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.     

SPA1, a component of phytochrome A signal transduction,regulates the light signaling current

Mutations in a component of phytochrome A (phyA)-specific light signal transduction, SPA1, result in enhanced responsiveness of Arabidopsis seedlings to red and far-red light. Here, we have examined the effects of spa1 mutations on the two known modes of phyA function, the high-irradiance responses (HIRs) to continuous irradiation with far-red light and the very-low-fluence responses (VLFRs) to inductive pulses of light that establish only a small proportion of active phyA. spa1 mutants exhibited an enhanced VLFR under hourly pulses of far-red light for hypocotyl growth inhibition, cotyledon unfolding, anthocyanin accumulation, block of greening in subsequent white light and negative regulation of phyB signaling. We provide evidence that the phenotype of spa1 mutants in red light is also caused by an increase in the VLFR. Taken together, our results indicate that light-induced hypocotyl growth inhibition in spa1 mutants is primarily due to a VLFR. While wild-type seedlings required hourly pulses of far-red light to induce a VLFR, infrequent irradiation with far-red pulses (every 12 h) was sufficient to induce a strong VLFR of hypocotyl elongation in spa1 mutants. This shows that the effect of the VLFR was more persistent in spa1 mutants than in the wild type. We, therefore, propose that SPA1 has an important function in reducing the persistence of phyA signaling. spa1 mutations also enhanced the HIRs of anthocyanin accumulation and of phyA-mediated responsivity amplification towards phyB. Thus, our results suggest that spa1 mutations amplify both the phyA-mediated VLFR and the HIR.     

Genetic identification of FIN2, a far red light-specific signaling component of Arabidopsis thaliana

Phytochrome A (PhyA) mediates most, if not all various plant responses to far-red (FR) light. Here, we report a novel genetic mutation that impairs a variety of responses in the PhyA-signaling pathway of Arabidopsis thaliana . The mutation was isolated by screening seedlings that show reduced sensitivity to continuous far-red (FRc) light irradiation, but not to continuous red (Rc) light irradiation. The mutation named fin2–1 is not allelic to a PHYA mutation. Furthermore, immunoblot analysis indicated that the amount of the phytochrome A apoprotein in the fin2–1 mutant was comparable to that in wild type. Seedling of the fin2–1 mutant showed defects in hypocotyl growth inhibition and apical hook and cotyledon opening in FRc light but not in Rc light. The results showed that the mutation occurred in a downstream signaling component potentially specific to PhyA. Other PhyAmediated responses such as FR-preconditioned blocking of greening, anthocyanin accumulation, reduction of gravitropic response, and expression of the CAB and CHS genes were impaired by the fin2–1 mutation: the degree of the mutant effect on the responses was variable. However, FR light-mediated seed germination and photoperiodic flowering responses were not affected significantly in the mutant. These results showed that FIN2 defines an upstream branch point in the PhyA signaling pathway.     

CP3 is involved in negative regulation of phytochrome A signalling in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Several mutants with altered phytochrome A (phyA) signalling have been identified in screenings under continuous far-red light (FR). The latter protocol could preclude the identification of mutants affected in the signalling pathway that operates even under transient phyA activation, compared to the high-irradiance response (HIR) pathway that requires continuous FR. Since some photomorphogenic mutants show shoot-height phenotypes, the screening was conducted on dwarf mutants of Arabidopsis thaliana (L.) Heynh. from the ABRC stocks grown under hourly FR pulses. The dwarf mutant cp3 (compacta 3) showed normal hypocotyl length and folded cotyledons in darkness but enhanced hypocotyl-growth inhibition and cotyledon unfolding under pulsed FR. The HIR and the response mediated by phyB were not affected. Under pulsed FR, seed germination and blocking of greening upon transfer to white light were enhanced in cp3. PHYA levels were normal in cp3. The phenotype under pulsed FR but not the adult phenotype required phyA. We propose that CP3 is involved in the negative regulation of the signalling pathway that saturates with transient activation of phyA.     

The serine-rich N-terminal domain of oat phytochrome a helps regulate light responses and subnuclear localization of the photoreceptor

Phytochrome (phy) A mediates two distinct photobiological responses in plants: the very-low-fluence response (VLFR), which can be saturated by short pulses of very-low-fluence light, and the high-irradiance response (HIR), which requires prolonged irradiation with higher fluences of far-red light (FR). To investigate whether the VLFR and HIR involve different domains within the phyA molecule, transgenic tobacco (Nicotiana tabacum cv Xanthi) and Arabidopsis seedlings expressing full-length (FL) and various deletion mutants of oat (Avena sativa) phyA were examined for their light sensitivity. Although most mutants were either partially active or inactive, a strong differential effect was observed for the Delta6-12 phyA mutant missing the serine-rich domain between amino acids 6 and 12. Delta6-12 phyA was as active as FL phyA for the VLFR of hypocotyl growth and cotyledon unfolding in Arabidopsis, and was hyperactive in the VLFR of hypocotyl growth and cotyledon unfolding in tobacco, and the VLFR blocking subsequent greening under white light in Arabidopsis. In contrast, Delta6-12 phyA showed a dominant-negative suppression of HIR in both species. In hypocotyl cells of Arabidopsis irradiated with FR phyA:green fluorescent protein (GFP) and Delta6-12 phyA:GFP fusions localized to the nucleus and coalesced into foci. The proportion of nuclei with abundant foci was enhanced by continuous compared with hourly FR provided at equal total fluence in FL phyA:GFP, and by Delta6-12 phyA mutation under hourly FR. We propose that the N-terminal serine-rich domain of phyA is involved in channeling downstream signaling via the VLFR or HIR pathways in different cellular contexts.     

Missense mutation in the PAS2 domain of phytochrome A impairs subnuclear localization and a subset of responses

Phytochrome A signaling shows two photobiologically discrete outputs: so-called very-low-fluence responses (VLFR) and high-irradiance responses (HIR). By modifying previous screening protocols, we isolated two Arabidopsis mutants retaining VLFR and lacking HIR. Phytochrome A negatively or positively regulates phytochrome B signaling, depending on light conditions. These mutants retained the negative but lacked the positive regulation. Both mutants carry the novel phyA-302 allele, in which Glu-777 (a residue conserved in angiosperm phytochromes) changed to Lys in the PAS2 motif of the C-terminal domain. The phyA-302 mutants showed a 50% reduction in phytochrome A levels in darkness, but this difference was compensated for by greater stability under continuous far-red light. phyA-302:green fluorescent protein fusion proteins showed normal translocation from the cytosol to the nucleus under continuous far-red light but failed to produce nuclear spots, suggesting that nuclear speckles could be involved in HIR signaling and phytochrome A degradation. We propose that the PAS2 domain of phytochrome A is necessary to initiate signaling in HIR but not in VLFR, likely via interaction with a specific partner.     

Missense mutation in the amino terminus of phytochrome A disrupts the nuclear import of the photoreceptor

Phytochromes are the red/far-red photoreceptors in higher plants. Among them, phytochrome A (PHYA) is responsible for the far-red high-irradiance response and for the perception of very low amounts of light, initiating the very-low-fluence response. Here, we report a detailed physiological and molecular characterization of the phyA-5 mutant of Arabidopsis (Arabidopsis thaliana), which displays hyposensitivity to continuous low-intensity far-red light and shows reduced very-low-fluence response and high-irradiance response. Red light-induced degradation of the mutant phyA-5 protein appears to be normal, yet higher residual amounts of phyA-5 are detected in seedlings grown under low-intensity far-red light. We show that (1) the phyA-5 mutant harbors a new missense mutation in the PHYA amino-terminal extension domain and that (2) the complex phenotype of the mutant is caused by reduced nuclear import of phyA-5 under low fluences of far-red light. We also demonstrate that impaired nuclear import of phyA-5 is brought about by weakened binding affinity of the mutant photoreceptor to nuclear import facilitators FHY1 (for FAR-RED ELONGATED HYPOCOTYL1) and FHL (for FHY1-LIKE). Finally, we provide evidence that the signaling and degradation kinetics of constitutively nuclear-localized phyA-5 and phyA are identical. Taken together, our data show that aberrant nucleo/cytoplasmic distribution impairs light-induced degradation of this photoreceptor and that the amino-terminal extension domain mediates the formation of the FHY1/FHL/PHYA far-red-absorbing form complex, whereby it plays a role in regulating the nuclear import of phyA.     

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.     

Transgenic Tobacco Plants Low in Phytochrome A Content

Tobacco (Nicotiana tabacum) plants were transformed with a construct encoding phytochrome A (PHYA) antisense RNA. The construct inserted into the tobacco genome contained squash PHYA cDNA in an antisense orientation under the cauliflower mosaic virus 35S promoter providing for gene expression in higher plant tissues. Using immunoblot analysis and Z3-B1 antibodies against PHYA, the authors demonstrated that the PHYA content of the transgenic plants was lower than that of the wild-type plants. The studies of PHYA-dependent inhibition of hypocotyl elongation by high-intensity far-red light showed a considerable decrease in light sensitivity of the transgenic hypocotyl characteristic for aphyAmutation.     

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.     

Interactions of phytochromes A,B1 and B2 in light-induced competence for adventitious shoot formation in hypocotyl of tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis> L.)

The interactions of phytochrome A (phyA), phytochrome B1 (phyB1) and phytochrome B2 (phyB2) in light-dependent shoot regeneration from the hypocotyl of tomato was analysed using all eight possible homozygous allelic combinations of the null mutants. The donor plants were pre-grown either in the dark or under red or far-red light for 8 days after sowing; thereafter hypocotyl segments (apical, middle and basal portions) were transferred onto hormone-free medium for culture under different light qualities. Etiolated apical segments cultured in vitro under white light showed a very high frequency of regeneration for all of the genotypes tested besides phyB1phyB2, phyAphyB1 and phyAphyB1phyB2 mutants. Evidence is provided of a specific interference of phyB2 with phyA-mediated HIR to far-red and blue light in etiolated explants. Pre-treatment of donor plants by growth under red light enhanced the competence of phyB1phyB2, phyAphyB1 and phyAphyB1phyB2 mutants for shoot regeneration, whereas pre-irradiation with far-red light enhanced the frequency of regeneration only in the phyAphyB1 mutant. Multiple phytochromes are involved in red light- and far-red light-dependent acquisition of competence for shoot regeneration. The position of the segments along the hypocotyl influenced the role of the various phytochromes and the interactions between them. The culture of competent hypocotyl segments under red, far-red or blue light reduced the frequency of explants forming shoots compared to those cultured under white light, with different genotypes having different response patterns.Abbreviations HIR: High irradiance response - LFR: Low fluence response - Pfr: Far-red absorbing form of phytochrome - phyA: Phytochrome A - phyB1: Phytochrome B1 - phyB2: Phytochrome B2 - phyA(B1, B2): Phytochrome mutant deficient in phyA (B1, B2) - phyAphyB1(B1B2,AB2): Double phytochrome mutant deficient in phyA and phyB1(B1, B2) - phyAphyB1phyB2: Triple mutant deficient in phyA, phyB1 and phyB2 - VLFR: Very low fluence response - WT: Wild-type tomato Communicated by R. Reski     

shl, a New set of Arabidopsis mutants with exaggerated developmental responses to available red, far-red, and blue light

The interaction of light perception with development is the subject of intensive genetic analysis in the model plant Arabidopsis. We performed genetic screens in low white light-a threshold condition in which photomorphogenetic signaling pathways are only partially active-for ethyl methane sulfonate-generated mutants with altered developmental phenotypes. Recessive mutants with exaggerated developmental responses were obtained in eight complementation groups designated shl for seedlings hyperresponsive to light. shl1, shl2, shl5, and shl3 shl4 (double mutant) seedlings showed limited or no phenotypic effects in darkness, but showed significantly enhanced inhibition of hypocotyl elongation in low-white, red, far-red, blue, and green light across a range of fluences. These results reflect developmental hyper-responsiveness to signals generated by both phytochrome and cryptochrome photoreceptors. The shl11 mutant retained significant phenotypic effects on hypocotyl length in both the phyA mutant and phyB mutant backgrounds but may be dependent on CRY1 for phenotypic expression in blue light. The shl2 phenotype was partially dependent on PHYB, PHYA, and CRY1 in red, far-red, and blue light, respectively. shl2 and, in particular, shl1 were partially dependent on HY5 activity for their light-hyperresponsive phenotypes. The SHL genes act (genetically) as light-dependent negative regulators of photomorphogenesis, possibly in a downstream signaling or developmental pathway that is shared by CRY1, PHYA, and PHYB and other photoreceptors (CRY2, PHYC, PHYD, and PHYE).     

Genetic and transgenic evidence that phytochromes A and B act to modulate the gravitropic orientation of Arabidopsis thaliana hypocotyls

Hypocotyls of Arabidopsis thaliana exhibit negative gravitropism in the dark, growing against the gravity vector. The direction of growth is randomized in red light (R). In single mutants lacking either phytochrome A or B randomization of hypocotyl orientation in R is retained. However, a double mutant lacks this response, indicating that either phytochrome A or B is capable of inducing randomization and phytochrome A and B are the only phytochromes involved in this process. The induction of randomization was confirmed using lines that express to different levels PHYA and PHYB cDNAs. Overexpression of PHYA cDNAs induced randomization of hypocotyl orientation in the dark. Dark randomization was also seen in the phyB-1 mutant but not in two other phyB alleles, suggesting that dark randomization in the phyB-1 line may be due to a second mutation. When germination was induced by gibberellin, rather than exposure to brief white light, randomization in the dark associated with phytochrome A overproduction was not observed but was retained in the phyB-1 mutant. Overexpression of PHYB cDNAs induced a light-dependent randomization of hypocotyl orientation that responded to R:far-red light ratio. We conclude that the default situation in Arabidopsis hypocotyls is, therefore, negative gravitropism, and either phytochrome A or phytochrome B can mediate randomization.     

FIN5 positively regulates far-red light responses in Arabidopsis thaliana

We report the characterization of a semi-dominant mutation fin5-1 (far-red insensitive 5-1) of Arabidopsis, which was isolated from genetic screening of phytochrome A (phyA) signaling components. Plants with the fin5-1 mutation exhibited a long hypocotyl phenotype when grown under far-red (FR) light, but not under red light. Physiological analyses implied that FIN5 might be differentially involved in diverse responses that are regulated by phyA under continuous FR light. Anthocyanin accumulation, gravitropic response of hypocotyl growth, and FR light-preconditioned blocking of greening were also impaired in the fin5-1 mutant, whereas photoperiodic floral induction was not, if at all, significantly affected. Moreover, light-regulated expression of the CHS, PORA and PsbS genes was attenuated in fin5-1 mutant plants, while the light-induced expression of CAB was normal. The mutation exhibited semi-dominance regarding control of hypocotyl growth in FR light. We suggest that FIN5 defines a novel branch in the network of phyA signaling in Arabidopsis.     

光敏色素A调控光响应基因表达及其翻译后修饰研究进展

光是调节植物生长发育最重要的环境信号因子之一。植物通过光受体感受自然环境中光的强度、方向以及光周期等信号的变化,从而调控其生长发育过程。光敏色素A (phytochrome A, PHYA)是植物中唯一的远红光受体蛋白,具有在黑暗下在细胞质中合成,而在照光后快速入核和降解的特性,并通过多种途径精确调节了植物光响应基因的转录网络。同时,蛋白质翻译后修饰在调节PHYA稳定性和活性的过程中发挥了重要的作用。该文论述了PHYA调节光响应基因表达以及PHYA翻译后修饰方向的研究进展,并展望了PHYA在农作物分子设计育种中的应用前景。