HYPERSENSITIVE TO RED AND BLUE 1 and its C-terminal regulatory function control FLOWERING LOCUS T expression

The red and far-red light-absorbing phytochromes and UV-A/blue light-absorbing cryptochromes regulate seedling de-etiolation and flowering responses. The signaling steps that mediate the photoreceptor regulation on key flowering genes remain largely unknown. We report that a previously identified photomorphogenic mutant, hypersensitive to red and blue 1 (hrb1), flowered late and showed attenuated expression of FLOWERING LOCUS T (FT) over both long days and short days. Transgenic plants that overexpress the full-length HRB1, or its C-terminal half, flowered early and accumulated more FT messages under short-day conditions. The transgenic plants also displayed hyposensitive de-etiolation phenotypes, and the expression of these phenotypes requires the action of PIF4. The double mutant of hrb1/cry2 showed a flowering phenotype and an FT expression pattern similar to hrb1 under long-day conditions, suggesting that HRB1 may function downstream of cry2 under long-day conditions. In contrast, hrb1/phyB-9 showed a flowering phenotype and an FT expression pattern similar to phyB-9 over both long days and short days, indicating a modulatory role of HRB1 in the flowering pathway mediated by phyB. Overexpression of HRB1 did not affect the expression of the central clock oscillators, TOC1 and CCA1. HRB1 therefore represents a signaling step that regulates FT expression downstream of red and blue light perception.     

A gain-of-function mutation of Arabidopsis cryptochrome1 promotes flowering

Plants use different classes of photoreceptors to collect information about their light environment. Cryptochromes are blue light photoreceptors that control deetiolation, entrain the circadian clock, and are involved in flowering time control. Here, we describe the cry1-L407F allele of Arabidopsis (Arabidopsis thaliana), which encodes a hypersensitive cryptochrome1 (cry1) protein. Plants carrying the cry1-L407F point mutation have elevated expression of CONSTANS and FLOWERING LOCUS T under short-day conditions, leading to very early flowering. These results demonstrate that not only the well-studied cry2, with an unequivocal role in flowering promotion, but also cry1 can function as an activator of the floral transition. The cry1-L407F mutants are also hypersensitive toward blue, red, and far-red light in hypocotyl growth inhibition. In addition, cry1-L407F seeds are hypersensitive to germination-inducing red light pulses, but the far-red reversibility of this response is not compromised. This demonstrates that the cry1-L407F photoreceptor can increase the sensitivity of phytochrome signaling cascades. Molecular dynamics simulation of wild-type and mutant cry1 proteins indicated that the L407F mutation considerably reduces the structural flexibility of two solvent-exposed regions of the protein, suggesting that the hypersensitivity might result from a reduced entropic penalty of binding events during downstream signal transduction. Other nonmutually exclusive potential reasons for the cry1-L407F gain of function are the location of phenylalanine-407 close to three conserved tryptophans, which could change cry1's photochemical properties, and stabilization of ATP binding, which could extend the lifetime of the signaling state of cry1.     

PIF4, a phytochrome-interacting bHLH factor,functions as a negative regulator of phytochrome B signaling in Arabidopsis

Plants sense and respond to red and far-red light using the phytochrome (phy) family of photoreceptors. However, the mechanism of light signal transduction is not well defined. Here, we report the identification of a new mutant Arabidopsis locus, srl2 (short under red-light 2), which confers selective hypersensitivity to continuous red, but not far-red, light. This hypersensitivity is eliminated in srl2phyB, but not srl2phyA, double mutants, indicating that this locus functions selectively and negatively in phyB signaling. The SRL2 gene encodes a bHLH factor, designated PIF4 (phytochrome-interacting factor 4), which binds selectively to the biologically active Pfr form of phyB, but has little affinity for phyA. Despite its hypersensitive morphological phenotype, the srl2 mutant displays no perturbation of light-induced expression of marker genes for chloroplast development. These data suggest that PIF4 may function specifically in a branch of the phyB signaling network that regulates a subset of genes involved in cell expansion. Consistent with this proposal, PIF4 localizes to the nucleus and can bind to a G-box DNA sequence motif found in various light-regulated promoters.     

拟南芥QUA1基因在光信号途径中的表达与功能分析

光信号在植物生长发育过程中具有非常重要的作用。不同的光信号通过调节植物下游基因的表达,进而影响细胞分化、结构和功能的改变,以及组织和器官的形成,参与植物光形态建成。QUA1 (QUASIMODO1)是拟南芥糖基转移酶家族中的一个成员,参与植物细胞壁中果胶的合成。本文以拟南芥qua1-1/cry1以及qua1-1/phyB双突变体为材料,对QUA1基因在光信号途径中的功能进行了分析。结果显示,qua1-1突变体在暗、蓝光、红光以及远红外光培养条件下下胚轴的伸长均受到抑制,QUA1基因的表达同样受到光信号的调节,而且突变体中多种光信号调节基因的表达也受到了影响。通过对qua1-1突变体下胚轴的观察发现,突变体下胚轴表皮细胞长度明显变短。与cry1以及phyB突变体相比,qua1-1/cry1和qua1-1/phyB双突变体下胚轴长度明显变短,而且双突变体中光信号调节基因的表达也有明显变化,表明QUA1可能参与了CRY1以及PHYB介导的蓝光及红光信号传导。以上结果表明QUA1影响了下胚轴细胞的伸长以及光信号调节基因的表达,并参与调控多种光信号传导途径。     

Nitric oxide regulates DELLA content and PIF expression to promote photomorphogenesis in Arabidopsis

The transition from etiolated to green seedlings involves a shift from hypocotyl growth-promoting conditions to growth restraint. These changes occur through a complex light-driven process involving multiple and tightly coordinated hormonal signaling pathways. Nitric oxide (NO) has been lately characterized as a regulator of plant development interacting with hormone signaling. Here, we show that Arabidopsis (Arabidopsis thaliana) NO-deficient mutant hypocotyls are longer than those from wild-type seedlings under red light but not under blue or far-red light. Accordingly, exogenous treatment with the NO donor sodium nitroprusside and mutant plants with increased endogenous NO levels resulted in reduced hypocotyl length. In addition to increased hypocotyl elongation, NO deficiency led to increased anthocyanin levels and reduced PHYB content under red light, all processes governed by phytochrome-interacting factors (PIFs). NO-deficient plants accordingly showed an enhanced expression of PIF3, PIF1, and PIF4. Moreover, exogenous NO increased the levels of the gibberellin (GA)-regulated DELLA proteins and shortened hypocotyls, likely through the negative regulation of the GA Insensitive Dwarf1 (GID1)-Sleepy1 (SLY1) module. Consequently, NO-deficient seedlings displayed up-regulation of SLY1, defective DELLA accumulation, and altered GA sensitivity, thus resulting in defective deetiolation under red light. Accumulation of NO in wild-type seedlings undergoing red light-triggered deetiolation and elevated levels of NO in the GA-deficient ga1-3 mutant in darkness suggest a mutual NO-GA antagonism in controlling photomorphogenesis. PHYB-dependent NO production promotes photomorphogenesis by a GID1-GA-SLY1-mediated mechanism based on the coordinated repression of growth-promoting PIF genes and the increase in the content of DELLA proteins.     

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.     

Conditional Synergism between Cryptochrome 1 and Phytochrome B Is Shown by the Analysis of phyA, phyB, and hy4 Simple, Double, and Triple Mutants in Arabidopsis

Wild-type or phyA, phyB, or hy4 mutant Arabidopsis seedlings lacking phytochrome A (phyA), phytochrome B (phyB), or cryptochrome 1 (cry1), respectively, and the double and triple mutants were used in combination with blue-light treatments given simultaneously with red or far-red light. We investigated the interaction between phytochromes and cry1 in the control of hypocotyl growth and cotyledon unfolding. Under conditions deficient for cry1 (short exposures to blue light) or phyB (far-red background), these photoreceptors acted synergistically: Under short exposures to blue light (3 h/d) added to a red-light background, cry1 activity required phyB (e.g. the hy4 mutant was taller than the wild type but the phyBhy4 mutant was not taller than the phyB mutant). Under prolonged exposures to blue light (24 h/d) added to a far-red light background, phyB activity required cry1 (e.g. the phyAphyB mutant was taller than the phyA mutant but the phyAphyBhy4 mutant was not taller than the phyAhy4 mutant). Under more favorable light inputs, i.e. prolonged exposures to blue light added to a red-light background, the effects of cry1 and phyB were independent. Thus, the synergism between phyB and cry1 is conditional. The effect of cry1 was not reduced by the phyA mutation under any tested light condition. Under continuous blue light the triple mutant phyAphyBhy4 showed reduced hypocotyl growth inhibition and cotyledon unfolding compared with the phyAphyB mutant. The action of cry1 in the phyAphyB double mutant was higher under the red-light than the far-red-light background, indicating a synergistic interaction between cry1 and phytochromes C, D, or E; however, a residual action of cry1 independent of any phytochrome is likely to occur.     

shl, a New set of Arabidopsis mutants with exaggerated developmental responses to available red, far-red, and blue light

The interaction of light perception with development is the subject of intensive genetic analysis in the model plant Arabidopsis. We performed genetic screens in low white light-a threshold condition in which photomorphogenetic signaling pathways are only partially active-for ethyl methane sulfonate-generated mutants with altered developmental phenotypes. Recessive mutants with exaggerated developmental responses were obtained in eight complementation groups designated shl for seedlings hyperresponsive to light. shl1, shl2, shl5, and shl3 shl4 (double mutant) seedlings showed limited or no phenotypic effects in darkness, but showed significantly enhanced inhibition of hypocotyl elongation in low-white, red, far-red, blue, and green light across a range of fluences. These results reflect developmental hyper-responsiveness to signals generated by both phytochrome and cryptochrome photoreceptors. The shl11 mutant retained significant phenotypic effects on hypocotyl length in both the phyA mutant and phyB mutant backgrounds but may be dependent on CRY1 for phenotypic expression in blue light. The shl2 phenotype was partially dependent on PHYB, PHYA, and CRY1 in red, far-red, and blue light, respectively. shl2 and, in particular, shl1 were partially dependent on HY5 activity for their light-hyperresponsive phenotypes. The SHL genes act (genetically) as light-dependent negative regulators of photomorphogenesis, possibly in a downstream signaling or developmental pathway that is shared by CRY1, PHYA, and PHYB and other photoreceptors (CRY2, PHYC, PHYD, and PHYE).     

Genetic dissection of blue-light sensing in tomato using mutants deficient in cryptochrome 1 and phytochromes A, B1 and B2

Several novel allelic groups of tomato (Solanum lycopersicum L.) mutants with impaired photomorphogenesis have been identified after gamma-ray mutagenesis of phyA phyB1 double-mutant seed. Recessive mutants in one allelic group are characterized by retarded hook opening, increased hypocotyl elongation and reduced hypocotyl chlorophyll content under white light (WL). These mutants showed a specific impairment in response to blue light (BL) resulting from lesions in the gene encoding the BL receptor cryptochrome 1 (cry1). Phytochrome A and cry1 are identified as the major photoreceptors mediating BL-induced de-etiolation in tomato, and act under low and high irradiances, respectively. Phytochromes B1 and B2 also contribute to BL sensing, and the relative contribution of each of these four photoreceptors differs according to the light conditions and the specific process examined. Development of the phyA phyB1 phyB2 cry1 quadruple mutant under WL is severely impaired, and seedlings die before flowering. The quadruple mutant is essentially blind to BL, but experiments employing simultaneous irradiation with BL and red light suggest that an additional non-phytochrome photoreceptor may be active under short daily BL exposures. In addition to effects on de-etiolation, cry1 is active in older, WL-grown plants, and influences stem elongation, apical dominance, and the chlorophyll content of leaves and fruit. These results provide the first mutant-based characterization of cry1 in a plant species other than Arabidopsis.     

A study of gibberellin homeostasis and cryptochrome-mediated blue light inhibition of hypocotyl elongation

Cryptochromes mediate blue light-dependent photomorphogenic responses, such as inhibition of hypocotyl elongation. To investigate the underlying mechanism, we analyzed a genetic suppressor, scc7-D (suppressors of cry1cry2), which suppressed the long-hypocotyl phenotype of the cry1cry2 (cryptochrome1/cryptochrome2) mutant in a light-dependent but wavelength-independent manner. scc7-D is a gain-of-expression allele of the GA2ox8 gene encoding a gibberellin (GA)-inactivating enzyme, GA 2-oxidase. Although scc7-D is hypersensitive to light, transgenic seedlings expressing GA2ox at a level higher than scc7-D showed a constitutive photomorphogenic phenotype, confirming a general role of GA2ox and GA in the suppression of hypocotyl elongation. Prompted by this result, we investigated blue light regulation of mRNA expression of the GA metabolic and catabolic genes. We demonstrated that cryptochromes are required for the blue light regulation of GA2ox1, GA20ox1, and GA3ox1 expression in transient induction, continuous illumination, and photoperiodic conditions. The kinetics of cryptochrome induction of GA2ox1 expression and cryptochrome suppression of GA20ox1 or GA3ox1 expression correlate with the cryptochrome-dependent transient reduction of GA(4) in etiolated wild-type seedlings exposed to blue light. Therefore we propose that in deetiolating seedlings, cryptochromes mediate blue light regulation of GA catabolic/metabolic genes, which affect GA levels and hypocotyl elongation. Surprisingly, no significant change in the GA(4) content was detected in the whole shoot samples of the wild-type or cry1cry2 seedlings grown in the dark or continuous blue light, suggesting that cryptochromes may also regulate GA responsiveness and/or trigger cell- or tissue-specific changes of the level of bioactive GAs.     

KIDARI, Encoding a Non-DNA Binding bHLH Protein, Represses Light Signal Transduction in Arabidopsis thaliana

Through activation tagging mutagenesis, we isolated a kidari-D (kdr-D) mutant, which exhibited a defect in blue and far-red light mediated photomorphogenesis. Under continuous blue light, the kdr-D mutant showed long hypocotyl phenotype, whereas it showed normal cotyledon opening and expansion. In addition, the kdr-D showed slightly longer hypocotyl under continuous far-red light, suggesting that KDR functions in a branch of cry signaling and mediates a cross-talk between cry and phyA. In the kdr-D mutant, a gene encoding a putative basic/Helix-Loop-Helix (bHLH) protein was overexpressed by the insertion of 35S enhancer into 10 kb upstream of the gene. Consistently, overexpression of this gene recapitulated the phenotype of kdr-D. KDR is composed of 94 amino acids with non-DNA binding HLH domain, a structure found in human Inhibitor of DNA binding 1 (Id-1) which functions as a negative regulator of bHLH proteins through heterodimerization with them. The KDR specifically interacted with HFR1, a bHLH protein regulating photomorphogenesis, in yeast two hybrid assay and the kdr-D was epistatic to 35S::HFR1 in the hypocotyl phenotype. Thus, it shows that KDR functions as a negative regulator of HFR1, similar to Id-1 in human. The KDR exhibited circadian expression pattern with an increase during the day. Taken together, our results suggest that KDR attenuates light mediated responses in day light condition through inhibition of the activity of bHLH proteins involved in light signaling.