Protein Differences between Two Isogenic Cultivars of Barley (Hordeum vulgare L.) that Differ in Sensitivity to Photoperiod and Far-Red Light

A photoperiodically sensitive cultivar of barley (Hordeum vulgare L. Shabet) (BMDR-8) and an isogenic, single-gene recessive mutant of this genotype that is insensitive to photoperiod (BMDR-1) were grown under continuous cool white light with or without supplemental far-red fluorescent light. BMDR-1 initiates flowers 6 days after germination, irrespective of light treatment, whereas BMDR-8 remains vegetative for at least a week longer, even in continuous light. When far-red light is added, the delay of flowering in BMDR-8 is overcome and both genotypes initiate floral primordia at the same time. Total phenol extracted proteins of seedlings of both genotypes were resolved by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No protein differences were found between the genotypes when isoelectric focusing gels were run in the first dimension. Two qualitative genotypic differences were found when nonequilibrium pH gradient gel electrophoresis was run in the first dimension. An 85-kilodalton polypeptide (A) and a 26-kilodalton polypeptide (B) were always present in BMDR-8 but never found in BMDR-1. The levels of A appeared to decrease from the BMDR-8 during the first 3 days of far-red treatment but did not disappear completely even after 6 days of growth in the presence of farred. Polypeptide B decreases rapidly in continuous cool white light but is stabilized by far-red. The phytochrome content of BMDR-1 was found to be greater than that for BMDR-8. This increase appears to be caused by the type I (etiolated-tissue abundant) phytochrome pool, even in plants grown in continuous light.     

Flowering responses to altered expression of phytochrome in mutants and transgenic lines of Arabidopsis thaliana (L.) Heynh.

The long-day plant Arabidopsis thaliana (L.) Heynh. flowers early in response to brief end-of-day (EOD) exposures to far-red light (FR) following a fluorescent short day of 8 h. FR promotion of flowering was nullified by subsequent brief red light (R) EOD exposure, indicating phytochrome involvement. The EOD response to R or FR is a robust measure of phytochrome action. Along with their wild-type (WT) parents, mutants deficient in either phytochrome A or B responded similarly to the EOD treatments. Thus, neither phytochrome A nor B exclusively regulated flowering, although phytochrome B controlled hypocotyl elongation. Perhaps a third phytochrome species is important for the EOD responses of the mutants and/or their flowering is regulated by the amount of the FR-absorbing form of phytochrome, irrespective of the phytochrome species. Overexpression of phytochrome A or phytochrome B resulted in differing photoperiod and EOD responses among the genotypes. The day-neutral overexpressor of phytochrome A had an EOD response similar to all of the mutants and WTs, whereas R EOD exposure promoted flowering in the overexpressor of phytochrome B and FR EOD exposure inhibited this promotion. The comparisons between relative flowering times and leaf numbers at flowering of the over-expressors and their WTs were not consistent across photoperiods and light treatments, although both phytochromes A and B contributed to regulating flowering of the transgenic plants.     

Effect of End-of-day far-red light exposures on fertility alteration and flowering in photoperiod-sensitive genic male-sterile rice

The rice photoperiod-sensitive genic male-sterile mutant (PGMR) is sterile under long days, but fertile in short days. Phytochrome is involved in the photoperiod-induced male-sterile process. To investigate the mechanisms, of phytochrome action in PGMR, end-of-day (EOD) experiments were carried out. Flowering in PGMR was delayed considerably by EOD far-red light exposures following a short day of 10 hr, whereas its fertility decreased to the same extent as the original line. This result suggests that photoperiod response mediating fertility alteration in PGMR somewhat differed from that in flowering,i.e., fertility alteration and flowering might be under the separate phytochrome signaling control.     

The kinetics of type 1 phytochrome in green,light-grown wheat (Triticum aestivum L.)

The kinetics of type 1 phytochrome were investigated in green, light-grown wheat. Phytochrome was measured by a quantitative sandwich enzyme-linked immunosorbent assay using monoclonal antibodies. The assay was capable of detecting down to 150 pg of phytochrome. In red light, rapid first-order destruction of the far-red-light-absorbing form of phytochrome (Pfr) with a half-life of 15 min was observed. Following white light terminated by red, phytochrome synthesis was delayed in darkness by about 15 h compared to plants given a terminal far-red treatment. Synthesis of the red-light-absorbing form of phytochrome (Pr) was zero-order in these experiments. Phytochrome synthesis in far-red light was approximately equal to synthesis in darkness in wheat although net destruction occurred in light-grown Avena sativa tissues in continuous far-red light, as has been reported for other monocotyledons. In wheat, destruction of Pfr apparently did not occur below a certain threshold level of Pfr or Pfr/total phytochrome. These results are consistent with an involvement of type 1 phytochrome in the photoperiodic control of flowering in wheat and other long-day plants.Abbreviations ELISA enzyme-linked immunosorbent assay - FR far-red light - HIR high-irradiance response - Pfr farred-light-absorbing form of phytochrome - Pr red-light-absorbing form of phytochrome - Ptot total phytochrome (Pr + Pfr) - R red light The authors wish to thank Prof. Daphne Vince-Prue (University of Reading) for many helpful discussions regarding this work. Hugh Carr-Smith was supported by a Science and Engineering Research Council studentship and Chris Plumpton by an Agricultural and Food Research Council (AFRC) studentship. B. Thomas and G. Butcher were supported by the AFRC.     

The function of phytochrome A

Knowledge of the photoperceptive function of phytochrome A has improved substantially thanks to the availability of mutants lacking phytochrome A and transgenic plants transformed with the PHYA gene in sense or anti-sense orientation. In imbibed seeds, phytochrome A mediates very-low-fluence responses. In etiolated seedlings, phytochrome A mediates very-low-fluence responses, high-irradiance responses under continuous far-red light, responsivity amplification to phytochrome B and red-light enhancement of the phototropic response to blue light. In light-grown seedings, phytochrome A modulates the extent of response to reductions in red/far-red ratio perceived by phytochrome B, perceives daylength extensions and night interruptions affecting flowering, and perceives light treatments resetting endogenous rhythms. Under natural radiation these abilities are manifested during seed germination and seedling de-etiolation under dense canopies or extremely low light fluences, and during early neighbour detection, but other processes await experimental evaluation. Phytochrome A affects growth and development throughout the whole life cycle of angiosperms.     

Interactions of phytochromes A,B1 and B2 in light-induced competence for adventitious shoot formation in hypocotyl of tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis> L.)

The interactions of phytochrome A (phyA), phytochrome B1 (phyB1) and phytochrome B2 (phyB2) in light-dependent shoot regeneration from the hypocotyl of tomato was analysed using all eight possible homozygous allelic combinations of the null mutants. The donor plants were pre-grown either in the dark or under red or far-red light for 8 days after sowing; thereafter hypocotyl segments (apical, middle and basal portions) were transferred onto hormone-free medium for culture under different light qualities. Etiolated apical segments cultured in vitro under white light showed a very high frequency of regeneration for all of the genotypes tested besides phyB1phyB2, phyAphyB1 and phyAphyB1phyB2 mutants. Evidence is provided of a specific interference of phyB2 with phyA-mediated HIR to far-red and blue light in etiolated explants. Pre-treatment of donor plants by growth under red light enhanced the competence of phyB1phyB2, phyAphyB1 and phyAphyB1phyB2 mutants for shoot regeneration, whereas pre-irradiation with far-red light enhanced the frequency of regeneration only in the phyAphyB1 mutant. Multiple phytochromes are involved in red light- and far-red light-dependent acquisition of competence for shoot regeneration. The position of the segments along the hypocotyl influenced the role of the various phytochromes and the interactions between them. The culture of competent hypocotyl segments under red, far-red or blue light reduced the frequency of explants forming shoots compared to those cultured under white light, with different genotypes having different response patterns.Abbreviations HIR: High irradiance response - LFR: Low fluence response - Pfr: Far-red absorbing form of phytochrome - phyA: Phytochrome A - phyB1: Phytochrome B1 - phyB2: Phytochrome B2 - phyA(B1, B2): Phytochrome mutant deficient in phyA (B1, B2) - phyAphyB1(B1B2,AB2): Double phytochrome mutant deficient in phyA and phyB1(B1, B2) - phyAphyB1phyB2: Triple mutant deficient in phyA, phyB1 and phyB2 - VLFR: Very low fluence response - WT: Wild-type tomato Communicated by R. Reski     

Inhibitory Action of Blue and Far-Red Light in the Flowering of Lemna paucicostata

In a new strain of short-day duckweed (Lemna paucicostata T-101), blue and far-red light-induced inhibition of flowering was investigated. Flowering of this strain failed to be induced under a short-day photoperiod of blue and far-red light, although it responded as a typical short-day plant in red and white light. When the short-day photoperiod of blue or far-red light was terminated by a 15 min red light pulse, flowering recovered completely. This inducing effect of red light was reversed by subsequent exposure to far-red light. Furthermore, it could be demonstrated that 30 min of blue light completely reversed the flowering inductive effect of 5 min red light and vice versa. Evidence is presented suggesting that the inhibitory action of blue and far red light may be due to the lowering of phytochrome P_fr levels below those required to start the dark reactions which lead to flowering. These results are discussed in relation to the time measurement system of photoperiodism.     

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.     

The effect of far-red light on the photoperiodic regulation of carbohydrate accumulation in Allium cepa L.

Carbohydrate accumulation was induced in Allium cepa L. cv. Dorata di Parma by the addition of far-red light throughout the 18 h day. Neither an 18 h-day without far-red nor the addition of far-red light to a 10 h photoperiod could induce the carbohydrate accumulation. The accumulation of reducing sugar takes place both in the leaf-blades and in the bulb; while there is accumulation of oligosaccharides only in the bulb. The results implicate phytochrome in the promotion of carbohydrate accumulation by far-red light.     

Involvement of cotton gene GhFPF1 in the regulation of shade avoidance responses in Arabidopsis thaliana

Phytochrome system perceives the reduction in the ratio of red to far-red light when plants are grown under dense canopy. This signal, regarded as a warning of competition, will trigger a series of phenotypic changes to avoid shade. Progress has been made for several phytochrome signaling intermediates acting as positive regulators of accelerated elongation growth and promotion of flowering in shade-avoidance has been identified. Recently, a FPF1 homolog GhFPF1 was identified in upland cotton. Our data supported that transgenic Arabidopsis of over-expressing GhFPF1 displayed a constitutive shade-avoiding phenotype resembling phyB mutants in several respects such as accelerated elongation of hypocotyl and petioles, upward of leaf movement, and promoted flowering. In this addendum, by dissection of GhFPF1 acting as a component of shade-avoidance responses we suppose that GhFPF1 might influence the timing of the floral transition independently of shade-mediated early flowering. Furthermore, the opposite changes of IAA content in transgenic leaves and stems suggested that alteration of IAA storage and release took place during shade-avoidance responses.     

Photocontrol of petiole elongation in light-grown strawberry plants

Summary The mode of phytochrome control of elongation growth was studied in fully-green strawberry (Fragaria x Ananassa Duch.) plants. Petiole growth showed two distinct types of response to light. In one, the end-of-day response, petioles were lengthened by low-intensity far-red irradiation for 1 h immediately following the 8 h photoperiod. The response was little or no greater with prolonged exposure and less when the start of far-red was delayed. It was already evident in the first leaf to emerge after treatment began. With the development of successive leaves a second, photoperiodic, type of response appeared, in which petioles lengthened following only prolonged exposure to red, far-red, mixtures of the two, or tungsten lighting, all at low levels of intensity. As with the inhibition of flowering in previous experiments, irradiation with red light during the second half of the otherwise long dark period gave the greatest response.Abbreviations and Symbols FR far-red light - HIR high irradiance response - R red light - P_r phytochrome in the red light absorbing form - P_fr phytochrome in the far-red light absorbing form - SDP short-day plant - LDP long-day plant - PAR photosynthetically active radiation     

Flowering responses to light-breaks in photomorphogenic mutants of Arabidopsis thaliana, a long-day plant

Flowering response and plant form of photomorphogenic mutants (hy1, hy2, hy3, hy4 and hy5) of Arabidopsis thaliana (L.), a long-day plant, were examined in long and short days. There were only slight differences among genotypes including Landsberg wild type with respect to the flowering time under long days. The effect of 1 h light-(night)-breaks of far-red, red, blue and white light given in the middle of the dark period of plants grown under short days, was studied. Effects of far-red light applied at the end or the beginning of the main photoperiod on flowering and plant form were also examined. The light-breaks with all the above mentioned light qualities promoted floral initiation of all the genotypes including the wild type in terms of both the flowering time and the number of rosette leaves. In general, far-red light was most effective. It is possible to classify the hy-mutants into 3 groups by their responses to light-breaks under short day conditions: (a) Mutants hy2 and hy3, which have a reduced number of rosette leaves, and flower early. Red light is as effective as far-red light. The wavelength of light-breaks is relatively unimportant for flowering response. (b) Mutants hy4, hy5 and Landsberg wild type, which have a greater number of rosette leaves, and flower relatively late. The effectiveness of light-breaks is in the following order, far-red, blue, and red light, which is in reverse order to the transformation of phytochrome to the P_fr form. (c) Mutant hy1, which behaves anomalously with respect to relations between flowering time and number of rosette leaves; late flowering with reduced number of rosette leaves. Red, blue and far-red light are effective, but white light is ineffective for reducing the number of rosette leaves. When far-red light was given in the middle of the night or at the end of the main photoperiod, it markedly reduced the number of rosette leaves compared to those grown under short days for all the genotypes, while when applied at the beginning of the main photoperiod far-red light did not affect the number of rosette leaves. Different effects on the plant form dependent on the time of treatment with far-red light-breaks are also discussed.     

The rosette habit of Arabidopsis thaliana is dependent upon phytochrome action: novel phytochromes control internode elongation and flowering time

A major function of phytochromes in light-grown plants involves the perception of changes in the relative amounts of red and far-red light (R:FR ratio) and the initiation of the shade-avoidance response. In Arabidopsis thaliana, this response is typified by increased elongation growth of petioles and accelerated flowering and can be fully induced by end-of-day far-red light (EOD FR) treatments. Phytochrome B-deficient (phyB) mutants, which have a constitutive elongated-petiole and early-flowering phenotype, do not display a petiole elongation growth response to EOD FR, but they do respond to EOD FR by earlier flowering. Seedlings deficient in both phytochrome A and phytochrome B (phyA phyB), have a greatly reduced stature compared with wild-type or either monogenic mutant. The phyA phyB double null mutants also respond to EOD FR treatments by flowering early, suggesting the operation of novel phytochromes. Contrary to the behaviour of wild-type or monogenic phyA or phyB seedlings, petiole elongation in phyA phyB seedlings is reduced in response to EOD FR treatments. This reduction in petiole elongation is accompanied by the appearance of elongated internodes such that under these conditions the plants no longer display a rosette habit.     

Photoresponses of Light-Grown phyA Mutants of Arabidopsis (Phytochrome A Is Required for the Perception of Daylength Extensions)

Several aspects of the photophysiology of wild-type Arabidopsis thaliana seedlings were compared with those of a phytochrome A null mutant, phyA-1, and a mutant, fhy1, that is putatively involved in the transduction of light signals from phytochrome A. Although phyA seedlings display a near wild-type phenotype when grown in white light (W), they nevertheless display several photomorphogenic abnormalities. Thus, whereas the germination of wild-type and fhy1 seeds is almost fully promoted by a pulse of red light (R) or by continuous far-red light (FR), phyA seed germination is responsive only to R. Following growth under day/night cycles, but not under continuous W, the hypocotyls of light-grown phyA and fhy1 seedlings are more elongated than those of wild-type seedlings. For seedlings grown under low red/far-red (R/FR) ratio light conditions, phyA and fhy1 seedlings display a more marked promotion of hypocotyl elongation than wild-type seedlings. Similarly, seedlings that are doubly null for phytochrome A and phytochrome B(phyA phyB) also have more elongated hypocotyls under low R/FR ratio conditions than phyB seedlings. This indicates that phytochrome A action in light-grown seedlings is antagonistic to the action of phytochrome B. Although wild-type, fhy1, and phyA seedlings flower at essentially the same time under both short-day and long-day conditions, an obvious consequence of phytochrome A deficiency is a pronounced late flowering under conditions where a short day of 8 h of fluorescent W is extended by 8 h of low-fluence-rate incandescent light. The evidence thus indicates that phytochrome A plays a role in seed germination, in the control of elongation growth of light-grown seedlings, and in the perception of daylength.     

Integral association of phytochrome with a membranous fraction fromAvena shoots: in vivo characterization and physiological significance

Phytochrome in the far-red light absorbing form (Pfr) was observed to disappear in vivo more rapidly from the non-cation-requiring pelletable phytochrome population than from the supernantant phytochrome population of oat seedlings given an increasing dark incubation after red irradiation. The amount of pelletable phytochrome in the red light absorbing form (Pr) remained relatively stable while supernatant Pr was lost. These observations indicated that supernant Pfr was subject to loss during the incubation, while pelletable Pfr was subject to both dark reversion and loss.During the incubation, the ability of far-red irradiation to reverse the red-induced increase in phytochrome pelletability was lost, with kinetics similar to those of the loss of pelletable Pfr.Far-red reversibility of the red-induced increase in coleoptile elongation correlated with the change intotal Pfr in both supernatant and pelletable phytochrome populations, but with the change in the ratio of Pfr to total phytochrome only in the pelletable phytochrome population.The possible significance of these results is discussed with reference to the action of phytochrome in the photocontrol of physiological growth responses.Abbreviations Pfr phytochrome in the far-red light absorbing form - Pr phytochrome in the red absorbing form - Ptot total phytochrome     

Reversible phytochrome regulation influenced the severity of ozone-induced visible foliar injuries in Trifolium subterraneum L.

In Trifolium subterraneum, oxidative stress caused by ozone has been shown to result in more severe visible foliar injuries when plants were kept in dim broadband white light during the night (i.e. a long photoperiod) compared to darkness during the night (a short photoperiod). As phytochrome signalling is involved in photoperiod sensing, the effect of night-time red and far-red illumination on the ozone-induced response was studied. T. subterraneum plants were treated with ozone enriched air (70?ppb) for either 1?h for a single day or 6?h for three consecutive days. After the first ozone exposure, plants were separated into six night-time light regimes during the two subsequent nights (10?h?day, 14?h night): (1) darkness, (2) far-red light (FR), (3) a short night-break of red followed by far-red light during an otherwise dark night (R FR), (4) a short night-break of red, far-red and finally red light during an otherwise dark night (R FR R), (5) dim white light (L) and (6) red light (R). The treatments L and R resulted in significantly more severe ozone-induced visible foliar injuries relative to D and FR treatments, indicating a phytochrome-mediated response. The night-breaks resulted in a photoreversible and significantly different ozone response depending on the light quality of the last light interval (R FR or R FR R), supporting a photoreversible (between P_r and P_fr) phytochrome signalling response. Thus, in T. subterraneum, the outcome of oxidative stress due to ozone appears to depend on the photoperiod mediated by the night-time conformation of phytochrome.     

Missense mutation in the PAS2 domain of phytochrome A impairs subnuclear localization and a subset of responses

Phytochrome A signaling shows two photobiologically discrete outputs: so-called very-low-fluence responses (VLFR) and high-irradiance responses (HIR). By modifying previous screening protocols, we isolated two Arabidopsis mutants retaining VLFR and lacking HIR. Phytochrome A negatively or positively regulates phytochrome B signaling, depending on light conditions. These mutants retained the negative but lacked the positive regulation. Both mutants carry the novel phyA-302 allele, in which Glu-777 (a residue conserved in angiosperm phytochromes) changed to Lys in the PAS2 motif of the C-terminal domain. The phyA-302 mutants showed a 50% reduction in phytochrome A levels in darkness, but this difference was compensated for by greater stability under continuous far-red light. phyA-302:green fluorescent protein fusion proteins showed normal translocation from the cytosol to the nucleus under continuous far-red light but failed to produce nuclear spots, suggesting that nuclear speckles could be involved in HIR signaling and phytochrome A degradation. We propose that the PAS2 domain of phytochrome A is necessary to initiate signaling in HIR but not in VLFR, likely via interaction with a specific partner.     

Kinetics and time dependence of the effect of far red light on the photoperiodic induction of flowering in wintex barley

Flowering in the long day plant Hordeum vulgare L. var. Wintex barley was enhanced by the addition of far red light to the main light portion of the photoperiod. Far red energy was provided to produce quantum flux ratios (660/730 nm) and phytochrome photoequilibria (Pfr/total phytochrome) equivalent to those reported both beneath a leaf canopy and outside a canopy at twilight. The photoperiodic requirement for long days can be completely eliminated by the addition of far red light. However, both the effect of extending the photoperiod without far red and the addition of far red to 12-hour photoperiods were suboptimal. Maximal stimulation was achieved only when far red was added to continuous light. The duration of the period of maximal apex elongation rate, as well as the reduction of the time required for floral initiation, were saturated by three inductive cycles. When far red energy was provided intermittently during 3 days of continuous light, the ability to respond varied in a circadian manner. This enhancement of flowering by far red appears to be mediated by the “high irradiance response” of phytochrome.     

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.     

Patterns of expression and normalized levels of the five Arabidopsis phytochromes

Using monoclonal antibodies specific for each apoprotein and full-length purified apoprotein standards, the levels of the five Arabidopsis phytochromes and their patterns of expression in seedlings and mature plants and under different light conditions have been characterized. Phytochrome levels are normalized to the DNA content of the various tissue extracts to approximate normalization to the number of cells in the tissue. One phytochrome, phytochrome A, is highly light labile. The other four phytochromes are much more light stable, although among these, phytochromes B and C are reduced 4- to 5-fold in red- or white-light-grown seedlings compared with dark-grown seedlings. The total amount of extractable phytochrome is 23-fold lower in light-grown than dark-grown tissues, and the percent ratios of the five phytochromes, A:B:C:D:E, are measured as 85:10:2:1.5:1.5 in etiolated seedlings and 5:40:15:15:25 in seedlings grown in continuous white light. The four light-stable phytochromes are present at nearly unchanging levels throughout the course of development of mature rosette and reproductive-stage plants and are present in leaves, stems, roots, and flowers. Phytochrome protein expression patterns over the course of seed germination and under diurnal and circadian light cycles are also characterized. Little cycling in response to photoperiod is observed, and this very low amplitude cycling of some phytochrome proteins is out of phase with previously reported cycling of PHY mRNA levels. These studies indicate that, with the exception of phytochrome A, the family of phytochrome photoreceptors in Arabidopsis constitutes a quite stable and very broadly distributed array of sensory molecules.