Far-red High Irradiance Response Research Articles

Reproductive Induction is a Far-Red High Irradiance Response that is Mediated by Phytochrome and PHYTOCHROME INTERACTING FACTOR in Marchantia polymorpha.

Land plants have evolved a series of photoreceptors to precisely perceive environmental information. Among these, phytochromes are the sole photoreceptors for red light (R) and far-red light (FR), and play pivotal roles in modulating various developmental processes. Most extant land plants possess multiple phytochromes that probably evolved from a single phytochrome in the common ancestor of land plants. However, the ancestral phytochrome signaling mechanism remains unknown due to a paucity of knowledge regarding phytochrome functions in basal land plants. It has recently been reported that Mpphy, a single phytochrome in the liverwort Marchantia polymorpha, regulates typical photoreversible responses collectively classified as low fluence response (LFR). Here, we show that Mpphy also regulates the gametangiophore formation analogous to the mode of action of the far-red high irradiance response (FR-HIR) in angiosperms. Our phenotypic analyses using mutant plants obtained by CRISPR/Cas9-based genome editing revealed that MpFHY1, an ortholog of FAR-RED ELONGATED HYPOCOTYL1, as well as Mpphy is critical for the FR-HIR signaling in M. polymorpha. In addition, knockout of MpPIF, a single PHYTOCHROME INTERACTING FACTOR gene in M. polymorpha, completely abolished the FR-HIR-dependent gametangiophore formation, while overexpression of MpPIF accelerated the response. FR-HIR-dependent transcriptional regulation was also disrupted in the Mppif mutant. Our findings suggest that plants had already acquired the FR-HIR signaling mediated by phytochrome and PIF at a very early stage during the course of land plant evolution, and that a single phytochrome in the common ancestor of land plants could mediate both LFR and FR-HIR.

Two GRAS Proteins, SCARECROW-LIKE21 and PHYTOCHROME A SIGNAL TRANSDUCTION1, Function Cooperatively in Phytochrome A Signal Transduction

Photoreceptors, especially the far-red light-absorbing phytochrome A, play a crucial role in early seedling development, triggering the transition from etiolated to photomorphogenic growth. Here, we describe the biological functions of two GRAS proteins from Arabidopsis (Arabidopsis thaliana), SCARECROW-LIKE21 (SCL21) and PHYTOCHROME A SIGNAL TRANSDUCTION1 (PAT1), which are specifically involved in phytochrome A signal transduction. Loss-of-function mutants show an elongated hypocotyl under far-red light and are impaired in other far-red high-irradiance responses. The SCL21 transcript itself is down-regulated by far-red light in a phytochrome A- and PAT1-dependent manner. Our results demonstrate that both SCL21 and PAT1 are positive regulators of phytochrome A signal transduction for several high-irradiance responses. Genetic and biochemical evidence suggest a direct interaction of the two proteins.

Tests of the Link between Functional Innovation and Positive Selection at Phytochrome A: The Phylogenetic Distribution of Far-Red High-Irradiance Responses in Seedling Development

We surveyed the phylogenetic distribution of far-red high-irradiance responses (FR-HIRs), which are important for early development of seedlings in shaded habitats, in exemplar species across seed plants to test whether the responses are unique to angiosperms and may have helped early angiosperms colonize the understory of Mesozoic forests dominated by ferns and gymnosperms. We show that dark-grown seedlings in most seed plants adopt an etiolated morphology and that being green in the dark is a derived condition in some conifers and gnetophytes. We also show that FR-HIR-induced seedling de-etiolation, mediated by phytochrome, is widely distributed in seed plants. Together, these data show that despite the ability of gymnosperms to green in the dark, most of them do not do so but link seedling development to light signals, as do angiosperms. Our data refute an obvious link between the origin of FR-HIRs and the signal of positive selection at the PHYA locus early in the history of angiosperms.

Arabidopsis Phytochrome A Is Modularly Structured to Integrate the Multiple Features That Are Required for a Highly Sensitized Phytochrome

Phytochrome is a red (R)/far-red (FR) light-sensing photoreceptor that regulates various aspects of plant development. Among the members of the phytochrome family, phytochrome A (phyA) exclusively mediates atypical phytochrome responses, such as the FR high irradiance response (FR-HIR), which is elicited under prolonged FR. A proteasome-based degradation pathway rapidly eliminates active Pfr (the FR-absorbing form of phyA) under R. To elucidate the structural basis for the phyA-specific properties, we systematically constructed 16 chimeric phytochromes in which each of four parts of the phytochrome molecule, namely, the N-terminal extension plus the Per/Arnt/Sim domain (N-PAS), the cGMP phosphodiesterase/adenyl cyclase/FhlA domain (GAF), the phytochrome domain (PHY), and the entire C-terminal half, was occupied by either the phyA or phytochrome B sequence. These phytochromes were expressed in transgenic Arabidopsis thaliana to examine their physiological activities. Consequently, the phyA N-PAS sequence was shown to be necessary and sufficient to promote nuclear accumulation under FR, whereas the phyA sequence in PHY was additionally required to exhibit FR-HIR. Furthermore, the phyA sequence in PHY alone substantially increased the light sensitivity to R. In addition, the GAF phyA sequence was important for rapid Pfr degradation. In summary, distinct structural modules, each of which confers different properties to phyA, are assembled on the phyA molecule.

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.

Phytochrome A Mediates Rapid Red Light–Induced Phosphorylation ofArabidopsisFAR-RED ELONGATED HYPOCOTYL1 in a Low Fluence Response

Phytochrome A (phyA) is the primary photoreceptor for mediating the far-red high irradiance response in Arabidopsis thaliana. FAR-RED ELONGATED HYPOCOTYL1 (FHY1) and its homolog FHY1-LIKE (FHL) define two positive regulators in the phyA signaling pathway. These two proteins have been reported to be essential for light-regulated phyA nuclear accumulation through direct physical interaction with phyA. Here, we report that FHY1 protein is phosphorylated rapidly after exposure to red light. Subsequent exposure to far-red light after the red light pulse reverses FHY1 phosphorylation. Such a phenomenon represents a classical red/far-red reversible low fluence response. The phosphorylation of FHY1 depends on functioning phyA but not on other phytochromes and cryptochromes. Furthermore, we demonstrate that FHY1 and FHL directly interact with phyA by bimolecular fluorescence complementation and that both FHY1 and FHL interact more stably with the Pr form of phyA in Arabidopsis seedlings by coimmunoprecipitation. Finally, in vitro kinase assays confirmed that a recombinant phyA is able to robustly phosphorylate FHY1. Together, our results suggest that phyA may differentially regulate FHY1 and FHL activity through direct physical interaction and red/far-red light reversible phosphorylation to fine-tune their degradation rates and resulting light responses.

Spatial-specific phytochrome responses during de-etiolation in

Photoreceptors exhibit complex regulation of many aspects of growth and development, including developmental-, spatial- and temporal-specific photoregulatory responses. Such diverse regulation has been noted for all major classes of photoreceptors in plants, including red/far-red (R/FR) absorbing phytochromes and blue/UV-A (B/UV-A) light-absorbing cryptochromes and phototropins. However, the most insight into spatiotemporal responses has been reported for phytochromes both at the physiological and, more recently, at the molecular levels. Through tissue-specific degradation of the phytochrome chromophore, my laboratory recently demonstrated that phytochromes exhibit light-dependent, spatiotemporal control over de-etiolation responses in Arabidopsis thaliana. Mesophyll-localized phytochrome A (phyA) controls numerous far-red high irradiance responses (FR-HIR) in Arabidopsis. Meristem- and/or leaf primordia-localized phytochromes are involved in the regulation of leaf development. In this addendum, I provide additional novel evidence for spatial-specific, blue-light-dependent signaling roles of phytochromes.

Detection of Spatial-Specific Phytochrome Responses Using Targeted Expression of Biliverdin Reductase in Arabidopsis

To regulate levels of holophytochrome in a spatial-specific manner and investigate the major sites of action of phytochromes during seedling development, we constructed transgenic Arabidopsis (Arabidopsis thaliana) plant lines expressing plastid-targeted mammalian biliverdin IXalpha reductase (pBVR) under regulatory control of CAB3 and MERI5 promoters. Comparative photobiological and phenotypic analyses indicated that spatial-specific expression of pBVR led to the disruption of distinct subsets of phytochrome-regulated responses for different promoters. pBVR expression in photosynthetic tissues (CAB3::pBVR lines) had intermediate effects on chlorophyll accumulation, carotenoid production, anthocyanin synthesis, and leaf development responses in white-light conditions. CAB3::pBVR expression, however, resulted in distinctive phenotypes in far-red (FR) conditions. A number of FR high irradiance responses were disrupted in CAB::pBVR lines, including FR-dependent inhibition of hypocotyl elongation and stimulation of anthocyanin accumulation. By contrast, preferential expression of pBVR in the shoot apical meristem in MERI5::pBVR lines resulted in a phytochrome-deficient, leaf development phenotype under short-day growth conditions. These results implicate leaf-localized phytochrome A as having a unique role in regulating FR-mediated hypocotyl elongation and meristem- and/or leaf primordia-localized phytochromes as having a novel role in phytochrome-dependent responses. Taken together, these studies demonstrate the efficacy of selectively inactivating distinct phytochrome-mediated responses by regulated expression of BVR in transgenic plants, a novel means to investigate the sites of phytochrome photoperception and to regulate specifically light-mediated plant growth and development.

Ginkgo biloba retains functions of both type I and type II flowering plant phytochrome.

While the photoreceptor systems of flowering plants have been well studied, the origins of these gene families and their functions are only partially understood. To begin to resolve the evolutionary origins of angiosperm photoreceptor function, we have studied the photomorphogenic responses of the early diverging gymnosperm Ginkgo biloba. Here, we describe the effects of continuous white light, red light, far-red light, and blue light on stem length, chlorophyll accumulation, Lhcb mRNA accumulation, and plastid development. Differences in the efficacy of these light regimes on de-etiolation in Ginkgo suggest separate but complementary roles for red and blue light-sensing systems. Additionally, the unique manner in which developmental regulation occurs in Ginkgo reveals a far-red high irradiance response different from both angiosperm and other gymnosperm species. We conclude from these data that Ginkgo contains a functional complement to both flowering plant type I and type II phytochromes, as well as independent blue light-sensing system(s). The implications of these findings are discussed with respect to the evolution of higher plant photoreceptors.

Antagonistic but complementary actions of phytochromes A and B allow seedling de-etiolation.

Using dichromatic radiation, we show that the actions of phytochromes A and B (phyA and phyB) in Arabidopsis thaliana are antagonistic in mediating red and far-red radiation effects on seedling de-etiolation and yet act in a complementary manner to regulate de-etiolation, irrespective of spectral composition. At low phytochrome photoequilibria inhibition of hypocotyl extension was strong, because of the action of a far-red high-irradiance response mediated by phyA. At high phytochrome photoequilibria inhibition of hypocotyl extension was also strong, because of the action of phyB. At intermediate photoequilibria hypocotyl inhibition was less strong. In their natural environment, this dual action will strongly retard hypocotyl growth and promote cotyledon opening and expansion both in open daylight and under dense vegetation. Overlapping action by phyA and phyB will substantially promote de-etiolation in sparse vegetation. The antagonistic and complementary actions of phyA and phyB, therefore, allow the optimum regulation of seedling growth after emergence from the soil.

Dominant negative suppression of arabidopsis photoresponses by mutant phytochrome A sequences identifies spatially discrete regulatory domains in the photoreceptor.

We used the exaggerated short hypocotyl phenotype induced by oat phytochrome A overexpression in transgenic Arabidopsis to monitor the biological activity of mutant phytochrome A derivatives. Three different mutations, which were generated by removing 52 amino acids from the N terminus (delta N52), the entire C-terminal domain (delta C617), or amino acids 617-686 (delta 617-686) of the oat molecule, each caused striking dominant negative interference with the ability of endogenous Arabidopsis phytochrome A to inhibit hypocotyl growth in continuous far-red light ("far-red high irradiance response" conditions). By contrast, in continuous white or red light, delta N52 was as active as the unmutagenized oat phytochrome A protein in suppressing hypocotyl elongation, while delta C617 and delta 617-686 continued to exhibit dominant negative behavior under these conditions. These data suggest that at least three spatially discrete molecular domains coordinate the photoregulatory activities of phytochrome A in Arabidopsis seedlings. The first is the chromophore-bearing N-terminal domain between residues 53 and 616 that is apparently sufficient for the light-induced initiation but not the completion of productive interactions with transduction chain components. The second is the C-terminal domain between residues 617 and 1129 that is apparently necessary for completion of productive interactions under all irradiation conditions. The third is the N-terminal 52 amino acids that are apparently necessary for completion of productive interactions only under far-red high irradiance conditions and are completely dispensable under white and red light regimes.

Arabidopsis HY8 locus encodes phytochrome A.

hy8 long hypocotyl mutants of Arabidopsis defective in responsiveness to prolonged far-red light (the so-called "far-red high-irradiance response") are selectively deficient in functional phytochrome A. To define the molecular lesion in these mutants, we sequenced the phytochrome A gene (phyA) in lines carrying one or other of two classes of hy8 alleles. The hy8-1 and hy8-2 mutants that express no detectable phytochrome A each have a single nucleotide change that inserts a translational stop codon in the protein coding sequence. These results establish that phyA resides at the HY8 locus. The hy8-3 mutant that expresses wild-type levels of photochemically active phytochrome A has a glycine-to-glutamate missense mutation at residue 727 in the C-terminal domain of the phyA sequence. Quantitative fluence rate response analysis showed that the mutant phytochrome A molecule produced by hy8-3 exhibited no detectable regulatory activity above that of the phyA-protein-deficient hy8-2 mutant. This result indicates that glycine-727, which is invariant in all sequenced phytochromes, has a function important to the regulatory activity of phytochrome A but not to photoperception.

Hy8, a new class of arabidopsis long hypocotyl mutants deficient in functional phytochrome A.

Emerging evidence suggests that individual members of the phytochrome family of photoreceptors may regulate discrete facets of plant photomorphogenesis. We report here the isolation of phytochrome A mutants of Arabidopsis using a novel screening strategy aimed at detecting seedlings with long hypocotyls in prolonged far-red light. Complementation analysis of 10 selected mutant lines showed that each represents an independent, recessive allele at a new locus, designated hy8. Immunoblot and spectrophotometric analyses of two of these lines, hy8-1 and hy8-2, showed that, whereas phytochromes B and C are expressed at wild-type levels, phytochrome A is undetectable, thus indicating that the long hypocotyl phenotype displayed by these mutants is caused by phytochrome A deficiency. A third allele, hy8-3, expresses wild-type levels of spectrally normal phytochrome A, suggesting a mutation that has resulted in loss of biological activity in an otherwise photochemically active photoreceptor molecule. Together with physiological experiments, these data provide direct evidence that endogenous phytochrome A is responsible for the "far-red high irradiance response" of etiolated seedlings, but does not play a major role in mediating responses to prolonged red or white light. Because the hy8 and the phytochrome B-deficient hy3 mutants exhibit reciprocal responsivity toward prolonged red and far-red light, respectively, the evidence indicates that phytochromes A and B have distinct photosensory roles in regulating seedling development.

Light-induced changes in the photoresponses of plant stems the loss of a high irradiance response to far-red light

De-etiolation results in phytochrome destruction, greening, and the loss of the far-red high irradiance responses (HIR). Evidence is presented against the hypothesis that the loss of the far-red HIR is a direct consequence of phytochrome destruction. Loss of the far-red HIR for the inhibition of elongation in hypocotyls of Raphanus sativus involves two different, but linked, actions of phytochrome. An induction reaction requires the far-red absorbing form of phytochrome for about 20 min after which accumulation of its product depends only on time. A second reaction requires continuous light or frequent short irradiations and involves cycling of the phytochrome system. This acts on the product of the induction reaction. It is proposed that in green plants an important mode of operation of phytochrome in the light depends on pigment cycling, and that during de-etiolation this system is established under phytochrome control.