Molecular and phenotypic specificity of an antisense PHYB gene in Arabidopsis

The family of phytochrome photoreceptors plays an essential role in regulating plant growth and development in response to the light environment. An antisense PHYB transgene has been introduced into wild-type Arabidopsis and shown to inhibit expression of the PHYB sense mRNA and the phyB phytochrome protein 4- to 5-fold. This inhibition is specific to phyB in that the levels of the four other phytochromes, notably the closely related phyD and phyE phytochromes, are unaffected in the antisense lines. Antisense-induced reduction in phyB causes alterations of red light effects on seedling hypocotyl elongation, rosette leaf morphology, and chlorophyll content, similar to the phenotypic changes caused by phyB null mutations. However, unlike the phyB mutants, the antisense lines do not flower early compared to the wild type. Furthermore, unlike the phyB mutants, the antisense lines do not show a reduction in phyC level compared to the wild type, making it possible to unequivocally associate several of the photomorphogenic effects seen in phyB mutants with phytochrome B alone. These results indicate that an antisense transgene approach can be used to specifically inhibit the expression and activity of a single member of the phytochrome family and to alter aspects of shade avoidance responses in a targeted manner.     

Interactive signalling by phytochromes and cryptochromes generates de-etiolation homeostasis in Arabidopsis thaliana

Single, double, triple and quadruple mutants of phyA, phyB, cry1 and cry2 were exposed to different sunlight irradiances and photoperiods to investigate the roll played by phytochrome A, phytochrome B, cryptochrome 1 and cryptochrome 2 during de-etiolation of Arabidopsis thaliana seedlings under natural radiation. Even the quadruple mutant retained some hypocotyl-growth inhibition by sunlight. Hypocotyl length was strongly affected by interactions among photoreceptors. Double phyA phyB, phyA cry1, and cry1 cry2 mutants were taller than expected from the additive action of single mutations. Some of these redundant interactions required the presence of phytochromes A and/or B. Interactions among photoreceptors resulted in a 44% reduction of the response to irradiance and a 70% reduction of the response to photoperiod. The complex network of interactions among photoreceptors is proposed to buffer de-etiolation against changes in irradiance and photoperiod, i.e light fluctuations not related to the positions of the shoot above or below soil level     

Biochemical characterization of Arabidopsis wild-type and mutant phytochrome B holoproteins.

Although phytochrome B (phyB) plays a particularly important role throughout the life cycle of a plant, it has not been studied in detail at the molecular level due to its low abundance. Here, we report on the expression, assembly with chromophore, and purification of epitope-tagged Arabidopsis phyB. In addition, we have reconstructed two missense mutations, phyB-4 and phyB-101, isolated in long hypocotyl screens. We show that mutant proteins phyB-4 and phyB-101 exhibit altered spectrophotometric and biochemical properties relative to the wild-type protein. In particular, we demonstrate that phyB-101 Pfr exhibits rapid nonphotochemical (dark) reversion to Pr that results in a lower photoequilibrium level of the active Pfr form. We conclude that this occurs in vivo as well because phyB-101 mutants are shown to lack an end-of-day-far-red hypocotyl elongation response that requires a stable Pfr species. We propose that this Pfr instability may be the primary molecular mechanism underlying the phyB-101 mutant phenotype.     

The light-induced reduction of the gravitropic growth-orientation of seedlings of Arabidopsis thaliana (L.) Heynh. is a photomorphogenic response mediated synergistically by the far-red-absorbing forms of phytochromes A and B

Hypocotyls of dark-grown seedlings of Ara bidosis thaliana exhibit a strong negative gravitropism, which is reduced by red and also by long-wavelength, far-red light treatments. Light treatments using phytochrome A (phyA)- and phytochrome B (phyB)-deficient mutants showed that this response is controlled by phyB in a red/far-red reversible way, and by phyA in a non-reversible, very-low-fluence response. Crosses of the previously analyzed phyB-1 allele (in the ecotype Landsberg erecta background) to the ecotype Nossen wild-type (WT) background resulted in a WT-like negative gravitropism in darkness, indicating that the previously described gravitropic randomization observed with phyB-1 in the dark is likely due to a second mutation independent of that in the PHYB gene.Abbreviations FR long-wavelength far-red light - phyA phytochrome A (holoprotein) - phyB phytochrome B (holoprotein) - Pr red-absorbing form of phytochrome - WT wild type We thank Dr. A. Nagatani (RIKEN Institute, Wako-City, Japan) and Dr. M. Furuya (Hitachi, Hatoyama, Japan) for the phyA-201/phyB-5 double mutant. The work was supported by Deutsche Forschungsgemeinschaft and Human Frontier Science Program grants to E.S.     

Nicotiana plumbaginifolia hlg mutants have a mutation in a PHYB-type phytochrome gene: they have elongated hypocotyls in red light, but are not elongated as adult plants

Two new allelic mutants of Nicotiana plumbaginifolia have been isolated which display a hypocotyl which is long (hlg) when seedlings are grown in continuous white light (W). This can be accounted for by the decreased response to red light (R) of the hypocotyl elongation rate in these mutants. Responses to other wavelengths are unaffected in the mutants. When grown in white light, mature hlg mutants are not elongated with respect to the wild-type; they also bolt and flower later. The shade-avoidance responses to red/far red ratio (R:FR) are intact in these mutants. Both mutants are deficient in a phyB-like polypeptide that is immunodetectable in the wild-type; both have wild-type levels of a phyA-like polypeptide. These alleles are inherited in a partially dominant manner, and correspond to single-base missense mutations in a gene highly homologous to N. tabacum PHYB, which codes for a phytochrome B-type photoreceptor. One allele, hlg-1, has an introduced amino acid substitution; this may define a residue essential for phytochrome protein stability. The other allele, hlg-2, has a stop codon introduced C-terminal to the chromophore binding domain. As these phyB mutants are unaffected in shade-avoidance responses, but deficient in perception of R, it is concluded that the phyB absent in these mutants is responsible for R perception in the N. plumbaginifolia seedling, but is not a R:FR sensor in light-grown plants.     

Effects of Gibberellins on Seed Germination of Phytochrome-Deficient Mutants of Arabidopsis thaliana

Experiments were carried out to explore the involvement of gibberellins(GAs) in the light-induced germination of Arabidopsis thaliana(L.) Heynh, using wild type (WT) and phytochrome-deficient mutants(phyA, phyB and phyAphyB deficient in phytochrome A, B and Aplus B, respectively). Seed germination of WT and phytochrome-deficientmutants was inhibited by uniconazole (an inhibitor of an earlystep in biosynthesis of GA, the oxidation of ent-kaurene) andprohexadione (an inhibitor of late steps, namely, 2rß-and 3rß-hydroxylation). This inhibition was overcomeby simultaneous application of 10^-5 M GA₄. The relative activityof GAs for promoting germination of uniconazole-treated seedswas GA₄>GA₁=GA₉>GA₂₀. The wild type and the phyA and phyBmutants had an increased response to a red light pulse in thepresence of GA₁, GA₄, GA₉, GA₂₀ and GA₂₄ but there were no significantdifferences in activity of each GA between the mutants. Therefore,neither phytochrome A nor hytochrome B appears to regulate GAbiosynthesis from GA₁₂ to GA₄ during seed germination, sincethe conversion of GA₁₂ to GA₉ is regulated by one enzyme (GA20-oxidase). However, GA responsiveness appears to be regulatedby phytochromes other than phytochromes A and B, since the phyAphyBdouble mutant retains the photoreversible increased responseto GAs after a red light pulse. (Received February 13, 1995; Accepted July 11, 1995)     

Regulation of growth and development in phytochrome mutants of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> by solar UV

Phytochrome mutants (phyA, phyB and phyAB) of Arabidopsis thaliana were grown under ambient and UV-excluded sunlight to understand their influence on growth and development by mutual exclusion. Phytochrome A and B played a complementary role in the regulation of germination. Suppression of hypocotyl length was predominantly under the control of phytochrome B; UV photoreceptors were active in suppression of hypocotyl growth only in phyB and phyAB mutants. Exclusion of UV promoted the number and the area of rosette leaves only in presence of phytochrome A and B. Phytochrome mutation reduced petiole length, whereas UV exclusion led to an increase. Requirement of long-day period for flowering was removed in the mutants. Under short-day conditions, flowering was predominantly under the control of phytochrome B, since phyB mutants flowered earlier than phyA mutants. Solar UV regulates the number of boltings and number of siliques per plant. Overall biomass of the plants is enhanced by the exclusion of UV only in the wild type. The interaction of phytochromes with UV photoreceptors is discussed in the paper.     

Suppression of late-flowering and semi-dwarf phenotypes in the arabidopsis clock mutant lhy-12;cca1-101 by phyB under continuous light

Photoperiodic flowering in Arabidopsis is controlled not only by floral activators such as GI, CO and FT, but also by repressors such as SVP and FLC. Double mutations in LHY and CCA1 (lhy;cca1) accelerated flowering under short days, mainly by the GI-CO dependent pathway. In contrast, lhy;cca1 showed delayed flowering under continuous light (LL), probably due to the GI-CO independent pathway. This late-flowering phenotype was suppressed by svp, flc and elf3. However, how SVP, FLC and ELF3 mediate LHY/CCA1 and flowering time is not fully understood. We found that lhy;cca1 exhibited short hypocotyls and petioles under LL, but the molecular mechanism for these effects has not been elucidated.To address these questions, we performed a screen for mutations that suppress either or both of the lhy;cca1 phenotypes under LL, using two different approaches. We identified two novel mutations, a dominant (del1) and a recessive (phyB-2511) allele of phyB. The flowering times of single mutants of three phyB alleles, hy3-1, del1 and phyB-2511, are almost the same and earlier than those of wild-type plants. A similar level of acceleration of flowering time was observed in all three phyB mutants tested when combined with the late-flowering mutations co-2 and SVPox. However, the effect of phyB-2511 on lhy;cca1 was different from those by hy3-1 or del1. svp-3 did not strongly enhance the early-flowering phenotypes of phyB-2511 or del1. These results suggest that light signaling via PhyB may affect factors downstream of the clock proteins, controlling flowering time and organ elongation. phyB mutations with different levels of effects on lhy;cca1-dependent late flowering would be useful to determine a specific role for PHYB in the flowering pathway controlled by lhy;cca1 under LL.Key words: Arabidopsis thaliana, CCA1, circadian clock, CO, FT, LHY, organ elongation, photoperiodic flowering, PHYB, SVP     

Co-action between phytochrome B and HY4 in Arabidopsis thaliana

A combination of physiological and genetic approaches was used to investigate whether phytochromes and blue light (BL) photoreceptors act in a fully independent manner during photomorphogenesis of Arabidopsis thaliana (L.) Heynh. Wild-type seedlings and phyA, phyBand hy4 mutants were daily exposed to 3 h BL terminated with either a red light (R) or a far-red light (FR) pulse. In wild-type and phyA-mutant seedlings, BL followed by an R pulse inhibited hypocotyl growth and promoted cotyledon unfolding. The effects of BL were reduced if exposure to BL was followed by an FR pulse driving phytochrome to the R-absorbing form (Pr). In the wild type, the effects of R versus FR pulses were small in seedlings not exposed to BL. Thus, maximal responses depended on the presence of both BL and the FR-absorbing form of phytochrome (Pfr) in the subsequent dark period. Impaired responses to BL and to R versus FR pulses were observed in phyB and hy4 mutants. Simultaneous irradiation with orange light indicated that BL, perceived by specific BL photoreceptors (i.e. not by phytochromes), required phytochrome B to display a full effect. These results indicate interdependent co-action between phytochrome B and BL photoreceptors, particularly the HY4 gene product. No synergism between phytochrome A (activated by continuous or pulsed FR) and BL photoreceptors was observed.Abbreviations BL blue light - D darkness - FR far-redlight - FRc continuous FR - Pfr FR-absorbing form of phytochrome - Pfr/P proportion of phytochrome as Pfr - phyA phytochrome A - phyB phytochrome B - R red light - WT wild type We thank Professors R.E. Kendrick and M. Koornneef (Wageningen Agricultural University, The Netherlands), Professor J. Chory (Salk Institute, Calif., USA) and the Arabidopsis Biological Resource Center (Ohio State University, Ohio, USA) for their kind provision of the original seed batches. This work was financially supported by CONICET, Universidad de Buenos Aires (AG 040) and Fundación Antorchas (A-12830/1 0000/9)     

The Phytochrome B/Phytochrome C Heterodimer Is Necessary for Phytochrome C-Mediated Responses in Rice Seedlings

Background

PhyC levels have been observed to be markedly lower in phyB mutants than in Arabidopsis or rice wild type etiolated seedlings, but the mechanism of this phenomenon has not been fully elucidated.

Results

In the present study, we investigated the mechanism by which phyB affects the protein concentration and photo-sensing abilities of phyC and demonstrated that rice phyC exists predominantly as phyB/phyC heterodimers in etiolated seedlings. PHYC-GFP protein was detected when expressed in phyA phyC mutants, but not in phyA phyB mutants, suggesting that phyC requires phyB for its photo-sensing abilities. Interestingly, when a mutant PHYB gene that has no chromophore binding site, PHYB(C364A), was introduced into phyB mutants, the phyC level was restored. Moreover, when PHYB(C364A) was introduced into phyA phyB mutants, the seedlings exhibited de-etiolation under both far-red light (FR) and red light (R) conditions, while the phyA phyB mutants were blind to both FR and R. These results are the first direct evidence that phyC is responsible for regulating seedling de-etiolation under both FR and R. These findings also suggest that phyB is indispensable for the expression and function of phyC, which depends on the formation of phyB/phyC heterodimers.

Significance

The present report clearly demonstrates the similarities and differences in the properties of phyC between Arabidopsis and rice and will advance our understanding of phytochrome functions in monocots and dicots.     

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.     

Sorghum Phytochrome B Inhibits Flowering in Long Days by Activating Expression of SbPRR37 and SbGHD7, Repressors of SbEHD1, SbCN8 and SbCN12

Light signaling by phytochrome B in long days inhibits flowering in sorghum by increasing expression of the long day floral repressors PSEUDORESPONSE REGULATOR PROTEIN (SbPRR37, Ma1) and GRAIN NUMBER, PLANT HEIGHT AND HEADING DATE 7 (SbGHD7, Ma6). SbPRR37 and SbGHD7 RNA abundance peaks in the morning and in the evening of long days through coordinate regulation by light and output from the circadian clock. 58 M, a phytochrome B deficient (phyB-1, ma3^R) genotype, flowered ∼60 days earlier than 100 M (PHYB, Ma3) in long days and ∼11 days earlier in short days. Populations derived from 58 M (Ma1, ma3^R, Ma5, ma6) and R.07007 (Ma1, Ma3, ma5, Ma6) varied in flowering time due to QTL aligned to PHYB/phyB-1 (Ma3), Ma5, and GHD7/ghd7-1 (Ma6). PHYC was proposed as a candidate gene for Ma5 based on alignment and allelic variation. PHYB and Ma5 (PHYC) were epistatic to Ma1 and Ma6 and progeny recessive for either gene flowered early in long days. Light signaling mediated by PhyB was required for high expression of the floral repressors SbPRR37 and SbGHD7 during the evening of long days. In 100 M (PHYB) the floral activators SbEHD1, SbCN8 and SbCN12 were repressed in long days and de-repressed in short days. In 58 M (phyB-1) these genes were highly expressed in long and short days. Furthermore, SbCN15, the ortholog of rice Hd3a (FT), is expressed at low levels in 100 M but at high levels in 58 M (phyB-1) regardless of day length, indicating that PhyB regulation of SbCN15 expression may modify flowering time in a photoperiod-insensitive manner.     

Mutations in the gene for the red/far-red light receptor phytochrome B alter cell elongation and physiological responses throughout Arabidopsis development.

Phytochromes are a family of plant photoreceptors that mediate physiological and developmental responses to changes in red and far-red light conditions. In Arabidopsis, there are genes for at least five phytochrome proteins. These photoreceptors control such responses as germination, stem elongation, flowering, gene expression, and chloroplast and leaf development. However, it is not known which red light responses are controlled by which phytochrome species, or whether the different phytochromes have overlapping functions. We report here that previously described hy3 mutants have mutations in the gene coding for phytochrome B (PhyB). These are the first mutations shown to lie in a plant photoreceptor gene. A number of tissues are abnormally elongated in the hy3(phyB) mutants, including hypocotyls, stems, petioles, and root hairs. In addition, the mutants flower earlier than the wild type, and they accumulate less chlorophyll. PhyB thus controls Arabidopsis development at numerous stages and in multiple tissues.     

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     

A role for ABCB19‐mediated polar auxin transport in seedling photomorphogenesis mediated by cryptochrome 1 and phytochrome B

During seedling establishment, blue and red light suppress hypocotyl growth through the cryptochrome 1 (cry1) and phytochrome B (phyB) photosensory pathways, respectively. How these photosensory pathways integrate with growth control mechanisms to achieve the appropriate degree of stem elongation was investigated by combining cry1 and phyB photoreceptor mutations with genetic manipulations of a multidrug resistance‐like membrane protein known as ABCB19 that influenced auxin distribution within the plant, as evidenced by a combination of reporter gene assays and direct auxin measurements. Auxin signaling and ABCB19 protein levels, hypocotyl growth rates, and apical hook opening were measured in mutant and wild‐type seedlings exposed to a range of red and blue light conditions. Ectopic/overexpression of ABCB19 (B19OE) greatly increased auxin in the hypocotyl, which reduced the sensitivity of hypocotyl growth specifically to blue light in long‐term assays and red light in high‐resolution, short‐term assays. Loss of ABCB19 partially suppressed the cry1 hypocotyl growth phenotype in blue light. Hypocotyl growth of B19OE seedlings in red light was very similar to phyB mutants. Altered auxin distribution in B19OE seedlings also affected the opening of the apical hook. The cry1 and phyB photoreceptor mutations both increased ABCB19 protein levels at the plasma membrane, as measured by confocal microscopy. The B19OE plant proved to be a useful tool for determining aspects of the mechanism by which light, acting through cry1 or phyB, influences the auxin transport process to control hypocotyl growth during de‐etiolation.     

Phytochromes influence stomatal conductance plasticity in Arabidopsis thaliana

The ability to sense and respond effectively to drought stress can be important for plant fitness. Here, phytochrome loss-of-function mutants were grown in dry and moist conditions to examine the role of three photoreceptors (phyA, phyB, and phyE) in stomatal conductance (g _ST) and abscisic acid (ABA) concentration. Overall, drought treatment plants had lower g _ST than moist treatment plants. However, the wild-type Landsberg erecta line had a less pronounced conductance response to drought treatment than the phytochrome mutants, suggesting a role for phytochrome in suppressing drought tolerance. Phytochrome gene effects were potentially additive for g _ST; however, PHYB and PHYE effects were nonadditive for ABA concentration.     

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.