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.     

Genetic identification of FIN2, a far red light-specific signaling component of Arabidopsis thaliana

Phytochrome A (PhyA) mediates most, if not all various plant responses to far-red (FR) light. Here, we report a novel genetic mutation that impairs a variety of responses in the PhyA-signaling pathway of Arabidopsis thaliana . The mutation was isolated by screening seedlings that show reduced sensitivity to continuous far-red (FRc) light irradiation, but not to continuous red (Rc) light irradiation. The mutation named fin2–1 is not allelic to a PHYA mutation. Furthermore, immunoblot analysis indicated that the amount of the phytochrome A apoprotein in the fin2–1 mutant was comparable to that in wild type. Seedling of the fin2–1 mutant showed defects in hypocotyl growth inhibition and apical hook and cotyledon opening in FRc light but not in Rc light. The results showed that the mutation occurred in a downstream signaling component potentially specific to PhyA. Other PhyAmediated responses such as FR-preconditioned blocking of greening, anthocyanin accumulation, reduction of gravitropic response, and expression of the CAB and CHS genes were impaired by the fin2–1 mutation: the degree of the mutant effect on the responses was variable. However, FR light-mediated seed germination and photoperiodic flowering responses were not affected significantly in the mutant. These results showed that FIN2 defines an upstream branch point in the PhyA signaling pathway.     

High-irradiance responses induced by far-red light in grass seedlings of the wild type or overexpressing phytochrome A

The occurrence of phytochrome-mediated highirradiance responses (HIR), previously characterised largely in dicotyledonous plants, was investigated in Triticum aestivum L., Zea mays L., Lolium multiflorum Lam. and in both wild-type Oryza sativa L. and in transgenic plants overexpressing oat phytochrome A under the control of a 35S promoter. Coleoptile growth was promoted (maize, ryegrass) or inhibited (wild-type rice) by continuous far-red light (FRc). However, at equal fluences, hourly pulses of far-red light (FRp) were equally effective, indicating that the growth responses to FRc were not true HIR. In contrast, in maize and rice, FRc increased anthocyanin content in the coleoptile in a fluence-rate dependent manner. This response was a true HIR as FRp had reduced effects. In maize, anthocyanin levels were significantly higher under FRc than under continuous red light. In rice, overexpression of phytochrome A increased the inhibition of coleoptile growth and the levels of anthocyanin under FRc but not under FRp or under continuous red light. The effect of FRc was fluence-rate dependent. In light-grown rice, overexpression of phytochrome A reduced leaf-sheath length, impaired the response to supplementary far-red light, but did not affect the response to canopy shade-light. In grasses, typical HIR, i.e. fluence-rate dependent responses showing reciprocity failure, can be induced by FRc. Under FRc, overexpressed phytochrome A operates through this action mode in transgenic rice.Abbreviations FR far-red light - FRc continuous far-red light - FRp pulses of far-red light - HIR high-irradiance responses - LFR low-fluence responses - OPHYA transgenic rice overexpressing oat phytochrome A - Pfr far-red light-absorbing form of phytochrome - phyA phytochrome A - R red light - Rc continuous red light - VLFR very low-fluence responses - WT wildtype We thank Marcelo J. Yanovsky for his help with the photographs and Professor Rodolfo A. Sanchez for providing a reprint of the paper by P.J.A.L. de Lint. This work was supported by grants from UBA (AG041) and Fundacion Antorchas (A-13218/1-15) to J.J.C.     

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)     

Dual effect of phytochrome A on hypocotyl growth under continuous red light

The role of phytochrome A in the control of hypocotyl growth under continuous red light (Rc) was investigated using phyA and phyB mutants of Arabidopsis thaliana, which lack phytochrome A (phyA) or phytochrome B (phyB), respectively, and transgenic seedlings of Nicotiana tabacum overexpressing Avena phyA, compared to the corresponding wild type (WT). In WT seedlings of A. thaliana, hypocotyl growth inhibition showed a biphasic response to the fluence rate of Rc, with a brake at 10^?2μmol m^?2 s^?1. At equal total fluence rate, hourly pulses of red light caused slightly more inhibition than Rc. The response to very low fluences of continuous or pulsed red light was absent in the phyA and phyA phyB mutants and present in the phyB mutant. The second part of the response was steeper in the phyA mutant than in the WT but was absent in the phyB mutant. In WT tobacco the response to Rc was biphasic. Overexpression of Avena phyA enhanced the response only at very low fluence rates of Rc (< 10^?2μmol m^?2 s^?1). In both species, the effect of hourly pulses of far-red light was similar to the maximum inhibition observed in the first phase of the response to Rc. Using reciprocity failure (i.e. higher inhibition under continuous than pulsed light) as the operational criterion, a ‘true’ high-irradiance reaction occurred under continuous far-red light but not under Rc or red plus far-red light mixtures. Native and overexpressed phyA are proposed to mediate very low fluence responses under Rc. In WT A. thaliana, this effect is counteracted by a negative action of phyA on phyB-mediated low-fluence responses.     

Overexpressed phytochrome C has similar photosensory specificity to phytochrome B but a distinctive capacity to enhance primary leaf expansion

Phytochrome C (phyC) is a low-abundance member of the five-membered phytochrome family of photoreceptors in Arabidopsis. Towards developing an understanding of the photosensory and physiological functions of phyC, transgenic Arabidopsis plants were generated that overexpress cDNA-encoded phyC and seedling responses to continuous white, red, or far-red light (Wc, Rc or FRc, respectively) were examined. Transgenic seedlings overexpressing phyC displayed enhanced inhibition of hypocotyl elongation in Rc, but were unchanged in responsiveness to FRc relative to wild-type. These data indicate that phyC has photosensory specificity that is similar to that of phyB and thus distinct from that of phyA. phyC overexpressors with levels only 3 to 4 times the level of endogenous phyC exhibited enhanced primary leaf expansion in Wc. This is in contrast to phyA or phyB overexpressors which respectively have levels that are 500-and 100-fold that of overexpressed phyC but showed no enhancement of primary leaf expansion. Therefore, phyC may have some physiological roles that are different to those of phyA and phyB in the control of seedling responses to light signals.     

Phytochrome A is an irradiance-dependent red light sensor

Plants perceive red (R) and far-red (FR) light signals using the phytochrome family of photoreceptors. In Arabidopsis thaliana, five phytochromes (phyA-phyE) have been identified and characterized. Unlike other family members, phyA is subject to rapid light-induced proteolytic degradation and so accumulates to relatively high levels in dark-grown seedlings. The insensitivity of phyA mutant seedlings to prolonged FR and wild-type appearance in R has led to suggestions that phyA functions predominantly as an FR sensor during the early stages of seedling establishment. The majority of published photomorphogenesis experiments have, however, used <50 micromol m(-2) sec(-1) of R when characterizing phytochrome functions. Here we reveal considerable phyA activity in R at higher (>160 micromol m(-2) sec(-1)) photon irradiances. Under these conditions, plant architecture was observed to be largely regulated by the redundant actions of phytochromes A, B and D. Moreover, quadruple phyBphyCphyDphyE mutants containing only functional phyA displayed R-mediated de-etiolation and survived to flowering. The enhanced activity of phyA in continuous R (Rc) of high photon irradiance correlates with retarded degradation of the endogenous protein in wild-type plants and prolonged epifluorescence of nuclear-localized phyA:YFP in transgenic lines. Such observations suggest irradiance-dependent 'photoprotection' of nuclear phyA in R, providing a possible explanation for the increased activity observed. The discovery that phyA can function as an effective irradiance sensor, even in light environments that establish a high Pfr concentration, raises the possibility that phyA may contribute significantly to the regulation of growth and development in daylight-grown plants.     

The GRAS protein SCL13 is a positive regulator of phytochrome-dependent red light signaling, but can also modulate phytochrome A responses

Phytochrome photoreceptors enable plants to perceive divergent light signals leading to adaptive changes in response to differing environmental conditions. However, the mechanism of light signal transduction is not fully understood. Here we report the identification of a new signaling intermediate from Arabidopsis thaliana, Scarecrow-like (SCL)13, which serves as a positive regulator of continuous red light signals downstream of phytochrome B (phyB). SCL13 antisense lines exhibit reduced sensitivity towards red light, but only a distinct subset of phyB-mediated responses is affected, indicating that SCL13 executes its major role in hypocotyl elongation during de-etiolation. Genetic evidence suggests that SCL13 is also needed to modulate phytochrome A (phyA) signal transduction in a phyB-independent way. The SCL13 protein is localized in the cytoplasm, but can also be detected in the nucleus. Overexpression of both a nuclear and cytoplasmic localized SCL13 protein leads to a hypersensitive phenotype under red light indicating that SCL13 is biologically active in both compartments. SCL13 is a member of the plant-specific GRAS protein family, which is involved in various different developmental and signaling pathways. A previously identified phytochrome A signaling intermediate, PAT1, belongs to the same subbranch of GRAS proteins as SCL13. Although both proteins are involved in phytochrome signaling, each is specific for a different light condition and regulates a different subset of responses.     

B-box protein BBX32 integrates light and brassinosteroid signals to inhibit cotyledon opening

Cotyledon opening is a key morphological change that occurs in seedlings during de-etiolation. Brassinosteroids (BRs) inhibit the opening of cotyledons in darkness while light promotes cotyledon opening. The molecular regulation of the interplay between light and BR to regulate cotyledon opening is not well understood. Here, we show the B-box protein BBX32 negatively regulates light signaling and promotes BR signaling to inhibit cotyledon opening in Arabidopsis (Arabidopsis thaliana). BBX32 is highly expressed in the cotyledons of seedlings during de-etiolation. bbx32 and 35S:BBX32 seedlings exhibit enhanced and reduced cotyledon opening, respectively, in response to both light and brassinazole treatment in dark, suggesting that BBX32 mediates cotyledon opening through both light and BR signaling pathways. BBX32 expression is induced by exogenous BR and is upregulated in bzr1-1D (BRASSINAZOLE RESISTANT1-1D). Our in vitro and in vivo interaction studies suggest that BBX32 physically interacts with BZR1. Further, we found that PHYTOCHROME-INTERACTING FACTOR 3 (PIF3) interacts with BBX32 and promotes BR-mediated cotyledon closure. BBX32, BZR1, and PIF3 regulate the expression of common target genes that modulate the opening and closing of cotyledons. Our work suggests BBX32 integrates light and BR signals to regulate cotyledon opening during de-etiolation.     

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.     

蓝光调节高粱突变体har1幼苗的去黄化反应

以航空诱变高粱突变体har1为材料,对其幼苗去黄化过程进行研究。萌发的种子在远红光下预培养6小时后,置于12小时蓝光/2小时黑暗条件下培养。测量幼苗的各器官伸长,结果表明,与野生型R111相比,harl的胚芽鞘、中胚轴、第一叶鞘以及第二叶鞘的伸长均受到蓝光的明显抑制,而蓝光对叶片生长影响不明显。3天龄har1黄化苗在连续蓝光下中胚轴花色素苷的积累明显增高,红光和远红光无此效应。此外,蓝光促进har1叶片叶绿体发育,且在蓝光照射24小时后叶片中叶绿素含量升高。Westernblot检测结果显示,7天龄R111和har1幼苗隐花色素SbCRY1b蛋白水平呈现蓝光下低、黑暗中高的变化趋势,har1的SbCRY1b蛋白水平在黑暗中高于R111。研究结果表明,高粱har1在去黄化过程中具有蓝光超敏感表型,SbCRY1b的作用值得进一步深入研究。