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Measuring the effects of mutation, natural variation or treatment on the development of plant form is often complicated by the shapes, dynamics or small size of the organismal structures under study. This limits accuracy and throughput of measurement and thereby limits progress toward understanding the underlying gene networks and signaling systems. A computer-vision platform based on electronic image capture and shape-analysis algorithms was developed as an alternative to the mostly manual methods of measuring seedling development currently in use. The spatial and temporal resolution of the method is in the range of microns and minutes, respectively. The algorithm simultaneously quantifies apical hook opening and inhibition of hypocotyl elongation during photomorphogenesis of Arabidopsis thaliana seedlings. It can determine when and where gravitropic curvature develops along the root axis in A. thaliana and Medicago truncatula seedlings. Novel features of gravitropic curvature development were discovered as a result of the high resolution. The computer-vision algorithms developed and demonstrated here could be used to study mutant phenotypes in detail, to form the basis of a high-throughput screening platform, or to quantify natural variation in a population of plants.

Responses to the plant hormone ethylene are mediated by a family of five receptors in Arabidopsis that act in the absence of ethylene as negative regulators of response pathways. In this study, we examined the rapid kinetics of growth inhibition by ethylene and growth recovery after ethylene withdrawal in hypocotyls of etiolated seedlings of wild-type and ethylene receptor-deficient Arabidopsis lines. This analysis revealed that there are two phases to growth inhibition by ethylene in wild type: a rapid phase followed by a prolonged, slower phase. Full recovery of growth occurs approximately 90 min after ethylene removal. None of the receptor null mutations tested had a measurable effect on the two phases of growth inhibition. However, loss-of-function mutations in ETR1, ETR2, and EIN4 significantly prolonged the time for recovery of growth rate after ethylene was removed. Plants with an etr1-6;etr2-3;ein4-4 triple loss-of-function mutation took longer to recover than any of the single mutants, while the ers1;ers2 double mutant had no effect on recovery rate, suggesting that receiver domains play a role in recovery. Transformation of the ers1-2;etr1-7 double mutant with wild-type genomic ETR1 rescued the slow recovery phenotype, while a His kinase-inactivated ETR1 construct did not. To account for the rapid recovery from growth inhibition, a model in which clustered receptors act cooperatively is proposed.

Rapid and measurable growth rate changes that occur in seedling stems upon illumination serve as an excellent means to analyze signal transduction. Growth kinetic studies have shown how red, far-red and blue light signals are transduced via the solitary and/or coordinated action of known plant photoreceptors. These reports are consistent with current findings describing light-induced photoreceptor interaction and compartmentation.

Previous evidence has suggested that SPA1 is a signal transduction component that appears to require phytochrome A for function in seedling photomorphogenesis. Using digital image analysis, we examined the time course of growth inhibition induced by red light in spa1 mutants to test the interpretation that SPA1 functions early in a phyA-specific signaling pathway. By comparing wild-type and mutant responses, we found that SPA1 caused an increase in hypocotyl growth rate after approximately 2 h of continuous red light, whereas the onset of phyA-mediated inhibition was detected within several minutes. Thus, SPA1-dependent growth promotion began after phyA started to inhibit growth. The action of SPA1 persisted for approximately 2 d of red light, a period well beyond the time when the phyA photoreceptor and its influence on growth have both decayed to undetectable levels. Also, SPA1 promoted growth for many hours in the complete absence of a light stimulus when red-light-grown seedlings were shifted to darkness. We propose that SPA1 functions in a light-induced mechanism that promotes growth and thereby counteracts growth inhibition mediated by phyA and phyB. Our finding that spa1 seedlings do not display growth promotion in response to end-of-day pulses of far-red light, even in a phyA-null background, supports this interpretation. Combined, these results lead us to the view that the rate of hypocotyl elongation in light is determined by at least two independent, opposing processes; an inhibition of growth by the phytochromes and a promotion of growth by light-activated SPA1.

Photoreceptor proteins of the phytochrome family mediate light-induced inhibition of stem (hypocotyl) elongation during the development of photoautotrophy in seedlings. Analyses of overt mutant phenotypes have established the importance of phytochromes A and B (phyA and phyB) in this developmental process, but kinetic information that would augment emerging molecular models of phytochrome signal transduction is absent. We have addressed this deficiency by genetically dissecting phytochrome-response kinetics, after having solved the technical issues that previously limited growth studies of small Arabidopsis seedlings. We show here, with resolution on the order of minutes, that phyA initiated hypocotyl growth inhibition upon the onset of continuous red light. This primary contribution of phyA began to decrease after 3 hr of irradiation, the same time at which immunochemically detectable phyA disappeared and an exclusively phyB-dependent phase of inhibition began. The sequential and coordinated actions of phyA and phyB in red light were not observed in far-red light, which inhibited growth persistently through an exclusively phyA-mediated pathway.

High fluence-rate blue light (BL) rapidly inhibits hypocotyl growth in Arabidopsis, as in other species, after a lag time of 30 s. This growth inhibition is always preceded by the activation of anion channels. The membrane depolarization that results from the activation of anion channels by BL was only 30% of the wild-type magnitude in hy4, a mutant lacking the HY4 BL receptor. High-resolution measurements of growth made with a computer-linked displacement transducer or digitized images revealed that BL caused a rapid inhibition of growth in wild-type and hy4 seedlings. This inhibition persisted in wild-type seedlings during more than 40 h of continuous BL. By contrast, hy4 escaped from the initial inhibition after approximately 1 h of BL and grew faster than wild type for approximately 30 h. Wild-type seedlings treated with 5-nitro-2-(3-phenylpropylamino)-benzoic acid, a potent blocker of the BL-activated anion channel, displayed rapid growth inhibition, but, similar to hy4, these seedlings escaped from inhibition after approximately 1 h of BL and phenocopied the mutant for at least 2.5 h. The effects of 5-nitro-2-(3-phenylpropylamino)-benzoic acid and the HY4 mutation were not additive. Taken together, the results indicate that BL acts through HY4 to activate anion channels at the plasma membrane, causing growth inhibition that begins after approximately 1 h. Neither HY4 nor anion channels appear to participate greatly in the initial phase of inhibition.

Phytochrome A (phyA) and phytochrome B photoreceptors have distinct roles in the regulation of plant growth and development. Studies using specific photomorphogenic mutants and transgenic plants overexpressing phytochrome have supported an evolving picture in which phyA and phytochrome B are responsive to continuous far-red and red light, respectively. Photomorphogenic mutants of Arabidopsis thaliana that had been selected for their inability to respond to continuous irradiance conditions were tested for their ability to carry out red-light-induced enhancement of phototropism, which is an inductive phytochrome response. We conclude that phyA is the primary photoreceptor regulating this response and provide evidence suggesting that a common regulatory domain in the phyA polypeptide functions for both high-irradiance and inductive phytochrome responses.

The phytochrome family of photoreceptors has dual molecular functions: photosensory, involving light signal perception, and regulatory, involving signal transfer to downstream transduction components. To define residues necessary specifically for the regulatory activity of phytochrome A (phyA), we undertook a genetic screen to identify Arabidopsis mutants producing wild-type levels of biologically defective but photochemically active and dimeric phyA molecules. Of eight such mutants identified, six contain missense mutations (including three in the same residue, glycine 727) clustered within a restricted segment in the C-terminal domain of the polypeptide. Quantitative photobiological analysis revealed retention of varying degrees of partial activity among the different alleles--a result consistent with the extent of conservation at the position mutated. Together with additional data, these results indicate that the photoreceptor subdomain identified here is critical to the regulatory activity of both phyA and phyB.

The phytochrome family of photoreceptors monitors the light environment and dictates patterns of gene expression that enable the plant to optimize growth and development in accordance with prevailing conditions. The enduring challenge is to define the biochemical mechanism of phytochrome action and to dissect the signaling circuitry by which the photoreceptor molecules relay sensory information to the genes they regulate. Evidence indicates that individual phytochromes have specialized photosensory functions. The amino-terminal domain of the molecule determines this photosensory specificity, whereas a short segment in the carboxyl-terminal domain is critical for signal transfer to downstream components. Heterotrimeric GTP-binding proteins, calcium-calmodulin, cyclic guanosine 5'-phosphate, and the COP-DET-FUS class of master regulators are implicated as signaling intermediates in phototransduction.

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.

hy8 long hypocotyl mutants of Arabidopsis defective in responsiveness to prolonged far-red light (the so-called "far-red high-irradiance response") are selectively deficient in functional phytochrome A. To define the molecular lesion in these mutants, we sequenced the phytochrome A gene (phyA) in lines carrying one or other of two classes of hy8 alleles. The hy8-1 and hy8-2 mutants that express no detectable phytochrome A each have a single nucleotide change that inserts a translational stop codon in the protein coding sequence. These results establish that phyA resides at the HY8 locus. The hy8-3 mutant that expresses wild-type levels of photochemically active phytochrome A has a glycine-to-glutamate missense mutation at residue 727 in the C-terminal domain of the phyA sequence. Quantitative fluence rate response analysis showed that the mutant phytochrome A molecule produced by hy8-3 exhibited no detectable regulatory activity above that of the phyA-protein-deficient hy8-2 mutant. This result indicates that glycine-727, which is invariant in all sequenced phytochromes, has a function important to the regulatory activity of phytochrome A but not to photoperception.

Emerging evidence suggests that individual members of the phytochrome family of photoreceptors may regulate discrete facets of plant photomorphogenesis. We report here the isolation of phytochrome A mutants of Arabidopsis using a novel screening strategy aimed at detecting seedlings with long hypocotyls in prolonged far-red light. Complementation analysis of 10 selected mutant lines showed that each represents an independent, recessive allele at a new locus, designated hy8. Immunoblot and spectrophotometric analyses of two of these lines, hy8-1 and hy8-2, showed that, whereas phytochromes B and C are expressed at wild-type levels, phytochrome A is undetectable, thus indicating that the long hypocotyl phenotype displayed by these mutants is caused by phytochrome A deficiency. A third allele, hy8-3, expresses wild-type levels of spectrally normal phytochrome A, suggesting a mutation that has resulted in loss of biological activity in an otherwise photochemically active photoreceptor molecule. Together with physiological experiments, these data provide direct evidence that endogenous phytochrome A is responsible for the "far-red high irradiance response" of etiolated seedlings, but does not play a major role in mediating responses to prolonged red or white light. Because the hy8 and the phytochrome B-deficient hy3 mutants exhibit reciprocal responsivity toward prolonged red and far-red light, respectively, the evidence indicates that phytochromes A and B have distinct photosensory roles in regulating seedling development.

The hy1 and hy2 long hypocotyl mutants of Arabidopsis contain normal levels of immunochemically detectable phytochrome A, but the molecule is photochemically nonfunctional. We have investigated the biochemical basis for this lack of function. When the hy1 and hy2 mutants were grown in white light on a medium containing biliverdin IX[alpha], a direct precursor to phytochromobilin, the phytochrome chromophore, the seedlings developed with a morphological phenotype indistinguishable from the light-grown wild-type control. Restoration of a light-grown phenotype in the hy1 mutant was also accomplished by using phycocyanobilin, a tetrapyrrole analog of phytochromobilin. Spectrophotometric and immunochemical analyses of the rescued hy1 and hy2 mutants demonstrated that they possessed wild-type levels of photochemically functional phytochrome that displayed light-induced conformational changes in the holoprotein indistinguishable from the wild type. Moreover, phytochrome A levels declined in vivo in response to white light in rescued hy1 and hy2 seedlings, indicative of biliverdin-dependent formation of photochemically functional phytochrome A that was then subject to normal selective turnover in the far-red-light-absorbing form. Combined, these data suggest that the hy1 and hy2 mutants are inhibited in chromophore biosynthesis at steps prior to the formation of biliverdin IX[alpha], thus potentially causing a global functional deficiency in all members of the phytochrome photoreceptor family.

The aurea locus mutant (au (w)) of tomato contains less than 5% of the level of phytochrome in wild-type tissue as measured by in vivo difference spectroscopy. Immunoblot analysis using antibodies directed against etiolated-oat phytochrome demonstrates that crude extracts of etiolated mutant tissue are deficient in a major immunodetectable protein (116 kDa) normally present in the parent wild type. Analyses of wild-type tissue extracts strongly indicate that the 116-kDa protein is phytochrome by showing that this protein: a) is degraded more rapidly in vitro after a brief far-red irradiation than after a brief red irradiation (Vierstra RD, Quail PH, Planta 156: 158-165, 1982); b) contains a covalently bound chromophore as detected by Zn-chromophore fluorescence on nitrocellulose blots; and c) has an apparent molecular mass comparable to phytochrome from other species on size exclusion chromatography under non-denaturing conditions. The demonstration that the aurea mutant is deficient in this 116-kDa phytochrome indicates that the lack of spectrally detectable phytochrome in this mutant is the result of a lesion which affects the abundance of the phytochrome molecule as opposed to its spectral integrity.

The axial (longitudinal) red light gradient (632 nanometers) of 4 day old dark-grown maize seedlings is increased by staining the peripheral cells of the coleoptile. The magnitude of increase in the light gradient is dependent solely on the light-absorbing qualities of the stain used. Metanil yellow has no effect on the axial red-light gradient, while methylene blue causes a large increase in this light gradient. These stains did not affect growth in darkness or the sensitivity of mesocotyl elongation to red light. However, mesocotyl elongation was altered for the dark-grown seedlings stained with methylene blue when these seedlings were transplanted, covered with soil, and permitted to emerge under natural lighting conditions. These observations are consistent with the idea that there is a single perceptive site below the coleoptilar node, and suggest that this perceptive site gives the actinic light which has traveled downward through the length of the shoot from an entry point in the plant tip region.

The hy 1 and hy 2 long hypocotyl mutants of Arabidopsis thaliana contain less than 20% (the detection limit) of the phytochrome in wild-type tissue as measured by in vivo difference spectroscopy. In contrast, spectral measurements for the hy 3, hy 4, and hy 5 long hypocotyl mutants indicate that they each contain levels of phytochrome equivalent to the wild-type parent. Immunoblot analysis using a monoclonal antibody directed against the chromophore-bearing region of etiolated-oat phytochrome demonstrates that extracts of all mutant and wild-type Arabidopsis tissues, prepared by extraction of proteins into hot SDS-containing buffer, have identical levels of one major immunodetectable protein (116 kDa). An assay involving controlled in vitro proteolysis, known to produce distinctive fragmentation patterns for Pr and Pfr (Vierstra RD, Quail PH, Planta 156: 158-165, 1982), indicates that the 116 kDa polypeptide from the wild-type parent represents Arabidopsis phytochrome. The 116 kDa protein from either hy 3, hy 4, or hy 5 displays the same fragmentation pattern found for the wild type. Together with the spectral data, these results indicate that the mutant phenotype of these variants does not involve lesions in the polypeptide sequence that lead to gross conformational aberrations, and suggest that the genetic lesions may affect steps in the transduction chain downstream of the photoreceptor. In contrast, this same analysis for hy 1 and hy 2 has revealed that the 116 kDa protein from either of these mutants is not degraded differently in response to the different wavelengths of irradiation given in vitro. Moreover, whereas immunoblot analysis of tissue extracts from light-grown wild-type seedlings show that the 116 kDa phytochrome protein level is greatly reduced relative to dark-grown tissue as expected, similar extracts of light-grown hy 1 and hy 2 seedlings contain the 116 kDa polypeptide in amounts equivalent to those of dark-grown tissue. Combined, these data indicate that the hy 1 and hy 2 mutants both produce normal levels of immunochemically detectable phytochrome that is photochemically nonfunctional.