Red light-induced phytochrome relocation into the nucleus in Adiantum capillus-veneris

Phytochromes in seed plants are known to move into nuclei in a red light-dependent manner with or without interacting factors. Here, we show phytochrome relocation to the nuclear region in phytochrome-dependent Adiantum capillus-veneris spore germination by partial spore-irradiation experiments. The nuclear or non-nuclear region of imbibed spores was irradiated with a microbeam of red and/or far-red light and the localization of phytochrome involved in spore germination was estimated from the germination rate. The phytochrome for spore germination existed throughout whole spore under darkness after imbibition, but gradually migrated to the nuclear region following red light irradiation. Intracellular distribution of PHY-GUS fusion proteins expressed in germinated spores by particle bombardment showed the migration of Acphy2, but not Acphy1, into nucleus in a red light-dependent manner, suggesting that Acphy2 is the photoreceptor for fern spore germination.     

Phytochrome A resets the circadian clock and delays tuber formation under long days in potato

Transgenic potatoes (Solanum tuberosum) with either increased (sense transformants) or reduced (antisense transformants) phytochrome A (phyA) levels were used, in combination with specific light treatments, to investigate the involvement of phyA in the perception of signals that entrain the circadian clock. Far-red or far-red plus red light treatments given during the night reset the circadian rhythm of leaf movements in wild-type plants and phyA over-expressors, but had little effect in phyA under-expressors. Far-red light was also able to reset the rhythm of leaf movement in wild-type Arabidopsis thaliana but was not effective in mutants without phyA. Blue light was necessary to reset the rhythm in phyA-deficient potato plants. Resetting of the rhythm by far-red plus red light was only slightly affected in transgenic plants with reduced levels of phytochrome B. The production of tubers was delayed by day extensions with far-red plus red light, but this effect was reduced in transgenic lines deficient in phyA. We conclude that phyA is involved in resetting the circadian clock controlling leaf movements and in photoperiod sensing in light-grown potato plants.     

Functional analyses of the Physcomitrella patens phytochromes in regulating chloroplast avoidance movement

Red light-induced chloroplast movement in Physcomitrella patens (Pp) is mediated by dichroic phytochrome in the cytoplasm. To analyze the molecular function of the photoreceptor in the cytoplasm, we developed a protoplast system in which chloroplast photomovement was exclusively dependent on the expression of phytochrome cDNA constructs introduced by polyethylene glycol (PEG) transformation. YFP was fused to the phytochrome constructs and their expression was detected by fluorescence. The chloroplast avoidance response was induced in the protoplasts expressing a YFP fusion of PHY1-PHY3, but not of PHY4 or YFP alone. Phy::yfp fluorescence was detected in the cytoplasm. No change in the location of phy1::yfp or phy2::yfp was revealed before and after photomovement. When phy1::yfp and phy2::yfp were targeted to the nucleus by fusing a nuclear localization signal to the constructs, red light avoidance was not induced. To determine the domains of PHY2 essential for avoidance response, various partially-deleted PHY2::YFP constructs were tested. The N-terminal extension domain (NTE) was found to be necessary but the C-terminal histidine kinase-related domain (HKRD) was dispensable. An avoidance response was not induced under expression of phytochrome N-terminal half domain [deleting both the PAS (Per, Arnt, Sim)-related domain (PRD) and HKRD]. GUS fusion of this N-terminal half domain, reported to be fully functional in Arabidopsis for several phyA- and phyB-regulated responses was not effective in chloroplast avoidance movement. Domain requirement and GUS fusion effect were also confirmed in PHY1. These results indicate that Pp phy1-Pp phy3 in the cytoplasm mediate chloroplast avoidance movement, and that NTE and PRD, but not HKRD, are required for their function.     

Overexpression of homologous phytochrome genes in tomato: exploring the limits in photoperception

Transgenic tomato [Lycopersicon esculentum (=Solanum lycopersicum)] lines overexpressing tomato PHYA, PHYB1, or PHYB2, under control of the constitutive double-35S promoter from cauliflower mosaic virus (CaMV) have been generated to test the level of saturation in individual phytochrome-signalling pathways in tomato. Western blot analysis confirmed the elevated phytochrome protein levels in dark-grown seedlings of the respective PHY overexpressing (PHYOE) lines. Exposure to 4 h of red light resulted in a decrease in phytochrome A protein level in the PHYAOE lines, indicating that the chromophore availability is not limiting for assembly into holoprotein and that the excess of phytochrome A protein is also targeted for light-regulated destruction. The elongation and anthocyanin accumulation responses of plants grown under white light, red light, far-red light, and end-of-day far-red light were used for characterization of selected PHYOE lines. In addition, the anthocyanin accumulation response to different fluence rates of red light of 4-d-old dark-grown seedlings was studied. The elevated levels of phyA in the PHYAOE lines had little effect on seedling and adult plant phenotype. Both PHYAOE in the phyA mutant background and PHYB2OE in the double-mutant background rescued the mutant phenotype, proving that expression of the transgene results in biologically active phytochrome. The PHYB1OE lines showed mild effects on the inhibition of stem elongation and anthocyanin accumulation and little or no effect on the red light high irradiance response. By contrast, the PHYB2OE lines showed a strong inhibition of elongation, enhancement of anthocyanin accumulation, and a strong amplification of the red light high irradiance response.     

APICAL GROWTH OF PROTONEMATA IN ADIANTUM CAPILLUSVENERIS. I. RED FAR-RED REVERSIBLE EFFECT ON GROWTH CESSATION IN THE DARK

Apical growth of individual protonemata in Adiantum capillus-veneris was microphotographically observed before, during and after light treatment. When single-celled protonemata precultured under continuous red light were transferred to darkness, the apical growth continued for the next 24 hr at a rate somewhat slower than that under continuous red light, but the rate significantly decreased thereafter and growth ceased within 72 hr in the dark. The growth in the dark was strongly inhibited by a brief irradiation with far-red light given immediately before the dark period, and the effect of far-red light was fully reversed by subsequent red light. This reversibility was repeatedly observed, suggesting the involvement of a phytochrome system.
The intracellular localization of the phytochrome system in the protonemata was studied, using a narrow-beam irradiator. The results showed that the photoreceptive sites of far-red light are not localized in any particular region of the cell.     

The fern Adiantum capillus-veneris lacks stomatal responses to blue light

We investigated the responses of stomata to light in the fern Adiantum capillus-veneris, a typical species of Leptosporangiopsida. Stomata in the intact leaves of the sporophytes opened in response to red light, but they did not open when blue light was superimposed on the red light. The results were confirmed in the isolated Adiantum epidermis. The red light-induced stomatal response was not affected by the mutation of phy3, a chimeric protein of phytochrome and phototropin in this fern. The lack of a blue light-specific stomatal response was observed in three other fern species of Leptosporangiopsida, i.e. Pteris cretica, Asplenium scolopendrium and Nephrolepis auriculata. Fusicoccin, an activator of the plasma membrane H(+)-ATPase, induced both stomatal opening and H(+) release in the Adiantum epidermis. Adiantum phototropin genes AcPHOT1 and AcPHOT2 were expressed in the fern guard cells. The transformation of an Arabidopsis phot1 phot2 double mutant, which lost blue light-specific stomatal opening, with AcPHOT1 restored the stomatal response to blue light. Taken together, these results suggest that ferns of Leptosporangiopsida lack a blue light-specific stomatal response, although the functional phototropin and plasma membrane H(+)-ATPase are present in this species.     

Intramolecular co-action of two independent photosensory modules in the fern phytochrome 3

Fern phytochrome3/neochrome1 (phy3/neo1) is a chimeric photoreceptor composed of a phytochrome-chromophore binding domain and an almost full-length phototropin. phy3 thus contains two different light-sensing modules; a red/far-red light receptor phytochrome and a blue light receptor phototropin. phy3 induces both red light- and blue light-dependent phototropism in phototropin-deficient Arabidopsis thaliana (phot1 phot2) seedlings. The red-light response is dependent on the phytochrome module of phy3, and the blue-light response is dependent on the phototropin module. We recently showed that both the phototropin-sensing module and the phytochrome-sensing module mediate the blue light-dependent phototropic response. Particularly under low-light conditions, these two light-sensing modules cooperate to induce the blue light-dependent phototropic response. This intramolecular co-action of two independent light-sensing modules in phy3 enhances light sensitivity, and perhaps allowed ferns to adapt to the low-light canopy conditions present in angiosperm forests.     

Action spectra for polarotropism and phototropism in protonemata of the fern Adiantum capillus-veneris

The action spectrum for polarotropism was determined, using the Okazaki large spectrograph, by brief irradiation with light between 260 nm and 850 nm in single-celled protonemata of the fern Adiantum capillus-veneris L., which had been cultured for 6 days in red light and then in the dark for 15 h. The action spectrum had a peak at around 680 nm. This effect was nullified by subsequent irradiaton with far-red light, and typical red/far-red reversibility was observed, indicating the involvement of phytochrome. Polarized ultraviolet or blue light had no effect on the direction of apical growth. The action spectrum for phototropism was also determined in the red light region by means of brief microbeam irradiation of a flank of the subapical region of the protonema. This spectrum showed a peak at 662 nm which was consistent with the absorption peak of phytochrome, but not with the peak of the action spectrum for polarotropism.     

Control of Mitosis by Phytochrome and a Blue-Light Receptor in Fern Spores

The first mitosis in spores of the fern A. capillus-veneris was observed under a microscope equipped with Nomarski optics with irradiation from a safelight at 900 nm, and under a fluorescent microscope after staining with 4[prime],6-diamidino-2-phenylindole. During imbibition the nucleus remained near one corner of each tetrahedron-shaped dormant spore, and asymmetric cell division occurred upon brief irradiation with red light. This red light-induced mitosis was photoreversibly prevented by subsequent brief exposure to far-red light and was photo-irreversibly prevented by brief irradiation with blue light. However, neither far-red nor blue light affected the germination rate when spores were irradiated after the first mitosis. Therefore, the first mitosis in the spores appears to be the crucial step for photoinduction of spore germination. Furthermore, experiments using a microbeam of red or blue light demonstrated that blue light was effective only when exposed to the nucleus, and no specific intracellular photoreceptive site for red light was found in the spores. Therefore, phytochrome in the far-red absorbing form induces the first mitosis in germinating spores but prevents the subsequent mitosis in protonemata, whereas a blue-light receptor prevents the former but induces the latter.     

Overexpression of rice phytochrome A partially complements phytochrome B deficiency in Arabidopsis

The red/far-red reversible phytochromes play a central role in regulating the development of plants in relation to their light environment. Studies on the roles of different members of the phytochrome family have mainly focused on light-labile, phytochrome A and light-stable, phytochrome B. Although these two phytochromes often regulate identical responses, they appear to have discrete photosensory functions. Thus, phytochrome A predominantly mediates responses to prolonged far-red light, as well as acting in a non-red/far-red-reversible manner in controlling responses to light pulses. In contrast, phytochrome B mediates responses to prolonged red light and acts photoreversibly under light-pulse conditions. However, it has been reported that rice (Oryza sativa L.) phytochrome A operates in a classical red/far-red reversible fashion following its expression in transgenic tobacco plants. Thus, it was of interest to determine whether transgenic rice phytochrome A could substitute for loss of phytochrome B in phyB mutants of Arabidopsis thaliana (L.) Heynh. We have observed that ectopic expression of rice phytochrome A can correct the reduced sensitivity of phyB hypocotyls to red light and restore their response to end-of-day far-red treatments. The latter is widely regarded as a hallmark of phytochrome B action. However, although transgenic rice phytochrome A can correct other aspects of elongation growth in the phyB mutant it does not restore other responses to end-of-day far-red treatments nor does it restore responses to low red:far-red ratio. Furthermore, transgenic rice phytochrome A does not correct the early-flowering phenotype of phyB seedlings. Received: 12 July 1998 / Accepted: 13 August 1998     

Characterization of a novel non-constitutive photomorphogenic cop1 allele

A specific light program consisting of multiple treatments with alternating red and far-red light pulses was used to isolate mutants in phytochrome A-dependent signal transduction in Arabidopsis seedlings. Because of their phenotype, the mutants were called eid (empfindlicher im dunkelroten Licht, which means hypersensitive in far-red light). One of the isolated mutants, eid6, is a novel recessive allele of the COP1 gene (constitutive photomorphogenic 1) that carries an amino acid transition in a conserved histidine residue of the RING finger domain. Mutant seedlings exhibited an extreme hypersensitivity towards all tested light qualities, but in contrast to known cop1 alleles, no constitutive photomorphogenic phenotype was detectable in darkness. Thus, the novel cop1eid6 allele seems to encode for a protein whose remaining activity is sufficient for the suppression of photomorphogenesis in dark-grown plants. In adult cop1eid6 plants, the development of the Cop1 phenotype is dominated by phytochrome B. Comparison of the phenotype of the novel cop1eid6 and the weak cop1-4 allele under continuous far-red light indicates that the RING finger and coiled-coil domains of COP1 are sufficient for some specific regulatory function in phytochrome A-dependent high irradiance responses.     

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.     

Downstream effectors of light- and phytochrome-dependent regulation of hypocotyl elongation in Arabidopsis thaliana

Arabidopsis, like most plants, exhibits tissue-specific, light-dependent growth responses. Cotyledon and leaf growth and the accumulation of photosynthetic pigments are promoted by light, whereas hypocotyl growth is inhibited. The identification and characterization of distinct phytochrome-dependent molecular effectors that are associated with these divergent tissue-specific, light-dependent growth responses are limited. To identify phytochrome-dependent factors that impact the photoregulation of hypocotyl length, we conducted comparative gene expression studies using Arabidopsis lines exhibiting distinct patterns of phytochrome chromophore inactivation and associated disparate hypocotyl elongation responses under far-red (FR) light. A large number of genes was misregulated in plants lacking mesophyll-specific phytochromes relative to constitutively-deficient phytochrome lines. We identified and characterized genes whose expression is impacted by light and by phyA and phyB that have roles in the photoregulation of hypocotyl length. We characterized the functions of several identified target genes by phenotyping of T-DNA mutants. Among these genes is a previously uncharacterized LHE (LIGHT-INDUCED HYPOCOTYL ELONGATION) gene, which we show impacts light- and phytochrome-mediated regulation of hypocotyl elongation under red (R) and FR illumination. We describe a new approach for identifying genes involved in light- and phytochrome-dependent, tissue-specific growth regulation and confirmed the roles of three such genes in the phytochrome-dependent photoregulation of hypocotyl length.     

FIN5 positively regulates far-red light responses in Arabidopsis thaliana

We report the characterization of a semi-dominant mutation fin5-1 (far-red insensitive 5-1) of Arabidopsis, which was isolated from genetic screening of phytochrome A (phyA) signaling components. Plants with the fin5-1 mutation exhibited a long hypocotyl phenotype when grown under far-red (FR) light, but not under red light. Physiological analyses implied that FIN5 might be differentially involved in diverse responses that are regulated by phyA under continuous FR light. Anthocyanin accumulation, gravitropic response of hypocotyl growth, and FR light-preconditioned blocking of greening were also impaired in the fin5-1 mutant, whereas photoperiodic floral induction was not, if at all, significantly affected. Moreover, light-regulated expression of the CHS, PORA and PsbS genes was attenuated in fin5-1 mutant plants, while the light-induced expression of CAB was normal. The mutation exhibited semi-dominance regarding control of hypocotyl growth in FR light. We suggest that FIN5 defines a novel branch in the network of phyA signaling in Arabidopsis.     

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.     

Negative phototropic response of rhizoid cells in the fern Adiantum capillus-veneris

In general, phototropic responses in land plants are induced by blue light and mediated by blue light receptor phototropins. In many cryptogam plants including the fern Adiantum capillus-veneris, however, red as well as blue light effectively induces a positive phototropic response in protonemal cells. In A. capillus-veneris, the red light effect on the tropistic response is mediated by phytochrome 3 (phy3), a chimeric photoreceptor of phytochrome and full-length phototropin. Here, we report red and blue light-induced negative phototropism in A. capillus-veneris rhizoid cells. Mutants deficient for phy3 lacked red light-induced negative phototropism, indicating that under red light, phy3 mediates negative phototropism in rhizoid cells, contrasting with its role in regulating positive phototropism in protonemal cells. Mutants for phy3 were also partially deficient in rhizoid blue light-induced negative phototropism, suggesting that phy3, in conjunction with phototropins, redundantly mediates the blue light response.     

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-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.     

Roles of different phytochromes in Arabidopsis photomorphogenesis

The red/far-red light-absorbing phytochromes play fundamental roles in photoperception of the light environment and the subsequent adaptation of plant growth and development. Higher plants possess multiple, discrete phytochromes, the apoproteins of which are the products of a family of divergent (PHY) genes. Arabidopsis thaliana has at least five PHY genes, encoding the apoproteins of phytochromes A-E. Through the analysis of mutants that are deficient in phytochrome A or B and the corresponding double mutant, it is becoming clear that these phytochromes perform both discrete and overlapping roles throughout plant development. Through analysis of the phyA phyB double mutant, it has been possible to define several responses that are mediated by other members of the phytochrome family. This article reviews some of the recent progress in the study of phytochrome-deficient mutants of the model plant Arabidopsis thaliana.     

Isolation and Initial Characterization of Arabidopsis Mutants That Are Deficient in Phytochrome A

Phytochrome, a red/far-red-light photoreceptor protein of plants, is encoded by a small gene family. Phytochrome A (PHYA), the product of the PHYA gene, is the predominant molecular species of phytochrome in etiolated tissue and has been best characterized biochemically. To define a role for PHYA, we isolated new mutants, designated fre1 (far-red elongated), in Arabidopsis thaliana that were specifically deficient in PHYA spectral activity and protein accumulation. These mutants were identified on the basis of their long hypocotyl phenotype under continuous far-red light. Although the fre1 mutants lacked the hypocotyl response to continuous far-red light, their responses to continuous white light and to end-of-day far-red-light treatments were normal. Thus, PHYA appears to play only a minor role in the regulation of hypocotyl elongation under natural conditions. In contrast, the fre1 mutation affected greening a fre1 mutant was less able than the wild type to deetiolate after growth in the dark. However, the potentiation effect of a red-light pulse on accumulation of chlorophyll was not changed significantly in the fre1 mutants. Thus, the function of PHYA might be highly specialized and restricted to certain phases of Arabidopsis development.