Phytochrome A regulates red-light induction of phototropic enhancement in Arabidopsis.

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

Nuclear accumulation of the phytochrome A photoreceptor requires FHY1

The phytochrome family of red/far-red (R/FR)-responsive photoreceptors plays a key role throughout the life cycle of plants . Arabidopsis has five phytochromes, phyA-phyE, among which phyA and phyB play the most predominant functions . Light-regulated nuclear accumulation of the phytochromes is an important regulatory step of this pathway, but to this date no factor specifically required for this event has been identified . Among all phyA signaling mutants, fhy1 and fhy3 (far-red elongated hypocotyl 1 and 3) have the most severe hyposensitive phenotype, indicating that they play particularly important roles . FHY1 is a small plant-specific protein of unknown function localized both in the nucleus and the cytoplasm . Here we show that FHY1 is specifically required for the light-regulated nuclear accumulation of phyA but not phyB. Moreover, phyA accumulation is only slightly affected in fhy3, indicating that the diminished nuclear accumulation of phyA observed in fhy1 seedlings is not simply a general consequence of reduced phyA signaling. By in vitro pull-down and yeast two-hybrid analyses, we demonstrate that FHY1 physically interacts with phyA, preferentially in its active Pfr form. Furthermore, FHY1 and phyA colocalize in planta. We therefore identify the first component required for light-regulated phytochrome nuclear accumulation.     

Functional analysis of yeast-derived phytochrome A and B phycocyanobilin adducts

Investigations of phytochrome mutants of Arabidopsis suggested that the expression of chalcone synthase ( chs ) and anthocyanin accumulation is predominantly controlled by phytochrome A. To test the functionality of phytochrome A and B at the molecular level recombinant, yeast-derived phytochrome-phycocyanobilin adducts (phyA*, phyB*) and oat phytochrome A (phyA) were microinjected into etiolated aurea tomato seedlings. Subsequent to microinjection anthocyanin and chlorophyll accumulation was monitored as well as β-glucuronidase (GUS) expression mediated by light-regulated promoters ( chs , chlorophyll a/b binding protein ( lhcb1 ) and ferredoxin NADP+ oxidoreductase ( fnr )). Microinjection of phyA* under white light conditions caused anthocyanin and chlorophyll accumulation and mediated chs —GUS, lhcb1 —GUS and fnr —GUS expression. Microinjection of phyB* under identical conditions induced chlorophyll accumulation and mediated lhcb1 —GUS and fnr —GUS expression but neither anthocyanin accumulation nor chs —GUS expression were observed. The characterization of Arabidopsis phytochrome mutants and the microinjection experiments suggested that phyB cannot induce the accumulation of juvenile anthocyanin. Microinjections under far-red light conditions demonstrated that phyA can act independently of other photoreceptors. By contrast, phyB* injections under red light conditions indicated that phyB* needs interactions with other photoreceptors to mediate a rapid and efficient de-etiolation signal.     

Mutant screen distinguishes between residues necessary for light-signal perception and signal transfer by phytochrome B

The phytochromes (phyA to phyE) are a major plant photoreceptor family that regulate a diversity of developmental processes in response to light. The N-terminal 651-amino acid domain of phyB (N651), which binds an open tetrapyrrole chromophore, acts to perceive and transduce regulatory light signals in the cell nucleus. The N651 domain comprises several subdomains: the N-terminal extension, the Per/Arnt/Sim (PAS)-like subdomain (PLD), the cGMP phosphodiesterase/adenyl cyclase/FhlA (GAF) subdomain, and the phytochrome (PHY) subdomain. To define functional roles for these subdomains, we mutagenized an Arabidopsis thaliana line expressing N651 fused in tandem to green fluorescent protein, beta-glucuronidase, and a nuclear localization signal. A large-scale screen for long hypocotyl mutants identified 14 novel intragenic missense mutations in the N651 moiety. These new mutations, along with eight previously identified mutations, were distributed throughout N651, indicating that each subdomain has an important function. In vitro analysis of the spectral properties of these mutants enabled them to be classified into two principal classes: light-signal perception mutants (those with defective spectral activity), and signaling mutants (those normal in light perception but defective in intracellular signal transfer). Most spectral mutants were found in the GAF and PHY subdomains. On the other hand, the signaling mutants tend to be located in the N-terminal extension and PLD. These observations indicate that the N-terminal extension and PLD are mainly involved in signal transfer, but that the C-terminal GAF and PHY subdomains are responsible for light perception. Among the signaling mutants, R110Q, G111D, G112D, and R325K were particularly interesting. Alignment with the recently described three-dimensional structure of the PAS-GAF domain of a bacterial phytochrome suggests that these four mutations reside in the vicinity of the phytochrome light-sensing knot.     

Phytochrome signalling modulates the SA-perceptive pathway in Arabidopsis

The interaction of phytochrome signalling with the SA signal transduction pathway has been investigated in Arabidopsis using single and multiple mutants affected in light perception (phyA and phyB deficient) and light-signal processing (psi2, phytochrome signalling). The induction of PR1 by SA and functional analogues has been found to strictly correlate with the activity of the signalling pathway controlled by both phyA and phyB photoreceptors. In darkness as well as dim light, and independently of a carbohydrate source, SA-induced PR gene expression as well as the hypersensitive response to pathogens (HR) are strongly reduced. Moreover, the initiation of HR also exhibits a strict dependence upon both the presence and the amplitude of a phytochrome-elicited signal. The growth of an incompatible strain of bacterial a pathogen (Pseudomonas syringae pv. tomato) was enhanced in phyA-phyB and decreased in psi2 mutants. While functional chloroplasts were found necessary for the development of an HR, the induction of PRs was strictly dependent on light, but independent of functional chloroplasts. Taken together, these data demonstrate that the light-induced signalling pathway interacts with the pathogen/SA-mediated signal transduction route. These results are summarized in a formalism that allows qualitative computer simulation.     

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.     

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.     

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.     

Transposing phytochrome into the nucleus

To control many physiological responses, phytochromes directly modulate gene expression. A key regulatory event in this signal transduction pathway is the light-controlled translocation of the photoreceptor from the cytoplasm into the nucleus. Recent publications are beginning to shed light on the molecular mechanisms underlying this central control point. Interestingly, there is a specific mechanism for phytochrome A (phyA) nuclear accumulation. The dedicated phyA nuclear import pathway might be important for the distinct photosensory specificity of this atypical phytochrome. Recent studies in the field also provide a starting point for investigating how the different subcellular pools of phytochrome can control distinct responses to light.     

PKS1 and PKS2 affect the phyA state in etiolated Arabidopsis seedlings.

Autophosphorylation of phytochrome A (phyA) and transphosphorylation of its reaction partners, phytochrome kinase substrate 1 (PKS1) in particular, might play important functions in signal transduction from phyA. It was shown that PKS1 and PKS2 physically interact with phyA and phyB in vitro, and that overexpression of PKS1 interferes with phytochrome signaling in vivo. Moreover, both pks1 and pks2 loss of function mutants are specifically defective for one branch of phyA signaling. We therefore used in vivo fluorescence spectroscopy to test whether mutations in pks1 and pks2 or overexpression of PKS1 (PKS1OX) have an effect on phyA and its subpopulations, phyA' and phyA'. It was found that the emission spectra of phyA in all the Arabidopsis lines are similar. The phyA content in the single mutants pks1 and pks2, and also in PKS1OX, was 1.2-1.5 times higher than in the wild type, whereas the phyA'/phyA' ratio remained practically unchanged (approx. 1.0). However, in the double mutant pks1pks2, the picture is reversed--the phyA concentration remained unchanged, while the phyA'/phyA' ratio shifted dramatically towards phyA'(0.3). This suggests that (i) the changes in PKS1 or PKS2 content may affect the total phyA concentration, (ii) PKS1, together with PKS2, could be critical for the formation of phyA', thus shifting the equilibrium towards phyA' in the double mutant and (iii) these variations in the phyA' and phyA' content may contribute to the mutant phenotype of pks1, pks2 and PKS1OX. The fact that in the single mutants there are only small changes in the phyA'/phyA' ratio, while in the double mutant the ratio is considerably affected, indicates that PKS1 or PKS2 act redundantly with each other in this regard.     

Variation in dynamics of phytochrome A in Arabidopsis ecotypes and mutants

Phytochromes are photoreceptors in plants which can exist in two different conformations: the red light‐absorbing form (P_r) and the far‐red light‐absorbing form (P_fr), depending on the light quality. The P_fr form is the physiologically active conformation. To attenuate the P_fr signal for phytochrome A (phyA), at least two different mechanisms exist: destruction of the molecule and dark reversion. Destruction is an active process leading to the degradation of P_fr. Dark reversion is the light‐independent conversion of physiologically active P_fr into inactive P_r. Here, we show that dark reversion is not only an intrinsic property of the phytochrome molecule but is modulated by cellular components. Furthermore, we demonstrate that dark reversion of phyA may be observed in Arabidopsis ecotype RLD but not in other Arabidopsis ecotypes. For the first time, we have identified mutants with altered dark reversion and destruction in a set of previously isolated loss of function PHYA alleles (Xu et al. Plant Cell 1995, 7, 1433–1443). Therefore, the dynamics of the phytochrome molecule itself need to be considered during the characterization of signal transduction mutants.     

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 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.     

Phytochromes are Pr-ipatetic kinases.

A series of new studies reveal how the red/far-red light photoreceptors called phytochromes act. Phytochrome A and phytochrome B each move to the nucleus when activated by light, and phytochrome A is a kinase. Phytochrome-interacting proteins provide candidate signal transduction components and a recent physiological study suggests how phyA may mediate responses to far-red light. Regulation of phytochrome nuclear localization and kinase activities creates multiple phytochrome species, which may each have different regulatory activities.     

Interaction of phytochromes A and B in the control of de-etiolation and flowering in pea

The interactions of phytochrome A (phyA) and phytochrome B (phyB) in the photocontrol of vegetative and reproductive development in pea have been investigated using null mutants for each phytochrome. White-light-grown phyA phyB double mutant plants show severely impaired de-etiolation both at the seedling stage and later in development, with a reduced rate of leaf production and swollen, twisted internodes, and enlarged cells in all stem tissues. PhyA and phyB act in a highly redundant manner to control de-etiolation under continuous, high-irradiance red light. The phyA phyB double mutant shows no significant residual phytochrome responses for either de-etiolation or shade-avoidance, but undergoes partial de-etiolation in blue light. PhyB is shown to inhibit flowering under both long and short photoperiods and this inhibition is required for expression of the promotive effect of phyA. PhyA is solely responsible for the promotion of flowering by night-breaks with white light, whereas phyB appears to play a major role in detection of light quality in end-of-day light treatments, night breaks and day extensions. Finally, the inhibitory effect of phyB is not graft-transmissible, suggesting that phyB acts in a different manner and after phyA in the control of flower induction.     

Photomorphogenic mutants of tomato

Photomorphogenesis of tomato (Lycopersicon esculentum Mill.) is being studied with the aid of mutants which are modified either in their photoreceptor composition or in their signal transduction chain(s). Phytochrome chromophore mutants, presumably deficient in all phytochromes, and mutants specifically deficient in phytochrome A (phy A) or B1 (phyB1) have been used to study the roles played by phytochromes in photomorphogenesis. In addition, other mutants, including transgenic lines overproducing phyA, exhibit exaggerated photomorphogenesis. Studies using these mutants are reviewed, with emphasis being placed on anthocyanin biosynthesis and plastid development as model systems for the dissection of the complex interactions between photoreceptors and to elucidate the nature of photoreceptor transduction chains. Recently, new mutants have been isolated by screening in a phyA, phyB1-deficient background. The novel phenotypes selected are candidates for mutants in additional photoreceptors or their transduction chains.     

Functional and biochemical analysis of the N-terminal domain of phytochrome A

Phytochrome A (phyA) is a versatile plant photoreceptor that mediates responses to brief light exposures (very low fluence responses, VLFR) as well as to prolonged irradiation (high irradiance responses, HIR). We identified the phyA-303 mutant allele of Arabidopsis thaliana bearing an R384K substitution in the GAF subdomain of the N-terminal half of phyA. phyA-303 showed reduced phyA spectral activity, almost normal VLFR, and severely impaired HIR. Recombinant N-terminal half oat of PHYA bearing the phyA-303 mutation showed poor incorporation of chromophore in vitro, despite the predicted relatively long distance (>13 A) between the mutation and the closest ring of the chromophore. Fusion proteins bearing the N-terminal domain of oat phyA, beta-glucuronidase, green fluorescent protein, and a nuclear localization signal showed physiological activity in darkness and mediated VLFR but not HIR. At equal protein levels, the phyA-303 mutation caused slightly less activity than the fusions containing the wild-type sequence. Taken together, these studies highlight the role of the N-terminal domain of phyA in signaling and of distant residues of the GAF subdomain in the regulation of phytochrome bilin-lyase activity.     

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.     

Phytochrome A affects stem growth, anthocyanin synthesis, sucrose-phosphate-synthase activity and neighbour detection in sunlight-grown potato

Phytochrome action in fully de-etiolated sunlight-grown potato (Solanum tuberosum L.) was studied by comparing wild-type (WT) plants and transgenic plants with either a sense or an anti-sense phytochrome A (phyA) construction. Radial stem growth, anthocyanin levels, and sucrose-phosphate-synthase activity were directly related to the levels of phyA (severely reduced in transgenics with anti-sense phyA, normal in WT and increased in transgenic with sense phyA). In contrast, longitudinal stem growth was inversely related to the levels of phyA. Phytochrome A influenced stem-extension growth responses to red/far-red ratios perceived by stable phytochrome[s]. First, far-red light reflected by non-shading neighbours promoted stem growth in WT plants but transgenic plants with either increased or reduced phyA levels failed to respond to this light signal. Second, plants with low phyA levels also showed impaired sensitivity to reductions in end-of-day red/far-red ratios. In addition, phyA appears to perceive changes in irradiance reaching the stem: lowering the amount of red plus far-red light reaching the stem promoted stem growth in WT plants. This effect was exaggerated in phyA overexpressors and absent in phyA underexpressors. Thus, phyA is active in fully de-etiolated, sunlight-grown plants. Received: 4 October 1997 / Accepted: 24 October 1997