Far-red High Irradiance 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.

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.

Light-Regulated Nuclear Import and Degradation of Arabidopsis Phytochrome-A N-Terminal Fragments

The photoreceptor phytochrome-A (phyA) regulates germination and seedling establishment by mediating very low fluence (VLFR) and far-red high irradiance (FR-HIR) responses in Arabidopsis thaliana. In darkness, phyA homodimers exist in the biologically inactive Pr form and are localized in the cytoplasm. Light induces formation of the biologically active Pfr form and subsequent rapid nuclear import. PhyA Pfr, in contrast to the Pr form, is labile and has a half-life of ∼30 min. We produced transgenic plants in a phyA-201 null background that express the PHYA-yellow fluorescent protein (YFP) or the PHYA686-YFP-dimerization domain (DD) and PHYA686-YFP-DD-nuclear localization signal (NLS) or PHYA686-YFP-DD-nuclear exclusion signal (NES) fusion proteins. The PHYA686-YFP fusion proteins contained the N-terminal domain of phyA (686 amino acid residues), a short DD and the YFP. Here we report that (i) PHYA686-YFP-DD fusion protein is imported into the nucleus in a light-dependent fashion; (ii) neither of the PHYA686 fusion proteins is functional in FR-HIR and nuclear VLFR; and (iii) the phyA-dependent, blue light-induced inhibition of hypocotyl growth is mediated by the PHYA686-YFP-DD-NES but not by the PHYA686-YFP-DD-NLS and PHYA686-YFP-DD fusion proteins. We demonstrate that (i) light induces degradation of all PHYA N-terminal-containing fusion proteins and (ii) these N-terminal domain-containing fusion proteins including the constitutively nuclear PHYA686-YFP-DD-NLS and predominantly cytoplasmic PHYA686-YFP-DD-NES degrade at comparable rates but markedly more slowly than PHYA-YFP, whereas (iii) light-induced degradation of the native phyA is faster compared with PHYA-YFP.

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.

Fluorescence and photochemical properties of phytochromes in wild‐type wheat and a transgenic line overexpressing an oat phytochrome A (PHYA) gene: functional implications

AbstractEtiolated seedlings of wild‐type wheat and a transgenic line overexpressing an oat PHYA gene were investigated by the use of in situ low‐temperature fluorescence spectroscopy. The red‐absorbing phytochrome form, Pr, was characterized by (1) fluorescence emission spectrum; (2) total phytochrome content, and (3) by the extent of the Pr → lumi‐R photoconversion at low temperature (γ1), and of the Pr → Pfr photoconversion at ambient temperature (γ2) as derived from emission data. All the characteristics were shown to be variable and to depend on (1) organ and tissue used; (2) seedling age; (3) transgenic wheat modification, and (4) continuous far‐red irradiation of seedlings during their growth. These variations were interpreted in terms of the existence in wheat seedlings of the two phenomenological Pr types: (a), Pr′– major longer wavelength (687/673 nm, emission/absorption maxima) variable and light‐labile with γ1 ≈ 0·5; and (b), Pr′′– minor, shorter wavelength (682/668 nm), relatively constant with its concentration not changing significantly with the increase of total phytochrome content in tissues and light‐stable with γ1 ≤ 0·05–0·1. Overexpression of oat phyA increases primarily the content of Pr′ suggesting that it is comprised of phyA (phyA′) whereas Pr′′ is believed to consist of the minor phyA fraction (phyA′′) and phyB. The transgenic wheat line has been demonstrated to have a modified phenotype – the appearance of the far‐red high irradiance reaction (FR‐HIR) (Shlumukov et al. Plant, Cell and Environment 24, 703–712). The increased content of phyA′ in the transgenic line, whereas the total [phyA′′ + phyB] remains the same as in the wild type, indicates that the phyA′ pool is primarily responsible for the observed modification of the phenotype and suggests that even in wild‐type plants the phyA′ component of the phyA pool may mediate the FR‐HIR.

Regulation of HEMA1 expression by phytochrome and a plastid signal during de‐etiolation in Arabidopsis thaliana

The synthesis of 5-aminolevulinic acid (ALA) is the rate-limiting step for the formation of all plant tetrapyrroles, including chlorophyll and heme, and regulation of ALA synthesis is therefore critical to plant development. Glutamyl-tRNA reductase (GluTR) is the first committed enzyme of this pathway and is encoded by a small family of nuclear HEMA genes. Here, we have used transgenic Arabidopsis (Arabidopsis thaliana L. Col) lines expressing chimeric HEMA1 promoter:gusA fusion genes, combined with RNA gel blot analyses, to characterise the light-mediated regulation of the Arabidopsis HEMA1 gene during de-etiolation. HEMA1 was expressed strongly, but not exclusively, in photosynthetic tissues and was shown to be light regulated at the transcriptional level by the phytochrome family of photoreceptors acting in both the far-red high irradiance and low fluence response modes. The HEMA2 gene, which is expressed only in roots of seedlings, was not light regulated. Analysis of truncated HEMA1 promoter constructs demonstrated that a -199/+252 promoter fragment was sufficient to confer full light-responsiveness to gusA expression. This fragment contained GT-1/I-box and CCA-1 binding sites that are implicated as the light-responsive cis elements. Both the full-length and truncated HEMA1 promoters required the presence of intact chloroplasts for full expression, consistent with previous indications that light and plastid factor signals converge to co-ordinately regulate expression of photosynthesis-related nuclear genes. These results provide the most comprehensive analysis to date of the light-regulation of a tetrapyrrole biosynthetic gene and support a direct link between regulation of HEMA1 transcription and chlorophyll accumulation during seedling de-etiolation.

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.

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.