Interaction of a plant 14-3-3 protein with the signal peptide of a thylakoid-targeted chloroplast precursor protein and the presence of 14-3-3 isoforms in the chloroplast stroma

The fhy3 mutation of Arabidopsis impairs phytochrome A (phyA)-mediated inhibition of hypocotyl growth without affecting the levels of phyA measured spectrophotometrically or immunochemically. We investigated whether the fhy3-1 mutation has similar effects on very low fluence responses (VLFR) and high irradiance responses (HIR) of phyA. When exposed to hourly pulses of far-red light, etiolated seedlings of the wild type or of the fhy3-1 mutant showed similar inhibition of hypocotyl growth, unfolding of the cotyledons, anthocyanin synthesis, and greening upon transfer to white light. In the wild type, continuous far-red light was significantly more effective than hourly far-red pulses (at equal total fluence). In the fhy3-1 mutant, hourly pulses were as effective as continuous far-red light, i.e. the failure of reciprocity typical of HIR was not observed. Germination was similarly promoted by continuous or pulsed far-red in wild-type and fhy3-1 seeds. Thus, for hypocotyl growth, cotyledon unfolding, greening, and seed germination, the fhy3-1 mutant retains VLFR but is severely impaired in HIR. These data are consistent with the idea that VLFR and HIR involve divergent signaling pathways of phyA.     

Dual effect of phytochrome A on hypocotyl growth under continuous red light

The role of phytochrome A in the control of hypocotyl growth under continuous red light (Rc) was investigated using phyA and phyB mutants of Arabidopsis thaliana, which lack phytochrome A (phyA) or phytochrome B (phyB), respectively, and transgenic seedlings of Nicotiana tabacum overexpressing Avena phyA, compared to the corresponding wild type (WT). In WT seedlings of A. thaliana, hypocotyl growth inhibition showed a biphasic response to the fluence rate of Rc, with a brake at 10^?2μmol m^?2 s^?1. At equal total fluence rate, hourly pulses of red light caused slightly more inhibition than Rc. The response to very low fluences of continuous or pulsed red light was absent in the phyA and phyA phyB mutants and present in the phyB mutant. The second part of the response was steeper in the phyA mutant than in the WT but was absent in the phyB mutant. In WT tobacco the response to Rc was biphasic. Overexpression of Avena phyA enhanced the response only at very low fluence rates of Rc (< 10^?2μmol m^?2 s^?1). In both species, the effect of hourly pulses of far-red light was similar to the maximum inhibition observed in the first phase of the response to Rc. Using reciprocity failure (i.e. higher inhibition under continuous than pulsed light) as the operational criterion, a ‘true’ high-irradiance reaction occurred under continuous far-red light but not under Rc or red plus far-red light mixtures. Native and overexpressed phyA are proposed to mediate very low fluence responses under Rc. In WT A. thaliana, this effect is counteracted by a negative action of phyA on phyB-mediated low-fluence responses.     

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.     

RSF1, an Arabidopsis locus implicated in phytochrome A signaling

In Arabidopsis, phytochrome A (phyA) is the major photoreceptor both for high irradiance responses to far-red light and broad spectrum very low fluence responses, but little is known of its signaling pathway(s). rsf1 was isolated as a recessive mutant with reduced sensitivity to far-red inhibition of hypocotyl elongation. At the seedling stage rsf1 mutants are affected, to various degrees, in all described phyA-mediated responses. However, in adult rsf1 plants, the photoperiodic flowering response is normal. The rsf1 mutant has wild-type levels of phyA suggesting that RSF1 is required for phyA signaling rather than phyA stability or biosynthesis. RSF1 thus appears to be a major phyA signaling component in seedlings, but not in adult, Arabidopsis plants.     

Light-induced nuclear import of phytochrome-A:GFP fusion proteins is differentially regulated in transgenic tobacco and Arabidopsis

Phytochromes (phy) are a family of photoreceptors that control various aspects of light-dependent plant development. Phytochrome A (phyA) is responsible for the very low fluence response (VLFR) under inductive light conditions and for the high irradiance response (HIR) under continuous far-red light. We have recently shown that nuclear import of rice phyA:GFP is regulated by VLFR in transgenic tobacco. The import is preceded by very fast, light-induced formation of sequestered areas of phyA:GFP in the cytosol. Here we report that expression of the Arabidopsis phyA:GFP fusion protein in phyA-deficient Arabidopsis plants complements the mutant phenotype. In these transgenic Arabidopsis lines, both light-dependent cytosolic formation of sequestered areas of the phyA:GFP as well as VLFR or HIR-mediated nuclear import of the fusion protein was observed. By contrast, light-dependent nuclear import of the same fusion protein was induced only by continuous far-red light (HIR) but not by pulses of far-red light (VLFR) in transgenic tobacco. These results demonstrate that photoregulation of intracellular partitioning of the Arabidopsis phyA:GFP differs significantly in different genetic backgrounds.     

Overexpression of rice phytochrome A in tobacco seedlings modifies responsiveness but not competence to phytochrome

To analyse the control of rice phytochrome A (phyA) overexpression (wild type or variously mutated) on gene regulation, transgenic tobacco lines overexpressing various rice phyA constructs were crossed with transgenic tobacco lines containing mustard Lhcb1 or Chs1 promoters fused to the uidA reporter gene (-glucuronidase). It was demonstrated that the temporal pattern of competence to respond to phytochrome was not altered by rice phyA overexpression. Also, overexpression of rice phyA did not change the spatial pattern of gene expression. The responsiveness to red and far-red light, on the other hand, depended on the type of overexpressed rice phyA in a structure-function relation: the serine-to-alanine mutant mediated an enhanced response both under continuous red and far-red light, whereas the N-terminal deletion mutant showed a dominant negative effect under continuous far-red light and even after red light pulses. However, the effectiveness of rice phyA overexpression depended on the promoter construct and the developmental stage of the seedlings. The Lhcb1 promoter also conferred -glucuronidase activity in etiolated seedlings. This dark expression could be decreased by a long-wavelength farred light pulse given early in development (24 h after sowing), indicating that this phenomenon is under the control of stable types of phytochrome.Abbreviations Chs1 chalcone synthase - GUS -glucuronidase - Lhcb1 type 1 light-harvesting chlorophyll a/b-binding protein - NTD N-terminal deletion mutant of rice phyA - phyA phytochrome A - phyB phytochrome B - Pfr far-red absorbing form of phytochrome - Pr red-absorbing form of phytochrome - RW rice wild-type phyA - S/A serine-to-alanine mutant of rice phyA - XAN wild-type tobacco cv. Xanthi We thank N.-H. Chua (Rockefeller Univ., New York, USA) and J. Stockhaus (Heinrich-Heine-Universität, Düsseldorf, Germany) for providing seeds from tobacco lines overexpressing the diverse rice phyA proteins. The work was supported by a grant from the Human Frontier Science Program and a grant from Deutsche Forschungsgemeinschaft (SFB 388). K.E. is a recipient of a Landesgraduierten-förderung fellowship     

eid1: a new Arabidopsis mutant hypersensitive in phytochrome A-dependent high-irradiance responses

To identify specific mutants for components of phytochrome A (phyA) signaling in Arabidopsis, we established a light program consisting of multiple treatments with alternating red and far-red light. In wild-type seedlings, irradiation with multiple red light pulses can reduce the amount of phyA, which in turn decreases the high-irradiance responses (HIRs) mediated by the subsequent treatments with far-red light. Our mutants were able to avoid this red light-dependent reduction of the HIR. Here, we describe eid1, a new recessive mutant with increased sensitivity to far-red light. The eid1 mutation maps to the top of chromosome 4. The mutants showed no change in phenotype in darkness or under continuous white light, but they exhibited an increased sensitivity to red light and an increased persistence of HIR during prolonged dark phases after multiple short pulses of far-red light. The eid1 seedlings accumulated normal amounts of phytochrome and showed no alterations in the degradation or de novo synthesis of phyA. The expression of the Eid1 phenotype requires the presence of phyA. Our data provide evidence that EID1 is a negatively acting component in the phyA-dependent HIR-signaling pathway.     

Ultraviolet B radiation enhances a phytochrome-B-mediated photomorphogenic response in Arabidopsis

Ultraviolet B radiation (UV-B, 290-315 nm) can cause damage and induce photomorphogenic responses in plants. The mechanisms that mediate the photomorphogenic effects of UV-B are unclear. In etiolated Arabidopsis seedlings, a daily exposure to 2.5 h of UV-B enhanced the cotyledon opening response induced by a subsequent red light (R) pulse. An R pulse alone, 2.5 h of UV-B terminated with a far-red pulse, or 2.5 h of continuous R caused very little cotyledon opening. The enhancing effect of UV-B increased with fluence rate up to approximately 7.58 micromol m(-2) s(-1); at higher fluence rates the response to UV-B was greatly reduced. The phyA, phyA cry1, and cry1 cry2 mutants behaved like the wild type when exposed to UV-B followed by an R pulse. In contrast, phyB, phyB cry1, and phyB phyA mutants failed to open the cotyledons. Thus, phytochrome B was required for the cotyledon opening response to UV-B --> R treatments, whereas phytochrome A and cryptochromes 1 and 2 were not necessary under the conditions of our experiments. The enhancing effect of low doses of UV-B on cotyledon opening in uvr1 uvr2 and uvr1 uvr3 mutants, deficient in DNA repair, was similar to that found in the wild type, suggesting that this effect of UV-B was not elicited by signals derived from UV-B-induced DNA lesions (cyclobutane pyrimidine dimers and 6-4 photoproducts). We conclude that low doses of UV-B, perceived by a receptor system different from phytochromes, cryptochromes, or DNA, enhance a de-etiolation response that is induced by active phytochrome B.     

SPA1: a new genetic locus involved in phytochrome A-specific signal transduction.

To identify mutants potentially defective in signaling intermediates specific to phytochrome A (phyA), we screened for extragenic mutations that suppress the morphological phenotype exhibited by a weak phyA mutant (phyA-105) of Arabidopsis. A new recessive mutant, designated spa1 (for suppressor of phyA-105), was isolated and mapped to the bottom of chromosome 2. spa1 phyA-105 double mutants exhibit restoration of several responses to limiting fluence rates of continuous far-red light that are absent in the parental phyA-105 mutant, such as deetiolation, anthocyanin accumulation, and a far-red light-induced inability of seedlings to green upon subsequent transfer to continuous white light. spa1 mutations do not cause a phenotype in darkness, indicating that the suppression phenotype is light dependent. Enhanced photoresponsiveness was observed in spa1 seedlings in a wild-type PHYA background as well as in the mutant phyA-105 background but not in a mutant phyA null background. These results indicate that phyA is necessary in a non-allele-specific fashion for the expression of the spa1 mutant phenotype and that phyB to phyE are not sufficient for this effect. Taken together, the data suggest that spa1 mutations specifically amplify phyA signaling and therefore that the SPA1 locus encodes a component that acts negatively early in the phyA-specific signaling pathway.     

eid1: A New Arabidopsis Mutant Hypersensitive in Phytochrome A–Dependent High-Irradiance Responses

To identify specific mutants for components of phytochrome A (phyA) signaling in Arabidopsis, we established a light program consisting of multiple treatments with alternating red and far-red light. In wild-type seedlings, irradiation with multiple red light pulses can reduce the amount of phyA, which in turn decreases the high-irradiance responses (HIRs) mediated by the subsequent treatments with far-red light. Our mutants were able to avoid this red light–dependent reduction of the HIR. Here, we describe eid1, a new recessive mutant with increased sensitivity to far-red light. The eid1 mutation maps to the top of chromosome 4. The mutants showed no change in phenotype in darkness or under continuous white light, but they exhibited an increased sensitivity to red light and an increased persistence of HIR during prolonged dark phases after multiple short pulses of far-red light. The eid1 seedlings accumulated normal amounts of phytochrome and showed no alterations in the degradation or de novo synthesis of phyA. The expression of the Eid1 phenotype requires the presence of phyA. Our data provide evidence that EID1 is a negatively acting component in the phyA-dependent HIR-signaling pathway.     

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     

Phytochrome E influences internode elongation and flowering time in Arabidopsis.

From a screen of M2 seedlings derived from gamma-mutagenesis of seeds doubly null for phytochromes phyA and phyB, we isolated a mutant lacking phyE. The PHYE gene of the selected mutant, phyE-1, was found to contain a 1-bp deletion at a position equivalent to codon 726, which is predicted to result in a premature stop at codon 739. Immunoblot analysis showed that the phyE protein was undetectable in the phyE-1 mutant. In the phyA- and phyB-deficient background, phyE deficiency led to early flowering, elongation of internodes between adjacent rosette leaves, and reduced petiole elongation. This is a phenocopy of the response of phyA phyB seedlings to end-of-day far-red light treatments. Furthermore, a phyE deficiency attenuated the responses of phyA phyB seedlings to end-of-day far-red light treatments. Monogenic phyE mutants were indistinguishable from wild-type seedlings. However, phyB phyE double mutants flowered earlier and had longer petioles than did phyB mutants. The elongation and flowering responses conferred by phyE deficiency are typical of shade avoidance responses to the low red/far-red ratio. We conclude that in conjunction with phyB and to a lesser extent with phyD, phyE functions in the regulation of the shade avoidance syndrome.     

The Phytochrome B/Phytochrome C Heterodimer Is Necessary for Phytochrome C-Mediated Responses in Rice Seedlings

Background

PhyC levels have been observed to be markedly lower in phyB mutants than in Arabidopsis or rice wild type etiolated seedlings, but the mechanism of this phenomenon has not been fully elucidated.

Results

In the present study, we investigated the mechanism by which phyB affects the protein concentration and photo-sensing abilities of phyC and demonstrated that rice phyC exists predominantly as phyB/phyC heterodimers in etiolated seedlings. PHYC-GFP protein was detected when expressed in phyA phyC mutants, but not in phyA phyB mutants, suggesting that phyC requires phyB for its photo-sensing abilities. Interestingly, when a mutant PHYB gene that has no chromophore binding site, PHYB(C364A), was introduced into phyB mutants, the phyC level was restored. Moreover, when PHYB(C364A) was introduced into phyA phyB mutants, the seedlings exhibited de-etiolation under both far-red light (FR) and red light (R) conditions, while the phyA phyB mutants were blind to both FR and R. These results are the first direct evidence that phyC is responsible for regulating seedling de-etiolation under both FR and R. These findings also suggest that phyB is indispensable for the expression and function of phyC, which depends on the formation of phyB/phyC heterodimers.

Significance

The present report clearly demonstrates the similarities and differences in the properties of phyC between Arabidopsis and rice and will advance our understanding of phytochrome functions in monocots and dicots.     

Photoresponses of transgenic Arabidopsis seedlings expressing introduced phytochrome B-encoding cDNAs: evidence that phytochrome A and phytochrome B have distinct photoregulatory functions

The photocontrol of hypocotyl elongation has been studied in two transgenic lines of Arabidopsis thaliana which contain elevated levels of phytochrome B encoded by either an introduced rice- or Arabidopsis -derived cDNA driven by the 35S CaMV promoter. Inhibition of hypocotyl growth in etiolated seedlings of the phyB -transformed lines was saturated at photon fluence rates of continuous red light (R) which were markedly lower than those required for inhibition of growth in seedlings of the isogenic wild-type (WT). Inhibition of hypocotyl growth in etiolated seedlings of the phyB -transgenic lines under continuous far-red irradiation (FR), however, showed the same relationship with fluence rate as WT. Light-grown seedlings of the phyB -transgenic lines responded to end-of-day FR by an acceleration of growth, in a manner comparable with WT. This response was unaltered when the end-of-day FR was extended from a 15 min pulse to 14 h of continuous irradiation. The response of light-grown, phyB -transformed seedlings to decreasing R:FR ratio was also qualitatively similar to WT, i.e. increased elongation growth of the hypocotyl and petioles occurred under low R:FR quantum ratio. However, absolute elongation growth was markedly less in the transgenic seedlings at all R:FR ratios tested than in WT. Together, these data indicate that seedlings over-expressing phytochrome B are more responsive to R than are WT, but are unaltered in their responsiveness to FR. By contrast, seedlings overexpressing phytochrome A are more responsive than WT to both R and FR; whereas the phytochrome B-deficient mutant hy3 is unresponsive to R while retaining WT-like responsiveness to FR. These data indicate that in WT etiolated seedlings phytochrome A mediates the effects of continuous FR, and phytochrome B the effects of continuous R. The evidence thus supports the conclusion that these two molecular species of the photoreceptor have differential regulatory roles in the plant.     

Phytochrome-regulated PIL1 derepression is developmentally modulated

We define the photoresponsiveness, during seedling de-etiolation,of PHYTOCHROME-INTERACTING FACTOR 3-LIKE 1 (PIL1), initiallyidentified by microarray analysis as an early-response genethat is robustly repressed by first exposure to light. We showthat PIL1 mRNA abundance declines rapidly, with a half-timeof 15 min, to a new steady-state level, 10-fold below the initialdark level, within 45 min of first exposure to red light. Analysisof phy-null mutants indicates that multiple phytochromes, includingphyA and phyB, impose this repression. Conversely, PIL1 expressionis rapidly derepressed by subsequent far-red irradiation ofpreviously red light-exposed seedlings. However, the magnitudeof this derepression is modulated over time, in a biphasic manner,in response to increasing duration of pre-exposure to continuousred light: (i) an early phase (up to about 6 h) of relativelyrapidly increasing effectiveness of far-red reversal of repression,as declining phyA levels relieve initial very low fluence suppressionof this response; and (ii) a second phase (beyond 6 h) of graduallydeclining effectiveness of far-red reversal, to only 20% ofmaximal derepression, within 36 h of continuous red light exposure,with no evidence of circadian modulation of this responsiveness,an observation in striking contrast to a previous report forentrained, green seedlings exposed to vegetative shade. Thesedata, together with analysis of phytochrome signaling mutantsand overexpressors with aberrant de-etiolation phenotypes, suggestthat the second-phase decline in robustness of PIL1 derepressionis an indirect consequence of the global developmental transitionfrom the etiolated to the de-etiolated state, and that circadiancoupling of derepression requires entrainment.     

The VLF loci, polymorphic between ecotypes Landsberg erecta and Columbia, dissect two branches of phytochrome A signal transduction that correspond to very-low-fluence and high-irradiance responses

Phytochromes play a key role in the perception of light signals by plants. In this study, the three classical phytochrome action modes, i.e. very-low-fluence responses (VLFR), low-fluence responses (LFR) and high-irradiance responses (HIR), were genetically dissected using phyA and phyB mutants of Arabidopsis thaliana (respectively lacking phytochrome A or phytochrome B) and a polymorphism between ecotypes Landsberg erecta and Columbia. Seed germination and potentiation of greening, hypocotyl growth inhibition and cotyledon unfolding in etiolated seedlings of the ecotype Landsberg erecta showed biphasic responses to the calculated proportion of active phytochrome established by one light pulse or repeated light pulses. The first phase, i.e. the VLFR, was absent in the phyA mutant, normal in the phyB mutant (both in the Landsberg erecta background) and severely deficient in Columbia. The second phase, i.e. the LFR, was present in the phyA mutant, deficient in the phyB mutant and normal in Columbia. Under continuous far-red light, HIR of etiolated seedlings were absent in phyA and normal in phyB and Columbia. The segregation of VLFR in recombinant inbred lines derived from a cross between Landsberg erecta and Columbia was analysed by MAPMAKER/QTL. Two quantitative trait loci, one on chromosome 2 ( VLF1 ) and another on chromosome 5 ( VLF2 ), were identified as responsible for the polymorphism. Phytochrome A is proposed to initiate two transduction pathways, VLFR and HIR, involving different cells and/or different molecular steps. This is the first application of the analysis of quantitative trait loci polymorphic between ecotypes to dissect transduction chains of environmental signals.     

Distinct and cooperative functions of phytochromes A, B, and C in the control of deetiolation and flowering in rice

We have isolated phytochrome B (phyB) and phyC mutants from rice (Oryza sativa) and have produced all combinations of double mutants. Seedlings of phyB and phyB phyC mutants exhibited a partial loss of sensitivity to continuous red light (Rc) but still showed significant deetiolation responses. The responses to Rc were completely canceled in phyA phyB double mutants. These results indicate that phyA and phyB act in a highly redundant manner to control deetiolation under Rc. Under continuous far-red light (FRc), phyA mutants showed partially impaired deetiolation, and phyA phyC double mutants showed no significant residual phytochrome responses, indicating that not only phyA but also phyC is involved in the photoperception of FRc in rice. Interestingly, the phyB phyC double mutant displayed clear R/FR reversibility in the pulse irradiation experiments, indicating that both phyA and phyB can mediate the low-fluence response for gene expression. Rice is a short-day plant, and we found that mutation in either phyB or phyC caused moderate early flowering under the long-day photoperiod, while monogenic phyA mutation had little effect on the flowering time. The phyA mutation, however, in combination with phyB or phyC mutation caused dramatic early flowering.