Regulation of phytochrome B signaling by phytochrome A and FHY1 in Arabidopsis thaliana

Phytochrome A (phyA) and phytochrome B (phyB) share the control of many processes but little is known about mutual signaling regulation. Here, we report on the interactions between phyA and phyB in the control of the activity of an Lhcb1*2 gene fused to a reporter, hypocotyl growth and cotyledon unfolding in etiolated Arabidopsis thaliana. The very-low fluence responses (VLFR) induced by pulsed far-red light and the high-irradiance responses (HIR) observed under continuous far-red light were absent in the phyA and phyA phyB mutants, normal in the phyB mutant, and reduced in the fhy1 mutant that is defective in phyA signaling. VLFR were also impaired in Columbia compared to Landsberg erecta. The low-fluence responses (LFR) induced by red-light pulses and reversed by subsequent far-red light pulses were small in the wild type, absent in phyB and phyA phyB mutants but strong in the phyA and fhy1 mutants. This indicates a negative effect of phyA and FHY1 on phyB-mediated responses. However, a pre-treatment with continuous far-red light enhanced the LFR induced by a subsequent red-light pulse. This enhancement was absent in phyA, phyB, or phyA phyB and partial in fhy1. The levels of phyB were not affected by the phyA or fhy1 mutations or by far-red light pre-treatments. We conclude that phyA acting in the VLFR mode (i.e. under light pulses) is antagonistic to phyB signaling whereas phyA acting in the HIR mode (i.e. under continuous far-red light) operates synergistically with phyB signaling, and that both types of interaction require FHY1.     

Sucrose control of phytochrome A signaling in Arabidopsis.

The expression of the Arabidopsis plastocyanin (PC) gene is developmentally controlled and regulated by light. During seedling development, PC gene expression is transiently induced, and this induction can be repressed by sucrose. In transgenic seedlings carrying a PC promoter-luciferase fusion gene, the luciferase-induced in vivo luminescence was similarly repressed by sucrose. From a mutagenized population of such transgenic seedlings, we selected for mutant seedlings that displayed a high luminescence level when grown on a medium with 3% sucrose. This screening of mutants resulted in the isolation of several sucrose-uncoupled (sun) mutants showing reduced repression of luminescence by sucrose. Analysis of the sun mutants revealed that the accumulation of PC and chlorophyll a/b binding protein (CAB) mRNA was also sucrose uncoupled, although the extent of uncoupling varied. The effect of sucrose on far-red light high-irradiance responses was studied in wild-type, sun1, sun6, and sun7 seedlings. In wild-type seedlings, sucrose repressed the far-red light-induced cotyledon opening and inhibition of hypocotyl elongation. sun7 seedlings showed reduced repression of these responses. Sucrose also repressed the far-red light-induced block of greening in wild-type seedlings, and both sun6 and sun7 were affected in this response. The results provide evidence for a close interaction between sucrose and light signaling pathways. Moreover, the sun6 and sun7 mutants genetically identify separate branches of phytochrome A-dependent signal transduction pathways.     

The protein phosphatase 7 regulates phytochrome signaling in Arabidopsis

The psi2 mutant of Arabidopsis displays amplification of the responses controlled by the red/far red light photoreceptors phytochrome A (phyA) and phytochrome B (phyB) but no apparent defect in blue light perception. We found that loss-of-function alleles of the protein phosphatase 7 (AtPP7) are responsible for the light hypersensitivity in psi2 demonstrating that AtPP7 controls the levels of phytochrome signaling. Plants expressing reduced levels of AtPP7 mRNA display reduced blue-light induced cryptochrome signaling but no noticeable deficiency in phytochrome signaling. Our genetic analysis suggests that phytochrome signaling is enhanced in the AtPP7 loss of function alleles, including in blue light, which masks the reduced cryptochrome signaling. AtPP7 has been found to interact both in yeast and in planta assays with nucleotide-diphosphate kinase 2 (NDPK2), a positive regulator of phytochrome signals. Analysis of ndpk2-psi2 double mutants suggests that NDPK2 plays a critical role in the AtPP7 regulation of the phytochrome pathway and identifies NDPK2 as an upstream element involved in the modulation of the salicylic acid (SA)-dependent defense pathway by light. Thus, cryptochrome- and phytochrome-specific light signals synchronously control their relative contribution to the regulation of plant development. Interestingly, PP7 and NDPK are also components of animal light signaling systems.     

The phytochrome A-specific signaling intermediate SPA1 interacts directly with COP1, a constitutive repressor of light signaling in Arabidopsis.

SPA1 is a phytochrome A (phyA)-specific signaling intermediate that acts as a light-dependent repressor of photomorphogenesis in Arabidopsis seedlings. It contains a WD-repeat domain that shows high sequence similarity to the WD-repeat region of the constitutive repressor of light signaling, COP1. Here, using yeast two-hybrid and in vitro interaction assays, we show that SPA1 strongly and selectively binds to COP1. Domain mapping studies indicate that the putative coiled-coil domain of SPA1 is necessary and sufficient for binding to COP1. Conversely, similar deletion analyses of the COP1 protein suggest that SPA1 interacts with the presumed coiled-coil domain of COP1. To further investigate SPA1 function in the phyA signaling pathway, we tested whether SPA1, like COP1, mediates changes in gene expression in response to light. We show that spa1 mutations increase the photoresponsiveness of certain light-regulated genes within 2 h of light treatment. Taken together, the results suggest that SPA1 may function to link the phytochrome A-specific branch of the light signaling pathway to COP1. Hence, our data provide molecular support for the hypothesis that COP1 is a convergence point for upstream signaling pathways dedicated to individual photoreceptors.     

Arabidopsis FHY3 defines a key phytochrome A signaling component directly interacting with its homologous partner FAR1

In Arabidopsis, phytochrome A (phyA) is the primary photoreceptor mediating various plant responses to far-red (FR) light. Here we show that phyA signaling involves a combinatorial action of downstream intermediates, which controls overlapping yet distinctive sets of FR responses. FHY3 is a prominent phyA signaling intermediate sharing structural similarity to FAR1, a previously identified phyA signaling component. The fhy3 and far1 mutants display similar yet distinctive defects in phyA signaling; however, overexpression of either FHY3 or FAR1 suppresses the mutant phenotype of both genes. Moreover, overexpression of partial fragments of FHY3 can cause a dominant-negative interference phenotype on phyA signaling that is stronger than those of the fhy3 or far1 null mutants. Further, we demonstrate that FHY3 and FAR1 are capable of homo- and hetero-interaction. Our data indicate that FHY3, together with FAR1, defines a key module in a signaling network underlying phyA-mediated FR light responses.     

The blue-light receptor cryptochrome 1 shows functional dependence on phytochrome A or phytochrome B in Arabidopsis thaliana

Blue-light responses in higher plants are mediated by specific photoreceptors, which are thought to be flavoproteins; one such flavin-type blue-light receptor, CRY1 (for cryptochrome), which mediates inhibition of hypocotyl elongation and anthocyanin biosynthesis, has recently been characterized. Prompted by classical photobiological studies suggesting possible co-action of the red/far-red absorbing photoreceptor phytochrome with blue-light photoreceptors in certain plant species, the role of phytochrome in CRY1 action in Arabidopsis was investigated. The activity of the CRY1 photoreceptor can be substantially altered by manipulating the levels of active phytochrome (Pfr) with red or far-red light pulses subsequent to blue-light treatments. Furthermore, analysis of severely phytochrome-deficient mutants showed that CRY1-mediated blue-light responses were considerably reduced, even though Western blots confirmed that levels of CRY1 photoreceptor are unaffected in these phytochrome-deficient mutant backgrounds. It was concluded that CRY1-mediated inhibition of hypocotyl elongation and anthocyanin production requires active phytochrome for full expression, and that this requirement can be supplied by low levels of either phyA or phyB.     

Overexpression of rice phytochrome A partially complements phytochrome B deficiency in Arabidopsis

The red/far-red reversible phytochromes play a central role in regulating the development of plants in relation to their light environment. Studies on the roles of different members of the phytochrome family have mainly focused on light-labile, phytochrome A and light-stable, phytochrome B. Although these two phytochromes often regulate identical responses, they appear to have discrete photosensory functions. Thus, phytochrome A predominantly mediates responses to prolonged far-red light, as well as acting in a non-red/far-red-reversible manner in controlling responses to light pulses. In contrast, phytochrome B mediates responses to prolonged red light and acts photoreversibly under light-pulse conditions. However, it has been reported that rice (Oryza sativa L.) phytochrome A operates in a classical red/far-red reversible fashion following its expression in transgenic tobacco plants. Thus, it was of interest to determine whether transgenic rice phytochrome A could substitute for loss of phytochrome B in phyB mutants of Arabidopsis thaliana (L.) Heynh. We have observed that ectopic expression of rice phytochrome A can correct the reduced sensitivity of phyB hypocotyls to red light and restore their response to end-of-day far-red treatments. The latter is widely regarded as a hallmark of phytochrome B action. However, although transgenic rice phytochrome A can correct other aspects of elongation growth in the phyB mutant it does not restore other responses to end-of-day far-red treatments nor does it restore responses to low red:far-red ratio. Furthermore, transgenic rice phytochrome A does not correct the early-flowering phenotype of phyB seedlings. Received: 12 July 1998 / Accepted: 13 August 1998     

Negative interference of endogenous phytochrome B with phytochrome A function in Arabidopsis

To study negative interactions between phytochromes, phytochrome B (phyB) overexpressor lines, the mutants phyA-201, phyB-4, phyB-5, phyD-1, phyA-201 phyB-5, phyA-201 phyD-1, and phyB-5 phyD-1 of Arabidopsis were used. Endogenous phyB, but not phytochrome D (phyD), partly suppressed phytochrome A (phyA)-dependent inhibition of hypocotyl elongation in far-red light (FR). Dichromatic irradiation demonstrated that the negative effect of phyB was largely independent of the photoequilibrium, i.e. far-red light absorbing form of phytochrome formation. Moreover, phyB-4, a mutant impaired in signal transduction, did not show a loss of inhibition of phyA by phyB. Overexpression of phyB, conversely, resulted in an enhanced inhibition of phyA function, even in the absence of supplementary carbohydrates. However, overexpression of a mutated phyB, which cannot incorporate the chromophore, had no detectable effect on phyA action. In addition to seedling growth, accumulation of anthocyanins in FR, another manifestation of the high irradiance response, was strongly influenced by phyB holoprotein. Induction of seed germination by FR, a very low fluence response, was suppressed by both endogenous phyB and phyD. In conclusion, we show that both classical response modes of phyA, high irradiance response, and very low fluence response are subject to an inhibitory action of phyB-like phytochromes. Possible mechanisms of the negative interference are discussed.     

Arabidopsis FHY3 specifically gates phytochrome signaling to the circadian clock

Circadian gating of light signaling limits the timing of maximum responsiveness to light to specific times of day. The fhy3 (for far-red elongated hypocotyl3) mutant of Arabidopsis thaliana is involved in independently gating signaling from a group of photoreceptors to an individual response. fhy3 shows an enhanced response to red light during seedling deetiolation. Analysis of two independent fhy3 alleles links enhanced inhibition of hypocotyl elongation in response to red light with an arrhythmic pattern of hypocotyl elongation. Both alleles also show disrupted rhythmicity of central-clock and clock-output gene expression in constant red light. fhy3 exhibits aberrant phase advances under red light pulses during the subjective day. Release-from-light experiments demonstrate clock disruption in fhy3 during the early part of the subjective day in constant red light, suggesting that FHY3 is important in gating red light signaling for clock resetting. The FHY3 gating function appears crucial in the early part of the day for the maintenance of rhythmicity under these conditions. However, unlike previously described Arabidopsis gating mutants that gate all light signaling, gating of direct red light-induced gene expression in fhy3 is unaffected. FHY3 appears to be a novel gating factor, specifically in gating red light signaling to the clock during daytime.     

Sustained but not transient phytochrome A signaling targets a region of an Lhcb1*2 promoter not necessary for phytochrome B action

Current evidence is inconclusive regarding the point of signaling convergence downstream from different members of the phytochrome family. In transgenic Arabidopsis, the activity of a reporter enzyme under the control of the -453 to +67 fragment of an Lhcb1*2 promoter shows very low fluence responses (VLFRs) and high-irradiance responses (HIRs) mediated by phytochrome A and low-fluence responses (LFRs) mediated by phytochrome B. A 5' deletion of the promoter to -134 abolished the HIR without affecting VLFR or LFR. In transgenic tobacco, VLFR and LFR were observed for the -176 to -31 or -134 to -31 fragments of Lhcb1*2 fused to 35S cauliflower mosaic virus minimal promoters, but only the largest fragment showed HIR. We propose that sustained activation of phytochrome A with far-red light initiates a signaling cascade that deviates from phytochrome B signaling and transient phytochrome A signaling and that this divergence extends as far as the Lhcb1*2 promoter.