iTRAQ-based proteome profiling revealed the role of Phytochrome A in regulating primary metabolism in tomato seedling.

In plants, during growth and development, photoreceptors monitor fluctuations in their environment and adjust their metabolism as a strategy of surveillance. Phytochromes (Phys) play an essential role in plant growth and development, from germination to fruit development. FR-light (FR) insensitive mutant (fri) carries a recessive mutation in Phytochrome A and is characterized by the failure to de-etiolate in continuous FR. Here we used iTRAQ-based quantitative proteomics along with metabolomics to unravel the role of Phytochrome A in regulating central metabolism in tomato seedlings grown under FR. Our results indicate that Phytochrome A has a predominant role in FR-mediated establishment of the mature seedling proteome. Further, we observed temporal regulation in the expression of several of the late response proteins associated with central metabolism. The proteomics investigations identified a decreased abundance of enzymes involved in photosynthesis and carbon fixation in the mutant. Profound accumulation of storage proteins in the mutant ascertained the possible conversion of sugars into storage material instead of being used or the retention of an earlier profile associated with the mature embryo. The enhanced accumulation of organic sugars in the seedlings indicates the absence of photomorphogenesis in the mutant.

Phytochrome A inhibits shade avoidance responses under strong shade through repressing the brassinosteroid pathway in Arabidopsis.

In dense canopy, a reduction in red to far-red (R/FR) light ratio triggers shade avoidance responses (SARs) in Arabidopsis thaliana, a shade avoiding plant. Two red/far-red (R/FR) light photoreceptors, PHYB and PHYA, were reported to be key negative regulators of the SARs. PHYB represses the SARs under normal light conditions; however, the role of PHYA in the SARs remains elusive. We set up two shade conditions: Shade and strong Shade (s-Shade) with different R/FR ratios (0.7 and 0.1), which allowed us to observe phenotypes dominated by PHYB- and PHYA-mediated pathway, respectively. By comparing the hypocotyl growth under these two conditions with time, we found PHYA was predominantly activated in the s-Shade after prolonged shade treatment. We further showed that under s-Shade, PHYA inhibits hypocotyl elongation partially through repressing the brassinosteroid (BR) pathway. COP1 and PIF4,5 act downstream of PHYA. After prolonged shade treatment, the nuclear localization of COP1 was reduced, while the PIF4 protein level was much lower in the s-Shade than that in Shade. Both changes occurred in a PHYA-dependent manner. We propose that under deep canopy, the R/FR ratio is extremely low, which promotes the nuclear accumulation of PHYA. Activated PHYA reduces COP1 nuclear speckle, which may lead to changes of downstream targets, such as PIF4,5 and HY5. Together, these proteins regulate the BR pathway through modulating BES1/BZR1 and the expression of BR biosynthesis and BR target genes.

Light controls stamen elongation via cryptochromes, phytochromes and COP1 through HY5 and HYH.

In Arabidopsis, stamen elongation, which ensures male fertility, is controlled by the auxin response factor ARF8, which regulates the expression of the auxin repressor IAA19. Here, we uncover a role for light in controlling stamen elongation. By an extensive genetic and molecular analysis we show that the repressor of light signaling COP1, through its targets HY5 and HYH, controls stamen elongation, and that HY5 - oppositely to ARF8 - directly represses the expression of IAA19 in stamens. In addition, we show that in closed flower buds, when light is shielded by sepals and petals, the blue light receptors CRY1/CRY2 repress stamen elongation. Coherently, at flower disclosure and in subsequent stages, stamen elongation is repressed by the red and far-red light receptors PHYA/PHYB. In conclusion, different light qualities - sequentially perceived by specific photoreceptors - and the downstream COP1-HY5/HYH module finely tune auxin-induced stamen elongation and thus male fertility.

Phytochrome A and B Negatively Regulate Salt Stress Tolerance of Nicotiana tobacum via ABA-Jasmonic Acid Synergistic Cross-Talk.

Light signaling and phytohormones play important roles in plant growth, development, and biotic and abiotic stress responses. However, the roles of phytochromes and cross-talk between these two signaling pathways in response to salt stress in tobacco plants remain underexplored. Here, we explored the defense response in phytochrome-defective mutants under salt stress. We monitored the physiological and molecular changes of these mutants under salt stress conditions. The results showed that phytochrome A (phyA), phytochrome B (phyB) and phyAphyB (phyAB) mutants exhibited improved salt stress tolerance compared with wild-type (WT) plants. The mutant plants had a lower electrolyte leakage (EL) and malondialdehyde (MDA) concentration than WT plants, and the effect was clearly synergistic in the phyAB double mutant plants. Furthermore, the data showed that the transcript levels of defense-associated genes and the activities of some antioxidant enzymes in the mutant plants were much higher than those in WT plants. Additionally, the results indicated that phytochrome signaling strongly modulates the expression of endogenous abscisic acid (ABA) and jasmonic acid (JA) of Nicotiana tobacum in response to salt stress. To illustrate further the relationship between phytochrome and phytohormone, we measured the expression of defense genes and phytochrome. The results displayed that salt stress and application of methyl jasmonate (MeJA) or ABA up-regulated the transcript levels of salt response-associated genes and inhibited the expression of NtphyA and NtphyB. Foliar application of inhibitors of ABA and JA further confirmed that JA co-operated with ABA in phytochrome-mediated salt stress tolerance.

Root-expressed phytochromes B1 and B2, but not PhyA and Cry2, regulate shoot growth in nature.

Although photoreceptors are expressed throughout all plant organs, most studies have focused on their function in aerial parts with laboratory-grown plants. Photoreceptor function in naturally dark-grown roots of plants in their native habitats is lacking. We characterized patterns of photoreceptor expression in field- and glasshouse-grown Nicotiana attenuata plants, silenced the expression of PhyB1/B2/A/Cry2 whose root transcripts levels were greater/equal to those of shoots, and by micrografting combined empty vector transformed shoots onto photoreceptor-silenced roots, creating chimeric plants with "blind" roots but "sighted" shoots. Micrografting procedure was robust in both field and glasshouse, as demonstrated by transcript accumulation patterns, and a spatially-explicit lignin visual reporter chimeric line. Field- and glasshouse-grown plants with PhyB1B2, but not PhyA or Cry2, -blind roots, were delayed in stalk elongation compared with control plants, robustly for two field seasons. Wild-type plants with roots directly exposed to FR phenocopied the growth of irPhyB1B2-blind root grafts. Additionally, root-expressed PhyB1B2 was required to activate the positive photomorphogenic regulator, HY5, in response to aboveground light. We conclude that roots of plants growing deep into the soil in nature sense aboveground light, and possibly soil temperature, via PhyB1B2 to control key traits, such as stalk elongation.

Evidence that phytochrome functions as a protein kinase in plant light signalling.

It has been suggested that plant phytochromes are autophosphorylating serine/threonine kinases. However, the biochemical properties and functional roles of putative phytochrome kinase activity in plant light signalling are largely unknown. Here, we describe the biochemical and functional characterization of Avena sativa phytochrome A (AsphyA) as a potential protein kinase. We provide evidence that phytochrome-interacting factors (PIFs) are phosphorylated by phytochromes in vitro. Domain mapping of AsphyA shows that the photosensory core region consisting of PAS-GAF-PHY domains in the N-terminal is required for the observed kinase activity. Moreover, we demonstrate that transgenic plants expressing mutant versions of AsphyA, which display reduced activity in in vitro kinase assays, show hyposensitive responses to far-red light. Further analysis reveals that far-red light-induced phosphorylation and degradation of PIF3 are significantly reduced in these transgenic plants. Collectively, these results suggest a positive relationship between phytochrome kinase activity and photoresponses in plants.

Lysine 206 in Arabidopsis phytochrome A is the major site for ubiquitin-dependent protein degradation.

Phytochrome A (phyA) is a light labile phytochrome that mediates plant development under red/far-red light condition. Degradation of phyA is initiated by red light-induced phyA-ubiquitin conjugation through the 26S proteasome pathway. The N-terminal of phyA is known to be important in phyA degradation. To determine the specific lysine residues in the N-terminal domain of phyA involved in light-induced ubiquitination and protein degradation, we aligned the amino acid sequence of the N-terminal domain of Arabidopsis phyA with those of phyA from other plant species. Based on the alignment results, phytochrome over-expressing Arabidopsis plants were generated. In particular, wild-type and mutant (substitutions of conserved lysines by arginines) phytochromes fused with GFP were expressed in phyA(-)211 Arabidopsis plants. Degradation kinetics of over-expressed phyA proteins revealed that degradation of the K206R phyA mutant protein was delayed. Delayed phyA degradation of the K206R phyA mutant protein resulted in reduction of red-light-induced phyA-ubiquitin conjugation. Furthermore, seedlings expressing the K206R phyA mutant protein showed an enhanced phyA response under far-red light, resulting in inhibition of hypocotyl elongation as well as cotyledon opening. Together, these results suggest that lysine 206 is the main lysine for rapid ubiquitination and protein degradation of Arabidopsis phytochrome A.

Phytochrome RNAi enhances major fibre quality and agronomic traits of the cotton Gossypium hirsutum L.

Simultaneous improvement of fibre quality, early-flowering, early-maturity and productivity in Upland cotton (G. hirsutum) is a challenging task for conventional breeding. The influence of red/far-red light ratio on the fibre length prompted us to examine the phenotypic effects of RNA interference (RNAi) of the cotton PHYA1 gene. Here we show a suppression of up to ~70% for the PHYA1 transcript, and compensatory overexpression of up to ~20-fold in the remaining phytochromes in somatically regenerated PHYA1 RNAi cotton plants. Two independent transformants of three generations exhibited vigorous root and vegetative growth, early-flowering, significantly improved upper half mean fibre length and an improvement in other major fibre characteristics. Small decreases in lint traits were observed but seed cotton yield was increased an average 10-17% compared with controls. RNAi-associated phenotypes were heritable and transferable via sexual hybridization. These results should aid in the development of early-maturing and productive Upland cultivars with superior fibre quality.

Overexpression of phytochrome A and its hyperactive mutant improves shade tolerance and turf quality in creeping bentgrass and zoysiagrass.

Phytochrome A (phyA) in higher plants is known to function as a far-red/shade light-sensing photoreceptor in suppressing shade avoidance responses (SARs) to shade stress. In this paper, the Avena PHYA gene was introduced into creeping bentgrass (Agrostis stolonifera L.) and zoysiagrass (Zoysia japonica Steud.) to improve turf quality by suppressing the SARs. In addition to wild-type PHYA, a hyperactive mutant gene (S599A-PHYA), in which a phosphorylation site involved in light-signal attenuation was removed, was also transformed into the turfgrasses. Phenotypic traits of the transgenic plants were compared to assess the suppression of SARs under a simulated shade condition and outdoor field conditions after three growth seasons. Under the shade condition, the S599A-PhyA transgenic creeping bentgrass plants showed shade avoidance-suppressing phenotypes with a 45 % shorter leaf lengths, 24 % shorter internode lengths, and twofold increases in chlorophyll concentrations when compared with control plants. Transgenic zoysiagrass plants overexpressing S599A-PHYA also showed shade-tolerant phenotypes under the shade condition with reductions in leaf length (15 %), internode length (30 %), leaf length/width ratio (19 %) and leaf area (22 %), as well as increases in chlorophyll contents (19 %) and runner lengths (30 %) compared to control plants. The phenotypes of transgenic zoysiagrass were also investigated in dense field habitats, and the transgenic turfgrass exhibited shade-tolerant phenotypes similar to those observed under laboratory shade conditions. Therefore, the present study suggests that the hyperactive phyA is effective for the development of shade-tolerant plants, and that the shade tolerance nature is sustained under field conditions.

Nuclear phytochrome A signaling promotes phototropism in Arabidopsis.

Phototropin photoreceptors (phot1 and phot2 in Arabidopsis thaliana) enable responses to directional light cues (e.g., positive phototropism in the hypocotyl). In Arabidopsis, phot1 is essential for phototropism in response to low light, a response that is also modulated by phytochrome A (phyA), representing a classical example of photoreceptor coaction. The molecular mechanisms underlying promotion of phototropism by phyA remain unclear. Most phyA responses require nuclear accumulation of the photoreceptor, but interestingly, it has been proposed that cytosolic phyA promotes phototropism. By comparing the kinetics of phototropism in seedlings with different subcellular localizations of phyA, we show that nuclear phyA accelerates the phototropic response, whereas in the fhy1 fhl mutant, in which phyA remains in the cytosol, phototropic bending is slower than in the wild type. Consistent with this data, we find that transcription factors needed for full phyA responses are needed for normal phototropism. Moreover, we show that phyA is the primary photoreceptor promoting the expression of phototropism regulators in low light (e.g., PHYTOCHROME KINASE SUBSTRATE1 [PKS1] and ROOT PHOTO TROPISM2 [RPT2]). Although phyA remains cytosolic in fhy1 fhl, induction of PKS1 and RPT2 expression still occurs in fhy1 fhl, indicating that a low level of nuclear phyA signaling is still present in fhy1 fhl.

GRAS proteins: the versatile roles of intrinsically disordered proteins in plant signalling.

IDPs (intrinsically disordered proteins) are highly abundant in eukaryotic proteomes and important for cellular functions, especially in cell signalling and transcriptional regulation. An IDR (intrinsically disordered region) within an IDP often undergoes disorder-to-order transitions upon binding to various partners, allowing an IDP to recognize and bind different partners at various binding interfaces. Plant-specific GRAS proteins play critical and diverse roles in plant development and signalling, and act as integrators of signals from multiple plant growth regulatory and environmental inputs. Possessing an intrinsically disordered N-terminal domain, the GRAS proteins constitute the first functionally required unfoldome from the plant kingdom. Furthermore, the N-terminal domains of GRAS proteins contain MoRFs (molecular recognition features), short interaction-prone segments that are located within IDRs and are able to recognize their interacting partners by undergoing disorder-to-order transitions upon binding to these specific partners. These MoRFs represent potential protein-protein binding sites and may be acting as molecular bait in recognition events during plant development. Intrinsic disorder provides GRAS proteins with a degree of binding plasticity that may be linked to their functional versatility. As an overview of structure-function relationships for GRAS proteins, the present review covers the main biological functions of the GRAS family, the IDRs within these proteins and their implications for understanding mode-of-action.

Phytochromes inhibit hypocotyl negative gravitropism by regulating the development of endodermal amyloplasts through phytochrome-interacting factors.

Phytochromes are red and far-red light photoreceptors that regulate various aspects of plant development. One of the less-understood roles of phytochromes is the inhibition of hypocotyl negative gravitropism, which refers to the loss of hypocotyl gravitropism and resulting random growth direction in red or far-red light. This light response allows seedlings to curve toward blue light after emergence from the soil and enhances seedling establishment in the presence of mulch. Phytochromes inhibit hypocotyl negative gravitropism by inhibiting four phytochrome-interacting factors (PIF1, PIF3, PIF4, PIF5), as shown by hypocotyl agravitropism of dark-grown pif1 pif3 pif4 pif5 quadruple mutants. We show that phytochromes inhibit negative gravitropism by converting starch-filled gravity-sensing endodermal amyloplasts to other plastids with chloroplastic or etioplastic features in red or far-red light, whereas PIFs promote negative gravitropism by inhibiting the conversion of endodermal amyloplasts to etioplasts in the dark. By analyzing transgenic plants expressing PIF1 with an endodermis-specific SCARECROW promoter, we further show that endodermal PIF1 is sufficient to inhibit the conversion of endodermal amyloplasts to etioplasts and hypocotyl negative gravitropism of the pif quadruple mutant in the dark. Although the functions of phytochromes in gravitropism and chloroplast development are normally considered distinct, our results indicate that these two functions are closely related.

Functional characterization of phytochrome autophosphorylation in plant light signaling.

Plant phytochromes, molecular light switches that regulate various aspects of plant growth and development, are phosphoproteins that are also known to be autophosphorylating serine/threonine kinases. Although a few protein phosphatases that directly interact with and dephosphorylate phytochromes have been identified, no protein kinase that acts on phytochromes has been reported thus far, and the exact site of phytochrome autophosphorylation has not been identified. In this study, we investigated the functional role of phytochrome autophosphorylation. We first mapped precisely the autophosphorylation sites of oat phytochrome A (phyA), and identified Ser8 and Ser18 in the 65 amino acid N-terminal extension (NTE) region as being the autophosphorylation sites. The in vivo functional roles of phytochrome autophosphorylation were examined by introducing autophosphorylation site mutants into phyA-deficient Arabidopsis thaliana. We found that all the transgenic plants expressing the autophosphorylation site mutants exhibited hypersensitive light responses, indicating an increase in phyA activity. Further analysis showed that these phyA mutant proteins were degraded at a significantly slower rate than wild-type phyA under light conditions, which suggests that the increased phyA activity of the mutants is related to their increased protein stability. In addition, protoplast transfection analyses with green fluorescent protein (GFP)-fused phyA constructs showed that the autophosphorylation site mutants formed sequestered areas of phytochrome (SAPs) in the cytosol much more slowly than did wild-type phyA. These results suggest that the autophosphorylation of phyA plays an important role in the regulation of plant phytochrome signaling through the control of phyA protein stability.

Phytochromes are the sole photoreceptors for perceiving red/far-red light in rice.

Phytochromes are believed to be solely responsible for red and far-red light perception, but this has never been definitively tested. To directly address this hypothesis, a phytochrome triple mutant (phyAphyBphyC) was generated in rice (Oryza sativa L. cv. Nipponbare) and its responses to red and far-red light were monitored. Since rice only has three phytochrome genes (PHYA, PHYB and PHYC), this mutant is completely lacking any phytochrome. Rice seedlings grown in the dark develop long coleoptiles while undergoing regular circumnutation. The phytochrome triple mutants also show this characteristic skotomorphogenesis, even under continuous red or far-red light. The morphology of the triple mutant seedlings grown under red or far-red light appears completely the same as etiolated seedlings, and they show no expression of the light-induced genes. This is direct evidence demonstrating that phytochromes are the sole photoreceptors for perceiving red and far-red light, at least during rice seedling establishment. Furthermore, the shape of the triple mutant plants was dramatically altered. Most remarkably, triple mutants extend their internodes even during the vegetative growth stage, which is a time during which wild-type rice plants never elongate their internodes. The triple mutants also flowered very early under long day conditions and set very few seeds due to incomplete male sterility. These data indicate that phytochromes play an important role in maximizing photosynthetic abilities during the vegetative growth stage in rice.

The histidine kinase-related domain of Arabidopsis phytochrome a controls the spectral sensitivity and the subcellular distribution of the photoreceptor.

Phytochrome A (phyA) is the primary photoreceptor for sensing extremely low amounts of light and for mediating various far-red light-induced responses in higher plants. Translocation from the cytosol to the nucleus is an essential step in phyA signal transduction. EID1 (for EMPFINDLICHER IM DUNKELROTEN LICHT1) is an F-box protein that functions as a negative regulator in far-red light signaling downstream of the phyA in Arabidopsis (Arabidopsis thaliana). To identify factors involved in EID1-dependent light signal transduction, pools of ethylmethylsulfonate-treated eid1-3 seeds were screened for seedlings that suppress the hypersensitive phenotype of the mutant. The phenotype of the suppressor mutant presented here is caused by a missense mutation in the PHYA gene that leads to an amino acid transition in its histidine kinase-related domain. The novel phyA-402 allele alters the spectral sensitivity and the persistence of far-red light-induced high-irradiance responses. The strong eid1-3 suppressor phenotype of phyA-402 contrasts with the moderate phenotype observed when phyA-402 is introgressed into the wild-type background, which indicates that the mutation mainly alters functions in an EID1-dependent signaling cascade. The mutation specifically inhibits nuclear accumulation of the photoreceptor molecule upon red light irradiation, even though it still interacts with FHY1 (for far-red long hypocotyl 1) and FHL (for FHY1-like protein), two factors that are essential for nuclear accumulation of phyA. Degradation of the mutated phyA is unaltered even under light conditions that inhibit its nuclear accumulation, indicating that phyA degradation may occur mostly in the cytoplasm.

Phytochrome A mediates rapid red light-induced phosphorylation of Arabidopsis FAR-RED ELONGATED HYPOCOTYL1 in a low fluence response.

Phytochrome A (phyA) is the primary photoreceptor for mediating the far-red high irradiance response in Arabidopsis thaliana. FAR-RED ELONGATED HYPOCOTYL1 (FHY1) and its homolog FHY1-LIKE (FHL) define two positive regulators in the phyA signaling pathway. These two proteins have been reported to be essential for light-regulated phyA nuclear accumulation through direct physical interaction with phyA. Here, we report that FHY1 protein is phosphorylated rapidly after exposure to red light. Subsequent exposure to far-red light after the red light pulse reverses FHY1 phosphorylation. Such a phenomenon represents a classical red/far-red reversible low fluence response. The phosphorylation of FHY1 depends on functioning phyA but not on other phytochromes and cryptochromes. Furthermore, we demonstrate that FHY1 and FHL directly interact with phyA by bimolecular fluorescence complementation and that both FHY1 and FHL interact more stably with the Pr form of phyA in Arabidopsis seedlings by coimmunoprecipitation. Finally, in vitro kinase assays confirmed that a recombinant phyA is able to robustly phosphorylate FHY1. Together, our results suggest that phyA may differentially regulate FHY1 and FHL activity through direct physical interaction and red/far-red light reversible phosphorylation to fine-tune their degradation rates and resulting light responses.

Phytochrome A regulates the intracellular distribution of phototropin 1-green fluorescent protein in Arabidopsis thaliana.

It has been known for decades that red light pretreatment has complex effects on subsequent phototropic sensitivity of etiolated seedlings. Here, we demonstrate that brief pulses of red light given 2 h prior to phototropic induction by low fluence rates of blue light prevent the blue light-induced loss of green fluorescent protein-tagged phototropin 1 (PHOT1-GFP) from the plasma membrane of cortical cells of transgenic seedlings of Arabidopsis thaliana expressing PHOT1-GFP in a phot1-5 null mutant background. This red light effect is mediated by phytochrome A and requires approximately 2 h in the dark at room temperature to go to completion. It is fully far red reversible and shows escape from photoreversibility following 30 min of subsequent darkness. Red light-induced inhibition of blue light-inducible changes in the subcellular distribution of PHOT1-GFP is only observed in rapidly elongating regions of the hypocotyl. It is absent in hook tissues and in mature cells below the elongation zone. We hypothesize that red light-induced retention of the PHOT1-GFP on the plasma membrane may account for the red light-induced increase in phototropic sensitivity to low fluence rates of blue light.

Phytochrome-mediated differential gene expression of plant Ran/TC4 small G-proteins.

Ran/TC4 is the only known member of the family of small GTP-binding proteins primarily localized inside the nucleus. We cloned a pea Ran gene (PsRan1) and characterized its expression in tissues, and under different light sources. PsRan1 is a member of a highly homologous multigene family, and it encodes a protein containing plant-specific amino acids in its sequence. It is ubiquitously expressed in pea tissues with high expression in radicles. The amount of total mRNA transcripts representing multiple Ran family members increased in response to very low-fluence R, while the amount of mRNA transcript encoding PsRan1 specifically was not affected by various light treatments. In addition, Ran genes in Arabidopsis were also differentially expressed in various mutants defective in phytochromes or the light-responding HY5 protein, such as phyA, phyB, and hy5. AtRan1 and AtRan3 gene expression was significantly reduced in the phyA mutant background compared to that in Ler-0 wild type plants. AtRan1 expression was also decreased in the phyB background. In contrast, the expression of AtRan2 did not vary in the hy5 and phytochrome mutant backgrounds examined. Interestingly, expression of AtRan1 was significantly reduced in hy5 plants, while AtRan3 expression was increased in the same plants. From these results, we conclude that Ran gene expression is differentially regulated by various light sources and phytochrome-mediated signaling pathways.

Phytochrome A is an irradiance-dependent red light sensor.

Plants perceive red (R) and far-red (FR) light signals using the phytochrome family of photoreceptors. In Arabidopsis thaliana, five phytochromes (phyA-phyE) have been identified and characterized. Unlike other family members, phyA is subject to rapid light-induced proteolytic degradation and so accumulates to relatively high levels in dark-grown seedlings. The insensitivity of phyA mutant seedlings to prolonged FR and wild-type appearance in R has led to suggestions that phyA functions predominantly as an FR sensor during the early stages of seedling establishment. The majority of published photomorphogenesis experiments have, however, used <50 micromol m(-2) sec(-1) of R when characterizing phytochrome functions. Here we reveal considerable phyA activity in R at higher (>160 micromol m(-2) sec(-1)) photon irradiances. Under these conditions, plant architecture was observed to be largely regulated by the redundant actions of phytochromes A, B and D. Moreover, quadruple phyBphyCphyDphyE mutants containing only functional phyA displayed R-mediated de-etiolation and survived to flowering. The enhanced activity of phyA in continuous R (Rc) of high photon irradiance correlates with retarded degradation of the endogenous protein in wild-type plants and prolonged epifluorescence of nuclear-localized phyA:YFP in transgenic lines. Such observations suggest irradiance-dependent 'photoprotection' of nuclear phyA in R, providing a possible explanation for the increased activity observed. The discovery that phyA can function as an effective irradiance sensor, even in light environments that establish a high Pfr concentration, raises the possibility that phyA may contribute significantly to the regulation of growth and development in daylight-grown plants.

Identification of an arsenic tolerant double mutant with a thiol-mediated component and increased arsenic tolerance in phyA mutants.

A genetic screen was performed to isolate mutants showing increased arsenic tolerance using an Arabidopsis thaliana population of activation tagged lines. The most arsenic-resistant mutant shows increased arsenate and arsenite tolerance. Genetic analyses of the mutant indicate that the mutant contains two loci that contribute to arsenic tolerance, designated ars4 and ars5. The ars4ars5 double mutant contains a single T-DNA insertion, ars4, which co-segregates with arsenic tolerance and is inserted in the Phytochrome A (PHYA) gene, strongly reducing the expression of PHYA. When grown under far-red light conditions ars4ars5 shows the same elongated hypocotyl phenotype as the previously described strong phyA-211 allele. Three independent phyA alleles, ars4, phyA-211 and a new T-DNA insertion allele (phyA-t) show increased tolerance to arsenate, although to a lesser degree than the ars4ars5 double mutant. Analyses of the ars5 single mutant show that ars5 exhibits stronger arsenic tolerance than ars4, and that ars5 is not linked to ars4. Arsenic tolerance assays with phyB-9 and phot1/phot2 mutants show that these photoreceptor mutants do not exhibit phyA-like arsenic tolerance. Fluorescence HPLC analyses show that elevated levels of phytochelatins were not detected in ars4, ars5 or ars4ars5, however increases in the thiols cysteine, gamma-glutamylcysteine and glutathione were observed. Compared with wild type, the total thiol levels in ars4, ars5 and ars4ars5 mutants were increased up to 80% with combined buthionine sulfoximine and arsenic treatments, suggesting the enhancement of mechanisms that mediate thiol synthesis in the mutants. The presented findings show that PHYA negatively regulates a pathway conferring arsenic tolerance, and that an enhanced thiol synthesis mechanism contributes to the arsenic tolerance of ars4ars5.