The phytochrome-interacting vascular plant one-zinc finger1 and VOZ2 redundantly regulate flowering in Arabidopsis.

The timing of the transition to flowering in plants is regulated by various environmental factors, including daylength and light quality. Although the red/far-red photoreceptor phytochrome B (phyB) represses flowering by indirectly regulating the expression of a key flowering regulator, FLOWERING LOCUS T (FT), the mechanism of phyB signaling for flowering is largely unknown. Here, we identified two Arabidopsis thaliana genes, VASCULAR PLANT ONE-ZINC FINGER1 (VOZ1) and VOZ2, which are highly conserved throughout land plant evolution, as phyB-interacting factors. voz1 voz2 double mutants, but neither single mutant, showed a late-flowering phenotype under long-day conditions, which indicated that VOZ1 and VOZ2 redundantly promote flowering. voz1 voz2 mutations suppressed the early-flowering phenotype of the phyB mutant, and FT expression was repressed in the voz1 voz2 mutant. Green fluorescent protein-VOZ2 signal was observed in the cytoplasm, and interaction of VOZ proteins with phyB was indicated to occur in the cytoplasm under far-red light. However, VOZ2 protein modified to localize constitutively in the nucleus promoted flowering. In addition, the stability of VOZ2 proteins in the nucleus was modulated by light quality in a phytochrome-dependent manner. We propose that partial translocation of VOZ proteins from the cytoplasm to the nucleus mediates the initial step of the phyB signal transduction pathway that regulates flowering.

A membrane trafficking pathway regulated by the plant-specific RAB GTPase ARA6.

Endosomal trafficking plays an integral role in various eukaryotic cell activities and serves as a basis for higher-order functions in multicellular organisms. An understanding of the importance of endosomal trafficking in plants is rapidly developing, but its molecular mechanism is mostly unknown. Several key regulators of endosomal trafficking, including RAB5, which regulates diverse endocytic events in animal cells, are highly conserved. However, the identification of lineage-specific regulators in eukaryotes indicates that endosomal trafficking is diversified according to distinct body plans and lifestyles. In addition to orthologues of metazoan RAB5, land plants possess a unique RAB5 molecule, which is one of the most prominent features of plant RAB GTPase organization. Plants have also evolved a unique repertoire of SNAREs, the most distinctive of which are diverse VAMP7-related longins, including plant-unique VAMP72 derivatives. Here, we demonstrate that a plant-unique RAB5 protein, ARA6, acts in an endosomal trafficking pathway in Arabidopsis thaliana. ARA6 modulates the assembly of a distinct SNARE complex from conventional RAB5, and has a functional role in the salinity stress response. Our results indicate that plants possess a unique endosomal trafficking network and provide the first indication of a functional link between a specific RAB and a specific SNARE complex in plants.

Luminescence detection of SNARE-SNARE interaction in Arabidopsis protoplasts.

Membrane associated proteins SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) provide the minimal fusion machinery necessary for cellular vesicles to fuse to target organelle membranes in eukaryotic cells. Despite the conserved nature of the fusion machinery in all eukaryotes, it still remains challenging to identify functional SNARE pairs in higher plants. We developed a method based on a split-luciferase complementation assay for detecting changes in SNARE-SNARE interaction by luminescence within Arabidopsis protoplasts that express recombinant proteins at physiological levels in 96-well plates. The reliability of the assay was confirmed by three experiments. First, reduction of the SNARE-SNARE interaction caused by a single amino acid substitution adjacent to the SNARE motif in endosome-localized AtVAM3/SYP22 (syntaxin of plant 22) was detected by a reduction of luminescence. Second, reduction of the interaction between plasma-membrane localized SYP121 and VAMP722 in response to sodium azide was detected in real-time. Third, the results of 21 SNARE pairs investigated by this method largely agreed with the results from previously reported co-immunoprecipitation assays. Using the method, we newly identified the interaction between SYP121 and VAMP722 was significantly increased when the protoplasts were incubated in the light. Microscopic observation of transgenic Arabidopsis expressing GFP-SYP121 (green fluorescent protein tagged SYP121) from its own promoter suggested that the plasma-membrane localization of GFP-SYP121 is maintained by light. These suggested that the vesicle trafficking pathway mediated by SYP121 might be regulated by light in Arabidopsis. In general, this article demonstrated the method that can generate new biological insight of the SNARE protein interactions in plant cells.