Sumire Fujiwara

Distinct Roles of GIGANTEA in Promoting Flowering and Regulating Circadian Rhythms in Arabidopsis

The circadian clock acts as the timekeeping mechanism in photoperiodism. In Arabidopsis thaliana, a circadian clock–controlled flowering pathway comprising the genes GIGANTEA (GI), CONSTANS (CO), and FLOWERING LOCUS T (FT) promotes flowering specifically under long days. Within this pathway, GI regulates circadian rhythms and flowering and acts earlier in the hierarchy than CO and FT, suggesting that GI might regulate flowering indirectly by affecting the control of circadian rhythms. We studied the relationship between the roles of GI in flowering and the circadian clock using late elongated hypocotyl circadian clock associated1 double mutants, which are impaired in circadian clock function, plants overexpressing GI (35S:GI), and gi mutants. These experiments demonstrated that GI acts between the circadian oscillator and CO to promote flowering by increasing CO and FT mRNA abundance. In addition, circadian rhythms in expression of genes that do not control flowering are altered in 35S:GI and gi mutant plants under continuous light and continuous darkness, and the phase of expression of these genes is changed under diurnal cycles. Therefore, GI plays a general role in controlling circadian rhythms, and this is different from its effect on the amplitude of expression of CO and FT. Functional GI:green fluorescent protein is localized to the nucleus in transgenic Arabidopsis plants, supporting the idea that GI regulates flowering in the nucleus. We propose that the effect of GI on flowering is not an indirect effect of its role in circadian clock regulation, but rather that GI also acts in the nucleus to more directly promote the expression of flowering-time genes.

SUMO E3 Ligase HIGH PLOIDY2 Regulates Endocycle Onset and Meristem Maintenance in Arabidopsis

Endoreduplication involves a doubling of chromosomal DNA without corresponding cell division. In plants, many cell types transit from the mitotic cycle to the endoreduplication cycle or endocycle, and this transition is often coupled with the initiation of cell expansion and differentiation. Although a number of cell cycle regulators implicated in endocycle onset have been identified, it is still largely unknown how this transition is developmentally regulated at the whole organ level. Here, we report that a nuclear-localized SUMO E3 ligase, HIGH PLOIDY2 (HPY2), functions as a repressor of endocycle onset in Arabidopsis thaliana meristems. Loss of HPY2 results in a premature transition from the mitotic cycle to the endocycle, leading to severe dwarfism with defective meristems. HPY2 possesses an SP-RING domain characteristic of MMS21-type SUMO E3 ligases, and we show that the conserved residues within this domain are required for the in vivo and in vitro function of HPY2. HPY2 is predominantly expressed in proliferating cells of root meristems and it functions downstream of meristem patterning transcription factors PLETHORA1 (PLT1) and PLT2. These results establish that HPY2-mediated sumoylation modulates the cell cycle progression and meristem development in the PLT-dependent signaling pathway.

Circadian Clock Proteins LHY and CCA1 Regulate SVP Protein Accumulation to Control Flowering in Arabidopsis

The floral regulators GIGANTEA (GI), CONSTANS (CO), and FLOWERING LOCUS T (FT) play key roles in the photoperiodic flowering responses of the long-day plant Arabidopsis thaliana. The GI-CO-FT pathway is highly conserved in plants. Here, we demonstrate that the circadian clock proteins LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK–ASSOCIATED1 (CCA1) not only repressed the floral transition under short-day and long-day conditions but also accelerated flowering when the plants were grown under continuous light (LL). LHY and CCA1 accelerated flowering in LL by promoting FT expression through a genetic pathway that appears to be independent of the canonical photoperiodic pathway involving GI and CO proteins. A genetic screen revealed that the late-flowering phenotype of the lhy;cca1 double mutant under LL was suppressed through mutations in SHORT VEGETATIVE PHASE (SVP), a MADS box transcription factor. Yeast two-hybrid analysis demonstrated an interaction between SVP and FLOWERING LOCUS C, and genetic analysis indicated that these two proteins act as partially redundant repressors of flowering time. SVP protein accumulated in lhy;cca1 plants under LL. We propose a model in which LHY and CCA1 accelerate flowering in part by reducing the abundance of SVP and thereby antagonizing its capacity to repress FT expression under LL.

Possible role of EARLY FLOWERING 3 (ELF3) in clock‐dependent floral regulation by SHORT VEGETATIVE PHASE (SVP) in Arabidopsis thaliana

Circadian clock proteins play key roles in adaptations of plants to diurnal environmental conditions. The photoperiodic flowering response is one of the mechanisms of adaptation to seasonal changes in the lengths of day and night. Double mutations in two clock genes, late elongated hypocotyl (LHY) and circadian clock associated 1 (CCA1), accelerated flowering under short days (SDs) but delayed flowering under continuous light (LL) in Arabidopsis thaliana. The mechanism underlying the late flowering of lhy;cca1 mutants under LL was investigated here. Late flowering of plants with overexpression of short vegetative phase (SVP) was much more pronounced under SDs and enhanced by constans 2 (co-2) under long days (LDs), suggesting that SVP and CO act independently in the photoperiodic flowering pathway. However, how SVP and flowering locus C (FLC) mediated the effects of LHY/CCA1 and thus influenced flowering time was not completely clear. A mutant line lhy;cca1 in the Landsberg erecta (Ler) background was established, ethyl methanesulfonate (EMS)-mutagenized and used to screen suppressors of late flowering of lhy;cca1 under LL. Mutations in the clock gene early flowering 3 (ELF3) were identified as suppressors. Overexpression and loss-of-function of ELF3 influenced SVP protein accumulation. Therefore, we propose that, as well as the classical GIGANTEA (GI)-CO pathway, LHY/CCA1 regulates a pathway negatively controlling flowering locus T (FT), possibly via ELF3-SVP/FLC.

Antisense suppression of the Arabidopsis PIF3 gene does not affect circadian rhythms but causes early flowering and increases FT expression.

Photoperiodic control of flowering is regulated by light and a circadian clock. Feedback regulation of the transcription of clock components is one of the most common and important mechanisms that control clock functions in animals, fungi, and plants. The Arabidopsis circadian clock is believed to involve two myb‐related proteins, LHY (late elongated hypocotyl) and CCA1 (circadian clock associated 1), which negatively regulate TOC1 (timing of cab expression 1) gene expression through direct binding to the TOC1 promoter. PIF3 (phytochrome‐interacting factor 3), a bHLH transcription factor binds promoter regions of the LHY and CCA1 genes, affecting the light induction of these genes, and interacts with TOC1 protein. Although the positive feedback regulation of clock components in plants has been predicted, and PIF3 has been assumed to be involved, the molecular nature of this process has not been elucidated. Here we demonstrate that the antisense suppression of the PIF3 gene causes higher levels of mRNA of floral activator genes CO (constans) and FT (flowering locus T) and results in early flowering under long days (LD). Neither the circadian rhythms of the clock‐controlled genes (CCGs) under constant conditions nor the diurnal rhythms of the CCGs under LD conditions are affected by the reduction in PIF3 gene expression. These results suggest that PIF3 may play an important role in the control of flowering through clock‐independent regulation of CO and FT gene expression in Arabidopsis.