Yasunobu Ogura

AUREOCHROME, a photoreceptor required for photomorphogenesis in stramenopiles

A blue light (BL) receptor was discovered in stramenopile algae Vaucheria frigida (Xanthophyceae) and Fucus distichus (Phaeophyceae). Two homologs were identified in Vaucheria; each has one basic region/leucine zipper (bZIP) domain and one light–oxygen–voltage (LOV)-sensing domain. We named these chromoproteins AUREOCHROMEs (AUREO1 and AUREO2). AUREO1 binds flavin mononucleotide via its LOV domain and forms a 390-nm-absorbing form, indicative of formation of a cysteinyl adduct to the C(4a) carbon of the flavin mononucleotide upon BL irradiation. The adduct decays to the ground state in ≈5 min. Its bZIP domain binds the target sequence TGACGT. The AUREO1 target binding was strongly enhanced by BL treatment, implying that AUREO1 functions as a BL-regulated transcription factor. The function of AUREO1 as photoreceptor for BL-induced branching is elucidated through RNAi experiments. RNAi of AUREO2 unexpectedly induces sex organ primordia instead of branches, implicating AUREO2 as a subswitch to initiate development of a branch, but not a sex organ. AUREO sequences are also found in the genome of the marine diatom Thalassiosira pseudonana (Bacillariophyceae), but are not present in green plants. AUREOCHROME therefore represents a BL receptor in photosynthetic stramenopiles.

Chloroplast Outer Envelope Protein CHUP1 Is Essential for Chloroplast Anchorage to the Plasma Membrane and Chloroplast Movement

Chloroplasts change their intracellular distribution in response to light intensity. Previously, we isolated the chloroplast unusual positioning1 (chup1) mutant of Arabidopsis (Arabidopsis thaliana). This mutant is defective in normal chloroplast relocation movement and shows aggregation of chloroplasts at the bottom of palisade mesophyll cells. The isolated gene encodes a protein with an actin-binding motif. Here, we used biochemical analyses to determine the subcellular localization of full-length CHUP1 on the chloroplast outer envelope. A CHUP1-green fluorescent protein (GFP) fusion, which was detected at the outermost part of mesophyll cell chloroplasts, complemented the chup1 phenotype, but GFP-CHUP1, which was localized mainly in the cytosol, did not. Overexpression of the N-terminal hydrophobic region (NtHR) of CHUP1 fused with GFP (NtHR-GFP) induced a chup1-like phenotype, indicating a dominant-negative effect on chloroplast relocation movement. A similar pattern was found in chloroplast OUTER ENVELOPE PROTEIN7 (OEP7)-GFP transformants, and a protein containing OEP7 in place of NtHR complemented the mutant phenotype. Physiological analyses of transgenic Arabidopsis plants expressing truncated CHUP1 in a chup1 mutant background and cytoskeletal inhibitor experiments showed that the coiled-coil region of CHUP1 anchors chloroplasts firmly on the plasma membrane, consistent with the localization of coiled-coil GFP on the plasma membrane. Thus, CHUP1 localization on chloroplasts, with the N terminus inserted into the chloroplast outer envelope and the C terminus facing the cytosol, is essential for CHUP1 function, and the coiled-coil region of CHUP1 prevents chloroplast aggregation and participates in chloroplast relocation movement.

LOV KELCH PROTEIN2 and ZEITLUPE repress Arabidopsis photoperiodic flowering under non‐inductive conditions, dependent on FLAVIN‐BINDING KELCH REPEAT F‐BOX1

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. Over-expression 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.

Anthocyanin production by over-expression of grape transcription factor gene VlmybA2 in transgenic tobacco and Arabidopsis

An myb-related transcription factor gene of the anthocyanin biosynthetic pathway, VlmybA2, from the Kyoho grape (Vitis labruscana) was introduced into tobacco and Arabidopsis under the control of the cauliflower mosaic virus 35S promoter. The 35S:VlmybA2-induced anthocyanin production was prominent in transformed tobacco calli, and the regenerated tobacco plants were completely purple. Except for the color, the transgenic plants were apparently not different from the control plants. During plant growth in pots, the purple color was not uniformly distributed but appeared as patches in the leaves, whereas the flowers showed intense pigmentation. In Arabidopsis, T1 transformants showing two prominent phenotypes: completely purple seedlings and seedlings with green leaves and purple roots. The partially purple seedlings grown in pots produced fertile and viable seeds of two distinguishable colors, purple and brown. VlmybA2 alone, without the aid of other myc-related genes, could induce complete pigmentation in tobacco and Arabidopsis, indicating its potential over other previously used myb- and myc-related genes.

Blue light diminishes interaction of PAS/LOV proteins, putative blue light receptors in Arabidopsis thaliana, with their interacting partners

The light, oxygen, or voltage (LOV) domain that belongs to the Per-ARNT-Sim (PAS) domain superfamily is a blue light sensory module. The Arabidopsis thalianaPAS/LOV PROTEIN (PLP) gene encodes three putative blue light receptor proteins, PLPA, PLPB, and PLPC, because of its mRNA splicing variation. PLPA and PLPB each contain one PAS domain at the N-terminal region and one LOV domain at the C-terminal region, while the LOV domain is truncated in PLPC. RNA gel blot analysis showed that PLP mRNA was markedly expressed after exposure to salt or dehydration stress. Yeast two-hybrid screening led to the isolation of VITAMIN C DEFECTIVE 2 (VTC2), VTC2-LIKE (VTC2L), and BEL1-LIKE HOMEODOMAIN 10 proteins (BLH10A and BLH10B) as PLP-interacting proteins. The molecular interaction of PLPA with VTC2L, BLH10A or BLH10B, and that of PLPB with VTC2L were diminished when yeasts were grown under blue light illumination. Furthermore, the possible binding of flavin chromophore to PLPA and PLPB was demonstrated. These results imply that the LOV domain of PLPA and PLPB functions as a blue light sensor, and suggest the applicability of these interactions to blue light-dependent switching in transcriptional regulation in yeast or other organisms.

Overexpression of Arabidopsis thaliana LOV KELCH REPEAT PROTEIN 2 promotes tuberization in potato (Solanum tuberosum cv. May Queen)

Potato tuberization is induced under short‐day conditions and repressed under long‐day conditions. In this study, we produced transgenic potatoes overexpressing either Arabidopsis thaliana LOV KELCH PROTEIN 2 (35S:LKP2) or CONSTANS fused with a transcription repressor motif (35S:CO‐Rep). In an in vitro tuberization assay, the average number of tubers per plant was greater in 35S:LKP2 plants than in vector‐control plants, but lower in 35S:CO‐Rep plants. Under long‐day conditions in soil, all 35S:LKP2 plants tuberized, whereas most control plants and 35S:CO‐Rep plants did not. These results suggest genes involved in flowering time regulation can be used to control potato tuber production.

Overexpression of the chimeric gene of the floral regulator CONSTANS and the EAR motif repressor causes late flowering in Arabidopsis

The transcription factor CONSTANS (CO) plays a central role in the photoperiod pathway by integrating the circadian clock and light signals into a control for flowering time. CO induces FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) expression, and thereby promotes flowering. The ethylene-responsive element-binding factor associated amphiphilic repression (EAR) motif was used to construct a CONSTANS-EAR motif repressor gene (CO-Rep), which was overexpressed in Arabidopsis under the control of the Cauliflower mosaic virus 35S promoter in order to test its potential for flowering time regulation under inductive long day conditions. Morphological abnormalities in the root and cotyledon formation, and dwarfness were frequently seen in the transgenic plants, suggesting that the proper timing, location, and/or level of CO-Rep expression are important for its application. In morphologically normal CO-Rep plants, both bolting and flowering times under inductive long day conditions were twofold greater than in controls. As a result of the delay in flowering, rosette leaf number at bolting, and rosette and cauline leaf number at flowering increased significantly in CO-Rep plants. RT-PCR analysis demonstrated that FT expression was greatly reduced in the CO-Rep plants, while endogenous CO and SOC1 expression levels were not markedly affected. Conservation of CO among a diverse range of plant species, and its involvement in a variety of photoperiodic responses including flowering, suggests a high potential for use of CO-Rep to manipulate such responses in an agronomically desirable manner.

PAS/LOV proteins: A proposed new class of plant blue light receptor

The light, oxygen, or voltage (LOV) domain belongs to the Per-ARNT-Sim (PAS) superfamily of domains, and functions with the flavin chromophore as a module for sensing blue light in plants and fungi. The Arabidopsis thaliana PAS/LOV proteins (PLPs), of unknown function, possess an N-terminal PAS domain and a C-terminal LOV domain. Our recent analysis using yeast two-hybrid and Escherichia coli protein production systems reveals that the interactions of Arabidopsis PLPs with several proteins diminish under blue light illumination and that the PLP LOV domain may bind to a flavin chromophore. These results suggest that PLP functions as a blue light receptor. Homologs of PLP exist in rice, tomato, and moss. The LOV domains of these PLP homologs form a distinct group in phylogenetic analysis. These facts suggest that PLP belongs to a new class of plant blue light receptor. Addendum to: Ogura Y, Komatsu A, Zikihara K, Nanjo T, Tokutomi S, Wada M, Kiyosue T. Blue light diminishes interaction of PAS/LOV proteins, putative blue light receptors in Arabidopsis thaliana, with their interacting partners. J Plant Res 2008; 121: 97–105.