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.     

CONSTANS delays Arabidopsis flowering under short days

Long days (LD) promote flowering of Arabidopsis thaliana compared with short days (SD) by activating the photoperiodic pathway. Here we show that growth under very‐SD (3 h) or darkness (on sucrose) also accelerates flowering on a biological scale, indicating that SD actively repress flowering compared with very‐SD. CONSTANS (CO) repressed flowering under SD, and the early flowering of co under SD required FLOWERING LOCUS T (FT). FT was expressed at a basal level in the leaves under SD, but these levels were not enhanced in co. This indicates that the action of CO in A. thaliana is not the mirror image of the action of its homologue in rice. In the apex, CO enhanced the expression of TERMINAL FLOWER 1 (TFL1) around the time when FT expression is important to promote flowering. Under SD, the tfl1 mutation was epistatic to co and in turn ft was epistatic to tfl1. These observations are consistent with the long‐standing but not demonstrated model where CO can inhibit FT induction of flowering by affecting TFL1 expression.     

CONSTANS activates SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 through FLOWERING LOCUS T to promote flowering in Arabidopsis

CONSTANS (CO) regulates flowering time by positively regulating expression of two floral integrators, FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), in Arabidopsis (Arabidopsis thaliana). FT and SOC1 have been proposed to act in parallel pathways downstream of CO based on genetic analysis using weak ft alleles, since ft soc1 double mutants showed an additive effect in suppressing the early flowering of CO overexpressor plants. However, this genetic analysis was inconsistent with the sequential induction pattern of FT and SOC1 found in inducible CO overexpressor plants. Hence, to identify genetic interactions of CO, FT, and SOC1, we carried out genetic and expression analyses with a newly isolated T-DNA allele of FT, ft-10. We found that ft-10 almost completely suppressed the early flowering phenotype of CO overexpressor plants, whereas soc1-2 partially suppressed the phenotype, suggesting that FT is the major output of CO. Expression of SOC1 was altered in gain- or loss-of-function mutants of FT, whereas expression of FT remained unchanged in gain- or loss-of-function mutants of SOC1, suggesting that FT positively regulates SOC1 to promote flowering. In addition, inactivation of FT caused down-regulation of SOC1 even in plants overexpressing CO, indicating that FT is required for SOC1 induction by CO. Taken together, these data suggest that CO activates SOC1 through FT to promote flowering in Arabidopsis.     

The role of cryptochrome 2 in flowering in Arabidopsis

We have investigated the genetic interactions between cry2 and the various flowering pathways in relation to the regulation of flowering by photoperiod and vernalization. For this, we combined three alleles of CRY2, the wild-type CRY2-Landsberg erecta (Ler), a cry2 loss-of-function null allele, and the gain-of-function CRY2-Cape Verde Islands (Cvi), with mutants representing the various photoreceptors and flowering pathways. The analysis of CRY2 alleles combined with photoreceptor mutants showed that CRY2-Cvi could compensate the loss of phyA and cry1, also indicating that cry2 does not require functional phyA or cry1. The analysis of mutants of the photoperiod pathway showed epistasis of co and gi to the CRY2 alleles, indicating that cry2 needs the product of CO and GI genes to promote flowering. All double mutants of this pathway showed a photoperiod response very much reduced compared with Ler. In contrast, mutations in the autonomous pathway genes were additive to the CRY2 alleles, partially overcoming the effects of CRY2-Cvi and restoring day length responsiveness. The three CRY2 alleles were day length sensitive when combined with FRI-Sf2 and/or FLC-Sf2 genes, which could be reverted when the delay of flowering caused by FRI-Sf2 and FLC-Sf2 alleles was removed by vernalization. In addition, we looked at the expression of FLC and CRY2 genes and showed that CRY2 is negatively regulated by FLC. These results indicate an interaction between the photoperiod and the FLC-dependent pathways upstream to the common downstream targets of both pathways, SOC1 and FT.     

Arabidopsis cryptochrome 1 is a soluble protein mediating blue light-dependent regulation of plant growth and development

Cryptochrome 1 (CRY1) is a flavin-type blue light receptor of Arabidopsis thaliana which mediates inhibition of hypocotyl elongation. In the work described in this report it is demonstrated that CRY1 is a soluble protein expressed in both young seedlings grown either in the dark or under light, and in different organs of adult plants. The functional role of CRY1 was further investigated using transgenic Arabidopsis plants overexpressing CRY1. It is demonstrated that overexpression of CRY1 resulted in hypersensitivity to blue, UV-A, and green light for the inhibition of hypocotyl elongation response. Transgenic plants overexpressing CRY1 also exhibited a dwarf phenotype with reduced size in almost every organ. This was in keeping with the previous observation of reciprocal alterations found in hy4 mutant plants and is consistent with a hypothesis that CRY1 mediates a light-dependent process resulting in a general inhibitory effect on plant growth. In addition, transgenic plants overexpressing CRY1 showed increased anthocyanin accumulation in response to blue, UV-A, and green light in a fluence rate-dependent manner. This increase in anthocyanin accumulation in transgenic plants was shown to be concomitant with increased blue light-induction of CHS gene expression. It is concluded that CRY1 is a photoreceptor mediating blue light-dependent regulation of gene expression in addition to its affect on plant growth.     

Conserved structure and function of the Arabidopsis flowering time gene CONSTANS in Brassica napus

The Arabidopsis thaliana CONSTANS (CO) gene which promotes flowering in long days was recently isolated by chromosome walking. The mapping of QTLs controlling flowering time in Brassica species has identified genomic regions that contain homologues of the CO gene. Four genes homologous to the Arabidopsis CO gene were isolated from a pair of homoeologous loci in each of two doubled-haploid Brassica napus lines displaying different flowering times, N-o-1 and N-o-9. The four genes, BnCOa1, BnCOa9, BnCOb1 and BnCOb9, are located on linkage groups N10 and N19, and are highly similar to each other and to the Arabidopsis CO gene. Two regions of the proteins are particularly well conserved, a N-terminal region with two putative zinc fingers and a C-terminal region which may contain a nuclear localization signal. All four genes appear to be expressed in B. napus. The BnCOa1 allele was shown to complement the co-2 mutation in Arabidopsis in a dosage-dependent manner causing earlier flowering than in wild type under both long- and short-day conditions.     

Blue light-induced phosphorylation of Arabidopsis cryptochrome 1 is essential for its photosensitivity

Plants possess two cryptochrome photoreceptors, cryptochrome 1 (CRY1) and cryptochrome 2 (CRY2), that mediate overlapping and distinct physiological responses. Both CRY1 and CRY2 undergo blue light-induced phosphorylation, but the molecular details of CRY1 phosphorylation remain unclear. Here we identify 19 in vivo phosphorylation sites in CRY1 using mass spectrometry and systematically analyze the physiological and photobiochemical activities of CRY1 variants with phosphosite substitutions. We demonstrate that nonphosphorylatable CRY1 variants have impaired phosphorylation, degradation, and physiological functions, whereas phosphomimetic variants mimic the physiological functions of phosphorylated CRY1 to constitutively inhibit hypocotyl elongation. We further demonstrate that phosphomimetic CRY1 variants exhibit enhanced interaction with the E3 ubiquitin ligase COP1 (CONSTITUTIVELY PHOTOMORPHOGENIC 1). This finding is consistent with the hypothesis that phosphorylation of CRY1 is required for COP1-dependent signaling and regulation of CRY1. We also determine that PHOTOREGULATORY PROTEIN KINASEs (PPKs) phosphorylate CRY1 in a blue light-dependent manner and that this phosphorylation is critical for CRY1 signaling and regulation. These results indicate that, similar to CRY2, blue light-dependent phosphorylation of CRY1 determines its photosensitivity.     

Hd1, a major photoperiod sensitivity quantitative trait locus in rice, is closely related to the Arabidopsis flowering time gene CONSTANS

A major quantitative trait locus (QTL) controlling response to photoperiod, Hd1, was identified by means of a map-based cloning strategy. High-resolution mapping using 1505 segregants enabled us to define a genomic region of approximately 12 kb as a candidate for Hd1. Further analysis revealed that the Hd1 QTL corresponds to a gene that is a homolog of CONSTANS in Arabidopsis. Sequencing analysis revealed a 43-bp deletion in the first exon of the photoperiod sensitivity 1 (se1) mutant HS66 and a 433-bp insertion in the intron in mutant HS110. Se1 is allelic to the Hd1 QTL, as determined by analysis of two se1 mutants, HS66 and HS110. Genetic complementation analysis proved the function of the candidate gene. The amount of Hd1 mRNA was not greatly affected by a change in length of the photoperiod. We suggest that Hd1 functions in the promotion of heading under short-day conditions and in inhibition under long-day conditions.