Altered dark- and photoconversion of phytochrome B mediate extreme light sensitivity and loss of photoreversibility of the phyB-401 mutant

The phyB-401 mutant is 10(3) fold more sensitive to red light than its wild-type analogue and shows loss of photoreversibility of hypocotyl growth inhibition. The phyB-401 photoreceptor displays normal spectral properties and shows almost no dark reversion when expressed in yeast cells. To gain insight into the molecular mechanism underlying this complex phenotype, we generated transgenic lines expressing the mutant and wild-type phyB in phyB-9 background. Analysis of these transgenic lines demonstrated that the mutant photoreceptor displays a reduced rate of dark-reversion but normal P(fr) to P(r) photoconversion in vivo and shows an altered pattern of association/dissociation with nuclear bodies compared to wild-type phyB. In addition we show (i) an enhanced responsiveness to far-red light for hypocotyl growth inhibition and CAB2 expression and (ii) that far-red light mediated photoreversibility of red light induced responses, including inhibition of hypocotyl growth, formation of nuclear bodies and induction of CAB2 expression is reduced in these transgenic lines. We hypothesize that the incomplete photoreversibility of signalling is due to the fact that far-red light induced photoconversion of the chromophore is at least partially uncoupled from the P(fr) to P(r) conformation change of the protein. It follows that the phyB-401 photoreceptor retains a P(fr)-like structure (P(r) (*)) for a few hours after the far-red light treatment. The greatly reduced rate of dark reversion and the formation of a biologically active P(r) (*) conformer satisfactorily explain the complex phenotype of the phyB-401 mutant and suggest that amino acid residues surrounding the position 564 G play an important role in fine-tuning phyB signalling.     

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.     

Coupling of phytochrome B to the control of hypocotyl growth in Arabidopsis

Etiolated seedlings of the wild-type (WT) and of the phyB-1 mutant of Arabidopsis thaliana (L.) Heynh. were exposed to red-light (R) and far-red light (FR) treatments to characterize the action of phytochrome B on hypocotyl extension growth. A single R or FR pulse had no detectable effects on hypocotyl growth. After 24-h pre-treatment with continuous FR (FRc) a single R, compared to FR pulse inhibited (more than 70%) subsequent hypocotyl growth in the WT but not in the phyB-1 mutant. This effect of FRc was fluence-rate dependent and more efficient than continuous R (Rc) or hourly FR pulses of equal total fluence. Hypocotyl growth inhibition by Rc was larger in WT than phyB-1 seedlings when chlorophyll screening was reduced either by using broadband Rc (maximum emission 610 nm) or by using narrow-band Rc (658 nm) over short periods (24 h) or with seedlings bleached with Norflurazon. Hourly R or R + FR pulses had similar effects in WT and phyB-1 mutant etiolated seedlings. It is concluded that phytochrome B is not the only photoreceptor of Rc and that the action of phytochrome B is enhanced by a FRc high-irradiance reaction. Complementary experiments with the phyA-201 mutant indicate that this promotion of a phytochrome B-mediated response occurs via co-action with phytochrome A.Abbreviations D darkness - FR far-red light - FRc continuous FR - Pfr FR-absorbing form of phytochrome - HIR high-irradiance reaction - Pfr/P proportion of phytochrome as Pfr - phyA phytochrome A - phyB phytochrome B - R red light - Rc continuous R - WT wild-type I thank Professors R.E. Kendrick and M. Koornneef (Wageningen Agricultural University, The Netherlands) and Professor J. Chory (Salk Institute, Calif., USA) for their kind provision of the original WT and phyB-1 and phyA-201 seed, respectively. This work was financially supported by grants PID and PID-BID from CONICET, AG 040 from Universidad de Buenos Aires and A 12830/1-000019 from Fundación Antorchas.     

The AT-hook-containing proteins SOB3/AHL29 and ESC/AHL27 are negative modulators of hypocotyl growth in Arabidopsis

SOB3 , which encodes a plant-specific AT-hook motif containing protein, was identified from an activation-tagging screen for suppressors of the long-hypocotyl phenotype of a weak phyB allele, phyB-4 . sob3-D ( suppressor of phyB-4#3 dominant ) overexpressing seedlings have shorter hypocotyls, and as adults develop larger flowers and leaves, and are delayed in senescence compared with wild-type plants. At the nucleotide level, SOB3 is closely related to ESCAROLA ( ESC ), which was identified in an independent activation-tagging screen. ESC overexpression also suppresses the phyB-4 long-hypocotyl phenotype, and confers an adult morphology similar to sob3-D , suggesting similar functions. Analysis of transgenic plants harboring SOB3:SOB3-GUS or ESC:ESC-GUS translational fusions, driven by their endogenous promoter regions, showed GUS activity in the hypocotyl and vasculature tissue in light- and dark-grown seedlings. A loss-of-function SOB3 allele ( sob3-4 ) was generated through an ethyl methanesulfonate intragenic suppressor screen of sob3-D phyB-4 plants, and this allele was combined with a predicted null allele, disrupting ESC ( esc-8 ), to examine potential genetic interactions. The sob3-4 esc-8 double mutant had a long hypocotyl in multiple fluence rates of continuous white, far-red, red and blue light. sob3-4 esc-8 phyB-9 and sob3-4 esc-8 cry-103 triple mutants also had longer hypocotyls than photoreceptor single mutants. In contrast, the sob3-4 esc-8 phyA-211 triple mutant was the same length as phyA-211 single mutants. Taken together, these data indicate that SOB3 and ESC act redundantly to modulate hypocotyl growth inhibition in response to light.     

Both phytochrome A and phytochrome B are required for the normal expression of phototropism in Arabidopsis thaliana seedlings

The role of phytochrome A (phyA) and phytochrome B (phyB) in phototropism was investigated by using the phytochrome-deficient mutants phyA-101 , phyB-1 and a phyA/phyB double mutant. The red-light-induced enhancement of phototropism, which is normally observed in wild-type seedlings, could not be detected in the phyA/phyB mutant at fluences of red light between 0.1 and 19 000 μmol m⁻². The loss of phyB has been shown to have no apparent effect on enhancement, while the loss of phyA resulted in a loss of enhancement only in the low fluence range (Janoudi et al. 1997). The conclusions of the aforementioned study can now be modified based on the current results which indicate that phototropic enhancement in the high fluence range is mediated by either phyA or phyB, and that other phytochromes have no role in enhancement. First positive phototropism was unaffected in phyA-101 and phyB-1 However, the magnitude of first positive phototropism in the phyA/phyB mutant was significantly lower than that of the wild-type Landsberg parent. Thus, the presence of either phyA or phyB is required for normal expression of first positive phototropism. The time threshold for second positive phototropism is unaltered in the phyA-101 and phyB mutants. However, the time threshold in the phyA/phyB mutant is about 2 h, approximately six times that of the wild type. Finally, the magnitude of second positive phototropism in both phyA-101 and phyB-1 is diminished in comparison with the wild-type response. Thus, phyA and phyB, acting independently or in combination, regulate the magnitude of phototropic curvature and the time threshold for second positive phototropism. We conclude that the presence of phyA and phyB is required, but not sufficient, for the expression of normal phototropism.     

Genetic and transgenic evidence that phytochromes A and B act to modulate the gravitropic orientation of Arabidopsis thaliana hypocotyls

Hypocotyls of Arabidopsis thaliana exhibit negative gravitropism in the dark, growing against the gravity vector. The direction of growth is randomized in red light (R). In single mutants lacking either phytochrome A or B randomization of hypocotyl orientation in R is retained. However, a double mutant lacks this response, indicating that either phytochrome A or B is capable of inducing randomization and phytochrome A and B are the only phytochromes involved in this process. The induction of randomization was confirmed using lines that express to different levels PHYA and PHYB cDNAs. Overexpression of PHYA cDNAs induced randomization of hypocotyl orientation in the dark. Dark randomization was also seen in the phyB-1 mutant but not in two other phyB alleles, suggesting that dark randomization in the phyB-1 line may be due to a second mutation. When germination was induced by gibberellin, rather than exposure to brief white light, randomization in the dark associated with phytochrome A overproduction was not observed but was retained in the phyB-1 mutant. Overexpression of PHYB cDNAs induced a light-dependent randomization of hypocotyl orientation that responded to R:far-red light ratio. We conclude that the default situation in Arabidopsis hypocotyls is, therefore, negative gravitropism, and either phytochrome A or phytochrome B can mediate randomization.     

Phosphorylation of Phytochrome B Inhibits Light-Induced Signaling via Accelerated Dark Reversion in Arabidopsis

The photoreceptor phytochrome B (phyB) interconverts between the biologically active Pfr (λ_max = 730 nm) and inactive Pr (λ_max = 660 nm) forms in a red/far-red–dependent fashion and regulates, as molecular switch, many aspects of light-dependent development in Arabidopsis thaliana. phyB signaling is launched by the biologically active Pfr conformer and mediated by specific protein–protein interactions between phyB Pfr and its downstream regulatory partners, whereas conversion of Pfr to Pr terminates signaling. Here, we provide evidence that phyB is phosphorylated in planta at Ser-86 located in the N-terminal domain of the photoreceptor. Analysis of phyB-9 transgenic plants expressing phospho-mimic and nonphosphorylatable phyB–yellow fluorescent protein (YFP) fusions demonstrated that phosphorylation of Ser-86 negatively regulates all physiological responses tested. The Ser86Asp and Ser86Ala substitutions do not affect stability, photoconversion, and spectral properties of the photoreceptor, but light-independent relaxation of the phyB^Ser86Asp Pfr into Pr, also termed dark reversion, is strongly enhanced both in vivo and in vitro. Faster dark reversion attenuates red light–induced nuclear import and interaction of phyB^Ser86Asp-YFP Pfr with the negative regulator PHYTOCHROME INTERACTING FACTOR3 compared with phyB–green fluorescent protein. These data suggest that accelerated inactivation of the photoreceptor phyB via phosphorylation of Ser-86 represents a new paradigm for modulating phytochrome-controlled signaling.     

Phytochrome B and shade signals regulate phytochrome A expression

The control of phytochrome A expression at the protein and mRNA levels was investigated in wild-type and phyB-1 mutant sorghum ( Sorghum bicolor [L.] Moench). PHYA mRNA abundance follows a diurnal rhythm in both genotypes, with maximal accumulation near the latter part of the light period. PHYA mRNA is more abundant in the phyB-1 mutant. The level of PHYA message correlates with both R : FR and photon flux density in wild-type, but only with photon flux density in the phyB-1 mutant. The differences in mRNA abundance are reflected in the level of phyA protein, which is elevated in the phyB-1 mutant and accumulates under low photon flux density. During de-etiolation, PHYA message accumulation is initially repressed solely by a very low fluence response (VLFR) presumably mediated by phyA. The phyB-mediated low fluence response maintains the repression of accumulation past the time controlled by the VLFR. With repetitive photoperiods, the transition from the etiolated growth form to autotrophic competency is accompanied by a transition from light-induced reduction of PHYA mRNA abundance to enhanced accumulation during the light period. The loss of phyB function allows partial de-repression of PHYA message accumulation under repetitive photoperiods, resulting in plants deficient in phyB but enriched in phyA. The modification of PHYA mRNA and protein levels in the phyB-1 mutant documented in this study may help clarify the molecular basis of the phyB-1 phenotype. The tailoring of phyA abundance in wild-type to the time of day and shade signals suggests a plastic role for this pigment in controlling development in light-grown plants.     

Functional interaction of cryptochrome 1 and phytochrome D

Arabidopsis thaliana wild-type and single, double and triple mutants lacking phytochrome A (phyA-201), phytochrome B (phyB-5), phytochrome D (phyD-1), phytochrome E (phyE-1), cryptochrome 1 (hy4-2.23n) and cryptochrome 2 (fha-1) were used to study the photoreceptor signal-transduction network. The inhibition of hypocotyl elongation was analysed using pulses of red light preceded by a pre-irradiation of white light. The interactions of phyA, phyB and cry1 have been studied in a series of previous papers. Here we focus on the signal transduction initiated by phyD. We observed that phyD can partly substitute for the loss of phyB. Specifically, in the phyB background, red pulses were only effective if both cry1 and phyD were present. The response to red pulses, enabled by the pre-irradiation of white light, was completely reversible by far-red light. Loss of reversibility occurred with an apparent half-life of 2 h, similar to the half-life of 3 h observed for the effect mediated by phyB. Furthermore, we could show that the response to an end-of-day far-red pulse in phyB depends on both phyD and cry1. In contrast to phyD, a functional interaction of phyE and cry1 could not be detected in Arabidopsis seedlings.     

phyB-1 sorghum maintains responsiveness to simulated shade, irradiance and red light: far-red light

The sorghum [Sorghum bicolor (L.) Moench] phyB-1 mutant exhibits a constitutive shade-avoidance phenotype including excessive shoot elongation. It was previously shown that this mutant also overproduces ethylene. Although phytochrome B (phyB) is assumed to be the pigment most important in sensing and transducing shade signals, the sorghum phyB-1 mutant still responds to light signals characteristic of shade. Specifically, it was determined that the leaf blade : leaf sheath elongation of phyB-1 is responsive to red : far red (R : FR), but this response is opposite that of wild type (WT). Reducing the photosynthetic photon flux density (PPFD) strongly reduced the leaf blade : leaf sheath of WT but did not affect phyB-1, demonstrating a role for phyB in sensing PPFD. Using light-emitting diode (LED) lighting, it was found that WT ethylene production was increased with low R : FR while PPFD had no effect. Conversely, phyB-1 ethylene production increased only with high PPFD, high R : FR which was the treatment resulting in the least ethylene production by WT. Elevated ethylene production inhibits shoot elongation, but may contribute to shade avoidance by reducing leaf blade : leaf sheath elongation. Ethylene responses to light treatments designed to promote or reduce phytochrome A (phyA) activity, and the analysis of PHYA levels in the two cultivars suggests that phyA could be involved in transducing shade signals in light-grown sorghum. Responses potentially tranduced by phyA are elevated in phyB-1 which also over-expresses PHYA.     

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.     

A deletion in the PHYD gene of the Arabidopsis Wassilewskija ecotype defines a role for phytochrome D in red/far-red light sensing.

The PHYD gene of the Wassilewskija (Ws) ecotype of Arabidopsis contains a 14-bp deletion (the phyD-1 mutation) beginning at amino acid 29 of the reading frame, resulting in translation termination at a nonsense codon 138 nucleotides downstream of the deletion end point. Immunoblot analyses showed that Ws lacks phyD but contains normal levels of phyA, phyB, and phyC. By backcrossing into the Ws and Landsberg erecta genetic backgrounds, we constructed sibling pairs of PHYD+ and phyD-1 lines and of phyB- PHYD+ and phyB- phyD- lines. Hypocotyl lengths after growth under white or red light increased sequentially in strains that were B+D+, B+D-, B-D+, and B-D-. In the Ws genetic background, an increase in petiole length, a reduction in cotyledon area and in anthocyanin accumulation in seedling stems, a diminished effect of an end-of-day pulse of far-red light on hypocotyl elongation, and a decrease in the number of rosette leaves at the onset of flowering were also seen sequentially in these lines. Thus, phyD, which is approximately 80% identical in amino acid sequence to phyB, acts in conjunction with phyB in regulating many shade avoidance responses. The existence of the apparently naturally occurring phyD-1 mutation indicates that phyD is not essential in some natural environments.     

PNZIP Is a Novel Mesophyll-Specific cDNA That Is Regulated by Phytochrome and a Circadian Rhythm and Encodes a Protein with a Leucine Zipper Motif

Single, double, and triple null combinations of Arabidopsis mutants lacking the photoreceptors phytochrome (phy) A (phyA-201), phyB (phyB-5), and cryptochrome (cry) 1 (hy4-2.23n) were examined for de-etiolation responses in high-fluence red, far-red, blue, and broad-spectrum white light. Cotyledon unhooking, unfolding, and expansion, hypocotyl growth, and the accumulation of chlorophylls and anthocyanin in 5-d-old seedlings were measured under each light condition and in the dark. phyA was the major photoreceptor/effector for most far-red-light responses, although phyB and cry1 modulated anthocyanin accumulation in a phyA-dependent manner. phyB was the major photoreceptor in red light, although cry1 acted as a phyA/phyB-dependent modulator of chlorophyll accumulation under these conditions. All three photoreceptors contributed to most blue light deetiolation responses, either redundantly or additively; however, phyB acted as a modulator of cotyledon expansion dependent on the presence of cry1. As reported previously, flowering time in long days was promoted by phyA and inhibited by phyB, with each suppressing the other's effect. In addition to the effector/modulator relationships described above, measurements of hypocotyls from blue-light-grown seedlings demonstrated phytochrome activity in blue light and cry1 activity in a phyAphyB mutant background.