Tomoyuki Takase

We recently demonstrated the circadian clock modulated water dynamics in the roots of a small model plant, Arabidopsis thaliana, by the Nuclear Magnetic Resonance (NMR) microimaging technique. Our developed technique was able to visualize the water distribution that depended on differences in the 1H signal among region in the shoot, such as the shoot apex, the hypocotyl and the root shoot junction. Water content in the shoot increased during periods of light in comparison with dark periods, and continued through the early stage of seedling growth until the dark period. When the water content changed, elongation and/or movement occurred in the hypocotyl, and these events were synchronized. The water dynamics of the shoot also displayed an opposite phase with the root water dynamics. © 2013 Landes Bioscience.

LOV KELCH PROTEIN2 (LKP2), ZEITLUPE (ZTL)/LOV KELCH PROTEIN1 (LKP1) and FLAVIN-BINDING KELCH REPEAT F-BOX1 (FKF1) constitute a family of Arabidopsis F-box proteins that regulate the circadian clock. Over-expression of LKP2 or ZTL causes arrhythmicity of multiple clock outputs under constant light and in constant darkness. Here, we show the significance of LKP2 and ZTL in the photoperiodic control of flowering time in Arabidopsis. In plants over-expressing LKP2, CO and FT expression was down-regulated under long-day conditions. LKP2 and ZTL physically interacted with FKF1, which was recruited from the nucleus into cytosolic speckles. LKP2 and ZTL inhibited the interaction of FKF1 with CYCLING DOF FACTOR 1, a ubiquitination substrate for FKF1 that is localized in the nucleus. The Kelch repeat regions of LKP2 and ZTL were sufficient for their physical interaction with FKF1 and translocation of FKF1 to the cytoplasm. Overexpression of LKP2 Kelch repeats induced late flowering under long-day conditions. lkp2 ztl double mutant plants flowered earlier than wild-type plants under short-day (non-inductive) conditions, and both CO and FT expression levels were up-regulated in the double mutant plants. The early flowering of lkp2 ztl was dependent on FKF1. LKP2, ZTL or both affected the accumulation of FKF1 protein during the early light period. These results indicate that an important role of LKP2 and ZTL in the photoperiodic pathway is repression of flowering under non-inductive conditions, and this is dependent on FKF1.

We have developed a plant growth system to analyze water dynamics in the roots of a small model plant, Arabidopsis thaliana, by nuclear magnetic resonance (NMR) microscopic imaging. Using the two-dimensional slice technique, we obtained a series of images with high signal-to-noise ratio indicating the water distribution in the root. To demonstrate light regulation of water transport in the root and involvement of aquaporin gene expression, we visualized the distribution of water in Arabidopsis roots under various light conditions and compared the data with the expression profiles of two aquaporin genes. (1)H-NMR imaging revealed that water content in Arabidopsis roots is lower in the light than in the dark. This diurnal variation in water content was clearly observed in the basal zone of the root. In addition, an autonomous rhythm of water dynamics was observed under continuous light (LL) and darkness (DD). However, the circadian oscillation in water dynamics was obscured in the early-flowering 3 (elf3) mutant under LL. The expression of both the aquaporin genes, AtPIP1;2 and AtPIP2;1, oscillated with the circadian rhythm under LL conditions in wild-type seedlings, but not in the elf3 mutant. These results demonstrate the advantages of our technique for monitoring water dynamics in roots of living Arabidopsis seedlings, and suggest that the circadian clock modulates water dynamics and aquaporin expression.

LOV KELCH PROTEIN 2 (LKP2) belongs to a group of blue light receptors in Arabidopsis thaliana that possess 1 light, oxygen, or voltage (LOV) domain, 1 F-box motif, and 1 kelch repeat domain. Members of this group regulate both the circadian clock and photoperiodic flowering. Transgenic Arabidopsis plants overexpressing a fusion of the genes for green fluorescent protein (GFP) and LKP2 exhibited long hypocotyls and arrhythmicity under constant light and late flowering under long-day conditions, as previously reported for LKP2-overexpressing Arabidopsis plants. The GFP-LKP2-overexpressing plants with elongated hypocotyls had more cortical cells than control plants but the same number of epidermal cells. Further, the cells in the hypocotyls of GFP-LKP2-overexpressing plants, especially in the middle regions of the epidermis and cortex, were longer and possessed larger nuclei, with increased DNA content. Therefore, LKP2 overexpression induces cell elongation and increases in cell number and ploidy in the Arabidopsis hypocotyls.

CONSTANS (CO) is a regulator of photoperiodic flowering that activates the transcription of a florigen gene, FLOWERING LOCUS T (FT), in Arabidopsis. CO belongs to the CO/CONSTANS-LIKE (COL) protein family, which consists of 17 proteins and is subclassified into three groups. Here, we characterized the gene for one of the group II members, COL8. The COL8 mRNA accumulated in seeds, leaves, flowers, and siliques. Transgenic Arabidopsis plants with a beta-glucuronidase (GUS) reporter gene driven by a 3-kb COL8 promoter displayed strong GUS activity in leaves. Both transiently and stably produced fluorescence-tagged COL8 proteins were localized in the nucleus. Transgenic Arabidopsis plants possessing COL8 cDNA driven by a cauliflower mosaic virus (CaMV) 35S promoter did not show any altered circadian rhythm under constant light conditions, but showed a late-flowering phenotype under long-day conditions. In these transgenic plants, CO mRNA did not decrease from zeitgeber time (zt) 12 to zt 16, whereas FT mRNA decreased from zt 8 to zt 24. The possible mechanisms for the late-flowering phenotype of the COL8 plants driven by the CaMV 35S promoter are discussed.