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.     

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.     

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.     

FHY1 and FHL act together to mediate nuclear accumulation of the phytochrome A photoreceptor

The phytochrome family of red/far-red photoreceptors is involved in the regulation of a wide range of developmental responses in plants. The Arabidopsis genome contains five phytochromes (phyA-E), among which phyA and phyB play the most important roles. Phytochromes localize to the cytosol in the dark and accumulate in the nucleus under light conditions, inducing specific phytochrome-mediated responses. Light-regulated nuclear accumulation of the phytochrome photoreceptors is therefore considered a key regulatory step of these pathways. In fact, one of the most severe phyA signaling mutants, fhy1 (far red elongated hypocotyl 1), is strongly affected in nuclear accumulation of phyA. The fhy1 fhl (fhy1 like) double mutant, lacking both FHY1 and its only close homolog FHL, is virtually blind to far-red light like phyA null seedlings. Here we show that FHL accounts for residual amounts of phyA in the nucleus in a fhy1 background and that nuclear accumulation of phyA is completely inhibited in an fhy1 FHL RNAi knock-down line. Moreover, we demonstrate that FHL and phyA interact with each other in a light-dependent manner and that they co-localize in light-induced nuclear speckles. We also identify a phyA-binding site at the C-terminus of FHY1 and FHL, and show that the N-terminal 406 amino acids of phyA are sufficient for the interaction with FHY1/FHL.     

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.     

A short amino-terminal part of Arabidopsis phytochrome A induces constitutive photomorphogenic response

Phytochrome A (phyA) is the dominant photoreceptor of far-red light sensing in Arabidopsis thaliana. phyA accumulates at high levels in the cytoplasm of etiolated seedlings, and light-induced phyA signaling is mediated by a complex regulatory network. This includes light- and FHY1/FHL protein-dependent translocation of native phyA into the nucleus in vivo. It has also been shown that a short N-terminal fragment of phyA (PHYA406) is sufficient to phenocopy this highly regulated cellular process in vitro. To test the biological activity of this N-terminal fragment of phyA in planta, we produced transgenic phyA-201 plants expressing the PHYA406-YFP (YELLOW FLUORESCENT PROTEIN)-DD, PHYA406-YFP-DD-NLS (nuclear localization signal), and PHYA406-YFP-DD-NES (nuclear export signal) fusion proteins. Here, we report that PHYA406-YFP-DD is imported into the nucleus and this process is partially light-dependent whereas PHYA406-YFP-DD-NLS and PHYA406-YFP-DD-NES display the expected constitutive localization patterns. Our results show that these truncated phyA proteins are light-stable, they trigger a constitutive photomorphogenic-like response when localized in the nuclei, and neither of them induces proper phyA signaling. We demonstrate that in vitro and in vivo PHYA406 Pfr and Pr bind COP1, a general repressor of photomorphogenesis, and co-localize with it in nuclear bodies. Thus, we conclude that, in planta, the truncated PHYA406 proteins inactivate COP1 in the nuclei in a light-independent fashion.     

The nuclear localization signal and the C-terminal region of FHY1 are required for transmission of phytochrome A signals

Plants use the family of phytochrome photoreceptors to sense their light environment in the red/far-red region of the spectrum. Phytochrome A (phyA) is the primary photoreceptor that regulates germination and early seedling development. This phytochrome mediates seedling de-etiolation for the developmental transition from heterotrophic to photoauxotrophic growth. High intensity far-red light provides a way to specifically assess the role of phyA in this process and was used to isolate phyA-signaling intermediates. fhy1 and pat3 (renamed fhy1-3) are independently isolated alleles of a gene encoding a phyA signal transduction component. FHY1 is a small 24 kDa protein that shows no homology to known functional motifs, besides a small conserved septin-related domain at the C-terminus, a putative nuclear localization signal (NLS) and a putative nuclear exclusion signal (NES). Here we demonstrate that the septin-related domain is important for FHY1 to transmit phyA signals. Moreover, the putative NLS and NES of FHY1 are indeed involved in its nuclear localization and exclusion. Nuclear localization of FHY1 is needed for it to execute responses downstream of phyA. Together with the results from global expression analysis, our findings point to an important role of FHY1 in phyA signaling through its nuclear translocation and induction of gene expression.     

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.     

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.     

Effects of missense mutation on structure and function of photoreceptor

Phytochromes (PHYs) are photoreceptors of the red (R ~660  nm) and far-red (FR ~730 nm) light, and they control a wide range of responses affecting crucial aspects of plant life. There are five genes PHYA-PHYE encoding for phytochromes of different but overlapping function. One of these, PHYA has the unique function controlling specific responses in high irradiance far-red, as well as in very weak light. Appropriate PHYA functioning requires not only the photoreversibility of molecule but also the proper nuclear localization and degradation of receptor. Recently, we identified and described a mutant PHYA allele (phyA-5) in Arabidopsis thaliana, which showed reduced binding affinity to FHY1/FHL, the proteins regulating its nuclear transport, resulting in impaired nuclear localization and altered signaling under certain conditions. We present here a hypothesis to explain how the identified amino acid substitution may lead to structural changes manifested as altered signaling and phenotype displayed by the phyA-5 mutant.     

RED AND FAR-RED INSENSITIVE 2, a RING-domain zinc finger protein, mediates phytochrome-controlled seedling deetiolation responses

Light is arguably the most important resource for plants, and an array of photosensory pigments enables plants to develop optimally in a broad range of ambient-light conditions. The red- and far-red-light-absorbing photosensory pigments or phytochromes (phy) regulate seedling deetiolation responses, photoperiodic flowering, and circadian rhythm. We have identified a long hypocotyl mutant under red and far-red light, rfi2-1 (red and far-red insensitive 2 to 1). rfi2-1 was also impaired in phytochrome-mediated end-of-day far-red light response, cotyledon expansion, far-red light block of greening, and light-induced expression of CHLOROPHYLL A/B BINDING PROTEIN 3 and CHALCONE SYNTHASE. Introduction of rfi2-1 mutation into phyB-9 or phyA-211 did not enhance or suppress the long hypocotyl phenotype of phyB-9 or phyA-211 under red or far-red light, respectively, and RFI2 likely functions downstream of phyB or phyA. RFI2 was identified through the segregation of two T-DNA insertions into different recombinant lines, genetic rescue, and phenotypic characterization of a second mutant allele rfi2-2. RFI2 encodes a protein with a C₃H₂C₃-type zinc finger or RING domain known to mediate protein-protein or protein-DNA interactions, and RFI2 is localized to the nucleus. RFI2 therefore reveals a signaling step that mediates phytochrome control of seedling deetiolation.     

Analysis of far-red light-regulated genome expression profiles of phytochrome A pathway mutants in Arabidopsis

Phytochrome A (phyA) is the primary photoreceptor responsible for various far-red (FR) light-mediated responses. Previous studies have identified multiple phyA signaling mutants, including both positive and negative regulators of the phyA-mediated responses. How these defined intermediates act to mediate FR light responses is largely unknown. Here a cDNA microarray was used to examine effects of those mutations on the far-red light control of genome expression. Clustering analysis of the genome expression profiles supports the notion that phyA signaling may entail a network with multiple paths, controlling overlapping yet distinct sets of gene expression. FHY1, FAR1 and FHY3 most likely act upstream in the phyA signaling network, close to the phyA photoreceptor itself. FIN219, SPA1 and REP1 most likely act somewhere more downstream in the network and control the expression of smaller sets of genes. Further, this study also provides genomics evidence for the partial functional redundancy between FAR1 and FHY3. These two homologous proteins control the expression of a largely overlapping set of genes, and likely act closely together in the phyA-mediated FR light responses.     

PNZIP Is a Novel Mesophyll-Specific cDNA That Is Regulated by Phytochrome and a Circadian Rhythm and Encodes a Protein with a Leucine Zipper Motif

Single, double, and triple null combinations of Arabidopsis mutants lacking the photoreceptors phytochrome (phy) A (phyA-201), phyB (phyB-5), and cryptochrome (cry) 1 (hy4-2.23n) were examined for de-etiolation responses in high-fluence red, far-red, blue, and broad-spectrum white light. Cotyledon unhooking, unfolding, and expansion, hypocotyl growth, and the accumulation of chlorophylls and anthocyanin in 5-d-old seedlings were measured under each light condition and in the dark. phyA was the major photoreceptor/effector for most far-red-light responses, although phyB and cry1 modulated anthocyanin accumulation in a phyA-dependent manner. phyB was the major photoreceptor in red light, although cry1 acted as a phyA/phyB-dependent modulator of chlorophyll accumulation under these conditions. All three photoreceptors contributed to most blue light deetiolation responses, either redundantly or additively; however, phyB acted as a modulator of cotyledon expansion dependent on the presence of cry1. As reported previously, flowering time in long days was promoted by phyA and inhibited by phyB, with each suppressing the other's effect. In addition to the effector/modulator relationships described above, measurements of hypocotyls from blue-light-grown seedlings demonstrated phytochrome activity in blue light and cry1 activity in a phyAphyB mutant background.