events which occur early in the transduction chain. The spectral characteristics of the LIACs are in accordance with a blue- and red-light absorbing flavosemiquinone which is generated upon light absorption by an oxidized flavin receptor. It is proposed that the flavosemiquinone functions itself as photoreceptor which mediates several red-light responses of Phycomyces. Received: 28 September 1998 / Accepted: 25 November 1998     

Light-Stimulated Apical Hook Opening in Wild-Type Arabidopsis thaliana Seedlings

Apical hook opening and cotyledon unfolding are characteristic responses that occur during deetiolation of dicotyledonous seedlings. Light-stimulated apical hook opening and cotyledon unfolding in etiolated Arabidopsis thaliana seedlings appears to involve the activities of multiple photosensory systems. Red, far-red, and blue light are all effective in stimulating these responses in Arabidopsis. Stimulation of hook opening by red light and low fluence blue light is inductive, far-red reversible, and exhibits reciprocity, as is characteristic of many low fluence-dependent phytochrome-mediated responses. Far-red and high-fluence blue light appear to stimulate hook opening and cotyledon unfolding through high-irradiance-response systems during long-term light treatments. Although a phytochrome high-irradiance-response system presumably mediates the responses in far-red light, the responses to high-fluence blue light may be mediated by a blue light-specific photosensory system.     

Acclimation of Arabidopsis thaliana to the light environment: regulation of chloroplast composition

The regulation by light of the composition of the photosynthetic apparatus was investigated in Arabidopsis thaliana (L.) Heynh. cv. Landsberg erecta. When grown in high- and low-irradiance white light, wild-type plants and photomorphogenic mutants showed large differences in their maximum photosynthetic rate and chlorophyll a/b ratios; such changes were abolished by growth in red light. Photosystem I (PSI) and PSII levels were measured in wild-type plants grown under a range of light environments; the results indicate that regulation of photosystem stoichiometry involves the specific detection of blue light. Supplementing red growth lights with low levels of blue light led to large increases in PSII content, while further increases in blue irradiance had the opposite effect; this latter response was abolished by the hy4 mutation, which affects certain events controlled by a blue-light receptor. Mutants defective in the phytochrome photoreceptors retained regulation of photosystem stoichiometry. We discuss the results in terms of two separate responses controlled by blue-light receptors: a blue-high-fluence response which controls photosystem stoichiometry; and a blue-low-fluence response necessary for activation of such control. Variation in the irradiance of the red growth light revealed that the blue-high-fluence response is attenuated by red light; this may be evidence that photosystem stoichiometry is controlled not only by photoreceptors, but also by photosynthetic metabolism.Abbreviations BHF blue-high-fluence - BLF blue-low-fluence - Chl chlorophyll - FR far-red light - LHCII light-harvesting complex of PSII - P_max maximum photosynthetic rate - R red light - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase This work was supported by Natural Environment Research Council Grant No. GR3/7571A. We would like to thank H. Smith (Botany Department, University of Leicester) and E. Murchie (INRA, Versailles) for helpful discussions.     

Bacterial bilin- and flavin-binding photoreceptors

The growing number of sequenced prokaryotic genomes reveals a wide distribution of open reading frames (ORFs) that putatively encode for red- and blue light sensing photoreceptors. They comprise the bilin-binding phytochromes and the flavin-binding cryptochromes, LOV and BLUF proteins, indicating that about 1/4 of bacteria do possess at least one of these photosensory proteins. The distribution of red- and blue-light sensors among different prokaryotic phyla and classes, and their functional activity as light-switched systems are the subject of this perspective. These photoreceptors were originally found in plants by following the associated physiological responses induced by the respective spectral irradiation. Genome-based approaches now require the assignment of a photochemical/physiological function to the heterologously expressed gene product. Database searches demonstrate in some cases several genes of one category in a certain prokaryot, indicating the presence of more than one type of red- or blue-light sensing properties, but also show a combination of proteins with both spectral sensitivities. Another interesting feature now "comes into light": according to their nature as biological sensors, these photoreceptors are equipped with signalling domains, initiating a cellular response, thereby constituting modular systems switchable by light. It is seen that many of these signalling domains, now found together with light-inducible sensing domains, were already described for other stimuli, e.g., osmo-regulation, oxygen, hydrogen, chemicals, or pH. In some cases, the same type of signalling domain can be found in a red- or a blue-light sensing photoreceptor. Following the characterization of their photochemistry, for several of these bacterial photoreceptors physiological functions are now assigned.     

A complement of ten essential and pleiotropic arabidopsis COP/DET/FUS genes is necessary for repression of photomorphogenesis in darkness.

Two genetic screens, one for mutations resulting in photomorphogenic development in darkness and the other for mutants with fusca phenotype, have thus far identified six pleiotropic Arabidopsis COP/DET/FUS genes. Here, we characterized representative mutants that define four additional pleiotropic photomorphogenic loci and a null mutant allele of the previously defined DET1 locus. Dark-grown seedlings homozygous for these recessive mutations exhibit short hypocotyls and expanded cotyledons and are lethal before reaching reproductive development. Dark-grown mutant seedlings also display characteristic photomorphogenic cellular differentiation and elevated expression of light-inducible genes. In addition, analyses of plastids from dark-grown mutants reveal partial chloroplast differentiation and absence of etioplast development. Root vascular bundle cells of light-grown mutant seedlings develop chloroplasts, suggesting that these FUS gene products are important for suppression of chloroplast differentiation in light-grown roots. Double-mutant analyses indicate that these pleiotropic cop/det/fus mutations are epistatic to mutations in phytochromes, a blue-light photoreceptor, and a downstream regulatory component, HY5. Therefore, there is a complement of at least 10 essential and pleiotropic Arabidopsis genes that are necessary for repression of photomorphogenic development.     

Keeping up with the neighbours: phytochrome sensing and other signalling mechanisms

Plants ‘forage’ for light in plant canopies using a variety of photosensory systems. Far-red radiation (FR) reflected by neighbours is an early signal of competition that elicits anticipatory shade-avoidance responses. In Arabidopsis and cucumber, perception of reflected FR requires phytochrome B. Horizontal blue (B) light gradients also guide plant shoots to canopy gaps in patchy vegetation, and these B light signals are perceived by specific photoreceptors. When plants are shaded by neighbours they undergo extensive reprogramming of their morphological development. Although phytochromes and B light receptors are certainly involved in these responses to shading, other sensory systems probably play important roles in the field. Recent studies of plant–plant signalling are unveiling a paradigm of sensory diversity and sophistication, which has important implications for understanding the functioning of plant populations and communities.     

CotB is essential for complete activation of green light-induced genes during complementary chromatic adaptation in Fremyella diplosiphon

The dramatic modifications of photosynthetic light harvesting antennae called phycobilisomes that occur during complementary chromatic adaptation in cyanobacteria are controlled by two separate photosensory systems. The first system involves the signal transduction components RcaE, RcaF and RcaC, which appear to make up a complex multistep phosphorelay. This system controls the light responsive expression of the cpcB2A2H2I2D2, cpeBA and cpeCDE operons, which encode phycobilisome proteins. The second system, which is not yet characterized, acts in concert with the first but only regulates the light responses of cpeBA and cpeCDE. We have generated and characterized a new mutant class, named the Tan mutants. In at least one member of this class, light-regulated RNA accumulation patterns are altered for cpeBA and cpeCDE, but not for cpcB2A2H2I2D2. Thus this mutant contains a lesion that may impair the operation of the second system. We demonstrate that several Tan mutants are the result of improper expression of the gene cotB. CotB has limited similarity to lyase class proteins, particularly those related to NblB, which is required for degradation of phycobilisomes in other cyanobacteria. Possible roles of CotB in the biogenesis of phycobilisomes are discussed.     

Phytochrome research in whole plant physiology. Wild-type seedlings versus mutants and transgenic plants

In this report I comment on a small selection of the many interesting papers presented at the European Symposium on Photomorphogenesis in Plants. My major concern arises from problems relating to classical phytochrome treatments versus mutants and transgenic plants as tools in photomorphogenic research on whole plant physiology. Plant responses to visible light are especially evident in etiolated seedlings, although they occur throughout the life cycle of the plant. These characteristics of plant photomorphogenesis in nature can be viewed as representing a continuous developmental series of graded responses to subsequent light exposures. Where the individual, photoautotrophic seedling is moving through this developmental gradient depends on its particular light environment, that is, on both the quality and the quantity of light to which the seedling is exposed. On the contrary, phytochrome treatment of mutants and transgenic seedlings having either deficiency or overexpression of photoreceptors will result in an aberrant kind of physiology.