Phytochrome A Mediates the Promotion of Seed Germination by Very Low Fluences of Light and Canopy Shade Light in Arabidopsis

Seeds of the wild type (WT) and of the phyA and phyB mutants of Arabidopsis thaliana were exposed to single red light (R)/far-red light (FR) pulses predicted to establish a series of calculated phytochrome photoequilibria (Pfr/P). WT and phyB seeds showed biphasic responses to Pfr/P. The first phase, i.e. the very-low-fluence response (VLFR), occurred below Pfr/P = 10-1%. The second phase, i.e. the low-fluence response, occurred above Pfr/P = 3%. The VLFR was similarly induced by either a FR pulse saturating photoconversion or a subsaturating R pulse predicted to establish the same Pfr/P. The VLFR was absent in phyA seeds, which showed a strong low-fluence response. In the field, even brief exposures to the very low fluences of canopy shade light (R/FR ratio < 0.05) promoted germination above dark controls in WT and phyB seeds but not in the phyA mutant. Seeds of the phyA mutant germinated normally under canopies providing higher R/FR ratios or under deep canopy shade light supplemented with R from light-emitting diodes. We propose that phytochrome A mediates VLFR of A. thaliana seeds.     

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.     

Tomato seed germination: regulation of different response modes by phytochrome B2 and phytochrome A

Lycopersicon esculentum seeds germinate after rehydration in complete darkness. This response was inhibited by a far-red light (FR) pulse, and the inhibition was reversed by a red light (R) pulse. Comparison of germination in phytochrome-deficient mutants (phyA, phyB1, phyB2, phyAB1, phyB1B2 and phyAB1B2) showed that phytochrome B2 (PhyB2) mediates both responses. The germination was inhibited by strong continuous R (38 micromol m(-2) s(-1)), whereas weak R (28 nmol m(-2) s(-1)) stimulated seed germination. Hourly applied R pulses of the same photon fluence partially replaced the effect of strong continuous R. This response was called 'antagonistic' because it counteracts the low fluence response (LFR) induced by a single R pulse. This antagonistic response might be an adaptation to a situation where the seeds sit on the soil surface in full sunlight (adverse for germination), while weak R might reflect that situation under a layer of soil. Unexpectedly, the effects of continuous R or repeated R pulses were mediated by phytochrome A (PhyA). We therefore suggest that low levels of PhyA in its FR-absorbing form (Pfr) cause inhibition of seed germination produced either by extended R irradiation (by degradation of PhyA-Pfr) or by extended FR irradiation [keeping a low Pfr/R-absorbing form (Pr) ratio].     

Phytochrome A-mediated inhibition of seed germination in tomato

Far-red light (FR) inhibition of seed germination of tomato (Solanum lycopersicum L.) was studied with the phytochrome (phy)-hypersensitive mutants, hp-1w, hp-1w,fri1, a phyA-deficient double mutant, and hp-1w,tri1, a phyB1-deficient double mutant. Seeds of all mutants germinated readily in the dark at 25 degrees C, and the germination was retarded by a single 100-s FR pulse given 1-3 h after sowing. The effect of an FR pulse was red-light reversible in all mutants used. After 24 h where a single FR pulse was no longer effective, prolonged FR exposure or hourly FR pulses suppressed germination in hp-1w and hp-1w,tri1, whereas in hp-1w,fri1 the suppressive effect of FR was almost absent. The effect of the prolonged FR was greater than that of the hourly 3-min FR pulses having equal photon fluence, and was fluencerate dependent. Thus we conclude that the germination inhibition by FR in tomato seed consists of a low-fluence response and a high irradiance response (HIR); the latter is controlled by phyA, but not phyB1. This is the first indication of phyA being involved in the HIR of seed germination inhibition.     

Distinct and cooperative functions of phytochromes A, B, and C in the control of deetiolation and flowering in rice

We have isolated phytochrome B (phyB) and phyC mutants from rice (Oryza sativa) and have produced all combinations of double mutants. Seedlings of phyB and phyB phyC mutants exhibited a partial loss of sensitivity to continuous red light (Rc) but still showed significant deetiolation responses. The responses to Rc were completely canceled in phyA phyB double mutants. These results indicate that phyA and phyB act in a highly redundant manner to control deetiolation under Rc. Under continuous far-red light (FRc), phyA mutants showed partially impaired deetiolation, and phyA phyC double mutants showed no significant residual phytochrome responses, indicating that not only phyA but also phyC is involved in the photoperception of FRc in rice. Interestingly, the phyB phyC double mutant displayed clear R/FR reversibility in the pulse irradiation experiments, indicating that both phyA and phyB can mediate the low-fluence response for gene expression. Rice is a short-day plant, and we found that mutation in either phyB or phyC caused moderate early flowering under the long-day photoperiod, while monogenic phyA mutation had little effect on the flowering time. The phyA mutation, however, in combination with phyB or phyC mutation caused dramatic early flowering.     

Negative interference of endogenous phytochrome B with phytochrome A function in Arabidopsis

To study negative interactions between phytochromes, phytochrome B (phyB) overexpressor lines, the mutants phyA-201, phyB-4, phyB-5, phyD-1, phyA-201 phyB-5, phyA-201 phyD-1, and phyB-5 phyD-1 of Arabidopsis were used. Endogenous phyB, but not phytochrome D (phyD), partly suppressed phytochrome A (phyA)-dependent inhibition of hypocotyl elongation in far-red light (FR). Dichromatic irradiation demonstrated that the negative effect of phyB was largely independent of the photoequilibrium, i.e. far-red light absorbing form of phytochrome formation. Moreover, phyB-4, a mutant impaired in signal transduction, did not show a loss of inhibition of phyA by phyB. Overexpression of phyB, conversely, resulted in an enhanced inhibition of phyA function, even in the absence of supplementary carbohydrates. However, overexpression of a mutated phyB, which cannot incorporate the chromophore, had no detectable effect on phyA action. In addition to seedling growth, accumulation of anthocyanins in FR, another manifestation of the high irradiance response, was strongly influenced by phyB holoprotein. Induction of seed germination by FR, a very low fluence response, was suppressed by both endogenous phyB and phyD. In conclusion, we show that both classical response modes of phyA, high irradiance response, and very low fluence response are subject to an inhibitory action of phyB-like phytochromes. Possible mechanisms of the negative interference are discussed.     

Phytochrome A enhances the promotion of hypocotyl growth caused by reductions in levels of phytochrome B in its far-red-light-absorbing form in light-grown Arabidopsis thaliana.

We sought to determine if phytochrome B (phyB)-mediated responses to the red light (R)/far-red light (FR) ratio are affected by phytochrome A (phyA) activity in light-grown seedlings of Arabidopsis thaliana. Pulses of FR delayed into the dark period were less effective than end-of-day (EOD) FR in promoting hypocotyl growth over a given period in darkness. White light minus blue light interposed instead of darkness between the end of the white-light photoperiod and the FR pulse was sufficient to maintain responsivity to the decrease in phyB in FR-light-absorbing form in wild-type (WT) seedlings, but not in the phyA mutant. Compared with EOD R, hourly R+FR pulses provided throughout the night caused a stronger promotion of stem growth than a single EOD R+FR pulse in WT Arabidopsis, cucumber, mustard, sunflower, tobacco, and tomato, but not in phyA Arabidopsis or in the aurea mutant of tomato. WT seedlings of Arabidopsis responded to a range of high EOD R/FR ratios, whereas the phyA mutant required stronger reductions in the EOD R/FR ratio. In sunlight, phyA seedlings of Arabidopsis showed no response to the "early warning" signals of neighboring vegetation, and hypocotyl-growth promotion occurred at higher plant densities than in the WT. Thus, under a series of light conditions, the sensitivity or responsivity to reductions in the R/FR ratio were larger in WT than in phyA seedlings. A product of phyA is therefore proposed to enhance the hypocotyl-growth response to decreases in phyB in FR-light-absorbing form in light grown seedlings.     

Functional diversity of phytochrome family in the control of light and gibberellin‐mediated germination in Arabidopsis

In several species, seed germination is regulated by light in a way that restricts seedling emergence to the environmental conditions that are likely to be favourable for the success of the new individual, and therefore, this behaviour is recognized to have adaptive value. The phytochromes are one of the most relevant photoreceptors involved in light perception by plants. We explored the redundancy and diversity functions of the phytochrome family in the control of seed responsiveness to light and gibberellins (GA) by using a set of phytochrome mutants of Arabidopsis. Our data show that, in addition to the well‐known role of phyB in the promotion of germination in response to high red to far‐red ratios (R/FR), phyE and phyD stimulate germination at very low R/FR ratios, probably by promoting the action of phyA. Further, we show that phyC regulates negatively the seed responsiveness to light, unravelling unexpected functions for phyC in seed germination. Finally, we find that seed responsiveness to GA is mainly controlled by phyB, with phyC, phyD and phyE having relevant roles when acting in a phyB‐deficient background. Our results indicate that phytochromes have multiple and complex roles during germination depending on the active photoreceptor background.     

Physiological interactions of phytochromes A, B1 and B2 in the control of development in tomato

The role of phytochrome B2 (phyB2) in the control of photomorphogenesis in tomato (Solanum lycopersicum L.) has been investigated using recently isolated mutants carrying lesions in the PHYB2 gene. The physiological interactions of phytochrome A (phyA), phytochrome B1 (phyB1) and phyB2 have also been explored, using an isogenic series of all possible mutant combinations and several different phenotypic characteristics. The loss of phyB2 had a negligible effect on the development of white-light-grown wild-type or phyA-deficient plants, but substantially enhanced the elongated pale phenotype of the phyB1 mutant. This redundancy was also seen in the control of de-etiolation under continuous red light (R), where the loss of phyB2 had no detectable effect in the presence of phyB1. Under continuous R, phyA action was largely independent of phyB1 and phyB2 in terms of the control of hypocotyl elongation, but antagonized the effects of phyB1 in the control of anthocyanin synthesis, indicating that photoreceptors may interact differently to control different traits. Irradiance response curves for anthocyanin synthesis revealed that phyB1 and phyB2 together mediate all the detectable response to high-irradiance R, and, surprisingly, that the phyA-dependent low-irradiance component is also strongly reduced in the phyB1 phyB2 double mutant. This is not associated with a reduction in phyA protein content or responsiveness to continuous far-red light (FR), suggesting that phyB1 and phyB2 specifically influence phyA activity under low-irradiance R. Finally, the phyA phyB1 phyB2 triple mutant showed strong residual responsiveness to supplementary daytime FR, indicating that at least one of the two remaining phytochromes plays a significant role in tomato photomorphogenesis.     

Overexpression of Arabidopsis phytochrome B inhibits phytochrome A function in the presence of sucrose

Overexpression of phytochrome B (phyB) in Arabidopsis has previously been demonstrated to result in dominant negative interference of phytochrome A (phyA)-mediated hypocotyl growth inhibition in far-red (FR) light. This phenomenon has been examined further in this study and has been found to be dependent on the FR fluence rate and on the availability of metabolizable sugars in the growth medium. Poorly metabolized sugars capable of activating the putative hexokinase sensory function were not effective in eliciting the phytochrome interference response. Overexpressed phyB lacking the chromophore-binding site was also effective at inhibiting the phyA response, especially at higher fluence rates of FR. Overexpressed phyB produces the dominant negative phenotype without any apparent effect on phyA abundance or degradation. It is possible that phyA and phyB interact with a common reaction partner but that either the energy state of the cell or a separate sugar-signaling mechanism modulates the phytochrome-signaling interactions.     

Light environment in pre- and post-dehiscent fruits affects seed germination in Calotropis procera

Fruits in Calotropis procera can be distinguished into five discrete but contiguous stages on the basis of diameter and seed color. Seeds from dehisced fruits at stage V germinated >80% on moist substratum in darkness. This was rather unexpected because the seeds developed and matured in an FR-enriched microenvironment (R:FR ratio ～0.3) of the chlorophyll-containing maternal tissue and displayed low-fluence response (LFR) mode of phytochrome action. In contrast to >80% dark-germinating seeds from dehisced fruits at stage V, about 50% seeds from undehisced fruits at that stage were dark germinating, whereas another 30% seeds required light for germination. The light-requiring fraction of the seed population did not only respond to a very low-fluence R and to a short FR pulse, but also lacked R–FR reversibility thereby indicating to a very low-fluence response (VLFR) mode of phytochrome action. The present study reporting VLFR to non-dormant seed state transition in C. procera suggested that the state of phytochrome and the subsequent seed germination response in dry-seeded species, besides being determined by the light environment immediately before maturation drying, might also be regulated by a post-dehiscence light signal.     

Different Phototransduction Kinetics of Phytochrome A and Phytochrome B in Arabidopsis thaliana

The kinetics of phototransduction of phytochrome A (phyA) and phytochrome B (phyB) were compared in etiolated Arabidopsis thaliana seedlings. The responses of hypocotyl growth, cotyledon unfolding, and expression of a light-harvesting chlorophyll a/b-binding protein of the photosystem II gene promoter fused to the coding region of β-glucuronidase (used as a reporter enzyme) were mediated by phyA under continuous far-red light (FR) and by phyB under continuous red light (R). The seedlings were exposed hourly either to n min of FR followed by 60 minus n min in darkness or to n min of R, 3 min of FR (to back-convert phyB to its inactive form), and 57 minus n min of darkness. For the three processes investigated here, the kinetics of phototransduction of phyB were faster than that of phyA. For instance, 15 min R h⁻¹ (terminated with a FR pulse) were almost as effective as continuous R, whereas 15 min of FR h⁻¹ caused less than 30% of the effect of continuous FR. This difference is interpreted in terms of divergence of signal transduction pathways downstream from phyA and phyB.     

Phytochrome A and Phytochrome B Have Overlapping but Distinct Functions in Arabidopsis Development

Plant responses to red and far-red light are mediated by a family of photoreceptors called phytochromes. In Arabidopsis thaliana, there are genes encoding at least five phytochromes, and it is of interest to learn if the different phytochromes have overlapping or distinct functions. To address this question for two of the phytochromes in Arabidopsis, we have compared light responses of the wild type with those of a phyA null mutant, a phyB null mutant, and a phyA phyB double mutant. We have found that both phyA and phyB mutants have a deficiency in germination, the phyA mutant in far-red light and the phyB mutant in the dark. Furthermore, the germination defect caused by the phyA mutation in far- red light could be suppressed by a phyB mutation, suggesting that phytochrome B (PHYB) can have an inhibitory as well as a stimulatory effect on germination. In red light, the phyA phyB double mutant, but neither single mutant, had poorly developed cotyledons, as well as reduced red-light induction of CAB gene expression and potentiation of chlorophyll induction. The phyA mutant was deficient in sensing a flowering response inductive photoperiod, suggesting that PHYA participates in sensing daylength. In contrast, the phyB mutant flowered earlier than the wild type (and the phyA mutant) under all photoperiods tested, but responded to an inductive photoperiod. Thus, PHYA and PHYB appear to have complementary functions in controlling germination, seedling development, and flowering. We discuss the implications of these results for possible mechanisms of PHYA and PHYB signal transduction.     

The Shade Avoidance Syndrome in Arabidopsis: The Antagonistic Role of Phytochrome A and B Differentiates Vegetation Proximity and Canopy Shade

Light limitation caused by dense vegetation is one of the greatest threats to plant survival in natural environments. Plants detect such neighboring vegetation as a reduction in the red to far-red ratio (R:FR) of the incoming light. The low R:FR signal, perceived by phytochromes, initiates a set of responses collectively known as the shade avoidance syndrome, intended to reduce the degree of current or future shade from neighbors by overtopping such competitors or inducing flowering to ensure seed production. At the seedling stage these responses include increased hypocotyl elongation. We have systematically analyzed the Arabidopsis seedling response and the contribution of phyA and phyB to perception of decreased R:FR, at three different levels of photosynthetically active radiation. Our results show that the shade avoidance syndrome, induced by phyB deactivation, is gradually antagonized by phyA, operating through the so-called FR-High Irradiance Response, in response to high FR levels in a range that simulates plant canopy shade. The data indicate that the R:FR signal distinguishes between the presence of proximal, but non-shading, neighbors and direct foliar shade, via a intrafamily photosensory attenuation mechanism that acts to suppress excessive reversion toward skotomorphogenic development under prolonged direct vegetation shade.     

CP3 is involved in negative regulation of phytochrome A signalling in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Several mutants with altered phytochrome A (phyA) signalling have been identified in screenings under continuous far-red light (FR). The latter protocol could preclude the identification of mutants affected in the signalling pathway that operates even under transient phyA activation, compared to the high-irradiance response (HIR) pathway that requires continuous FR. Since some photomorphogenic mutants show shoot-height phenotypes, the screening was conducted on dwarf mutants of Arabidopsis thaliana (L.) Heynh. from the ABRC stocks grown under hourly FR pulses. The dwarf mutant cp3 (compacta 3) showed normal hypocotyl length and folded cotyledons in darkness but enhanced hypocotyl-growth inhibition and cotyledon unfolding under pulsed FR. The HIR and the response mediated by phyB were not affected. Under pulsed FR, seed germination and blocking of greening upon transfer to white light were enhanced in cp3. PHYA levels were normal in cp3. The phenotype under pulsed FR but not the adult phenotype required phyA. We propose that CP3 is involved in the negative regulation of the signalling pathway that saturates with transient activation of phyA.     

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.     

Is the far-red-absorbing form of Avena phytochrome A that is present at the end of the day able to sustain stem-growth inhibition during the night in transgenic tobacco and tomato seedlings?

Avena phytochrome A (phyA) overexpressed in tobacco (Nicotiana tabacum L.) and tomato (Lycopersicon sculentum Mill) was functionally characterised by comparing wild-type (WT) and transgenic seedlings. Different proportions of phytochrome in its far-red-absorbing form (Pfr/P) were provided by end-of-day (EOD) light pulses. Stem-length responses occurred largely in the range of low Pfr/P (3–61%) for WT seedlings and in the range of high Pfr/P (61–87%) for transgenic seedlings. A similar shift was observed when the photoperiod was interrupted by short light pulses providing different Pfr/P ratios and followed by 1 h dark incubation. In other experiments, Avena phyA was allowed to re-accumulate in darkness and subsequently phototransformed to Pfr but no extra inhibition of stem extension growth was observed. In transgenic tomato seedlings the response to EOD far-red light was faster and the response to a far-red light pulse delayed into darkness was larger than in the WT. Avena phyA Pfr remaining at the end of the photoperiod appears intrinsically unable to sustain growth inhibition in subsequent darkness. Avena phyA modifies the sensitivity and the kinetics of EOD responses mediated by native phytochrome.Abbreviations EOD end-of-day - FR far-red light - Pfr/P pro-portion of phytochrome in its FR-absorbing form - phyA phyto-chrome A - phyB phytochrome B - R red light - RFR R to FR ratio - WT wild type We thank Dr Brian Thomas for providing the antibodies used in this work, and Federico Guerendiain for his excellent technical assistance. This work was financially supported by grants UBA AG 040 and Fundacion Antorchas A-12830/1-19 (both to J.J.C.), PID-CONICET (to R.A.S. and J.J.C.), United States Department of Energy DE-FG02-88ER13968 (to R.D.V.).     

Regulation of phytochrome B signaling by phytochrome A and FHY1 in Arabidopsis thaliana

Phytochrome A (phyA) and phytochrome B (phyB) share the control of many processes but little is known about mutual signaling regulation. Here, we report on the interactions between phyA and phyB in the control of the activity of an Lhcb1*2 gene fused to a reporter, hypocotyl growth and cotyledon unfolding in etiolated Arabidopsis thaliana. The very-low fluence responses (VLFR) induced by pulsed far-red light and the high-irradiance responses (HIR) observed under continuous far-red light were absent in the phyA and phyA phyB mutants, normal in the phyB mutant, and reduced in the fhy1 mutant that is defective in phyA signaling. VLFR were also impaired in Columbia compared to Landsberg erecta. The low-fluence responses (LFR) induced by red-light pulses and reversed by subsequent far-red light pulses were small in the wild type, absent in phyB and phyA phyB mutants but strong in the phyA and fhy1 mutants. This indicates a negative effect of phyA and FHY1 on phyB-mediated responses. However, a pre-treatment with continuous far-red light enhanced the LFR induced by a subsequent red-light pulse. This enhancement was absent in phyA, phyB, or phyA phyB and partial in fhy1. The levels of phyB were not affected by the phyA or fhy1 mutations or by far-red light pre-treatments. We conclude that phyA acting in the VLFR mode (i.e. under light pulses) is antagonistic to phyB signaling whereas phyA acting in the HIR mode (i.e. under continuous far-red light) operates synergistically with phyB signaling, and that both types of interaction require FHY1.