In plants, seasonal changes in day length are perceived in leaves, which initiate long-distance signaling that induces flowering at the shoot apex. The identity of the long-distance signal has yet to be determined. In Arabidopsis , activation of FLOWERING LOCUS T ( FT ) transcription in leaf vascular tissue (phloem) induces flowering. We found that FT messenger RNA is required only transiently in the leaf. In addition, FT fusion proteins expressed specifically in phloem cells move to the apex and move long distances between grafted plants. Finally, we provide evidence that FT does not activate an intermediate messenger in leaves. We conclude that FT protein acts as a long-distance signal that induces Arabidopsis flowering.

SummaryWe have produced 22 090 primary transgenic rice plants that carry a T-DNA insertion, which has resulted in 18 358 fertile lines. Genomic DNA gel-blot and PCR analyses have shown that approximately 65% of the population contains more than one copy of the inserted T-DNA. Hygromycin resistance tests revealed that transgenic plants contain an average of 1.4 loci of T-DNA inserts. Therefore, it can be estimated that approximately 25 700 taggings have been generated. The binary vector used in the insertion contained the promoterless b-glucuronidase (GUS) reporter gene with an intron and multiple splicing donors and acceptors immediately next to the right border. Therefore, this gene trap vector is able to detect a gene fusion between GUS and an endogenous gene, which is tagged by T-DNA. Histochemical GUS assays were carried out in the leaves and roots from 5353 lines, mature¯owers from 7026 lines, and developing seeds from 1948 lines. The data revealed that 1.6±2.1% of tested organs were GUS-positive in the tested organs, and that their GUS expression patterns were organ-or tissue-speci®c or ubiquitous in all parts of the plant. The large population of T-DNA-tagged lines will be useful for identifying insertional mutants in various genes and for discovering new genes in rice.

The transcriptional regulator CONSTANS (CO) promotes flowering of Arabidopsis under long summer days (LDs) but not under short winter days (SDs). Post-translational regulation of CO is crucial for this response by stabilizing the protein at the end of a LD, whereas promoting its degradation throughout the night under LD and SD. We show that mutations in CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), a component of a ubiquitin ligase, cause extreme early flowering under SDs, and that this is largely dependent on CO activity. Furthermore, transcription of the CO target gene FT is increased in cop1 mutants and decreased in plants overexpressing COP1 in phloem companion cells. COP1 and CO interact in vivo and in vitro through the C-terminal region of CO. COP1 promotes CO degradation mainly in the dark, so that in cop1 mutants CO protein but not CO mRNA abundance is dramatically increased during the night. However, in the morning CO degradation occurs independently of COP1 by a phytochrome B-dependent mechanism. Thus, COP1 contributes to day length perception by reducing the abundance of CO during the night and thereby delaying flowering under SDs.

We recently identified multivesicular bodies (MVBs) as prevacuolar compartments (PVCs) in the secretory and endocytic pathways to the lytic vacuole in tobacco (Nicotiana tabacum) BY-2 cells. Secretory carrier membrane proteins (SCAMPs) are post-Golgi, integral membrane proteins mediating endocytosis in animal cells. To define the endocytic pathway in plants, we cloned the rice (Oryza sativa) homolog of animal SCAMP1 and generated transgenic tobacco BY-2 cells expressing yellow fluorescent protein (YFP)-SCAMP1 or SCAMP1-YFP fusions. Confocal immunofluorescence and immunogold electron microscopy studies demonstrated that YFP-SCAMP1 fusions and native SCAMP1 localize to the plasma membrane and mobile structures in the cytoplasm of transgenic BY-2 cells. Drug treatments and confocal immunofluorescence studies demonstrated that the punctate cytosolic organelles labeled by YFP-SCAMP1 or SCAMP1 were distinct from the Golgi apparatus and PVCs. SCAMP1-labeled organelles may represent an early endosome because the internalized endocytic markers FM4-64 and AM4-64 reached these organelles before PVCs. In addition, wortmannin caused the redistribution of SCAMP1 from the early endosomes to PVCs, probably as a result of fusions between the two compartments. Immunogold electron microscopy with high-pressure frozen/freeze-substituted samples identified the SCAMP1-positive organelles as tubular-vesicular structures at the trans-Golgi with clathrin coats. These early endosomal compartments resemble the previously described partially coated reticulum and trans-Golgi network in plant cells.

Rice contains several MADS box genes. It has been demonstrated previously that one of these genes, OsMADS1 (for Oryza sativa MADS box gene1 ), is expressed preferentially in flowers and causes early flowering when ectopically expressed in tobacco plants. In this study, we demonstrated that ectopic expression of OsMADS1 in rice also results in early flowering. To further investigate the role of OsMADS1 during rice flower development, we generated transgenic rice plants expressing altered OsMADS1 genes that contain missense mutations in the MADS domain. There was no visible alteration in the transgenic plants during the vegetative stage. However, transgenic panicles typically exhibited phenotypic alterations, including spikelets consisting of elongated leafy paleae and lemmas that exhibit a feature of open hull, two pairs of leafy palea-like and lemma-like lodicules, a decrease in stamen number, and an increase in the number of carpels. In addition, some spikelets generated an additional floret from the same rachilla. These characteristics are very similar to those of leafy hull sterile1 ( lhs1 ). The map position of OsMADS1 is closely linked to that of lhs1 on chromosome 3. Examination of lhs1 revealed that it contains two missense mutations in the OsMADS1 MADS domain. A genetic complementation experiment showed that the 11.9-kb genomic DNA fragment containing the wild-type OsMADS1 gene rescued the mutant phenotypes. In addition, ectopic expression of the OsMADS1 gene isolated from the lhs1 line resulted in lhs1 -conferred phenotypes. These lines of evidence demonstrate that OsMADS1 is the lhs1 gene. INTRODUCTIONIn response to floral induction, the inflorescence meristem becomes committed to flowering. LEAFY ( LFY ) and APE-TALA1 ( AP1 ) in Arabidopsis and FLORICAULA ( FLO ) and SQUAMOSA ( SQUA ) in Antirrhinum are responsible for promoting the specification of floral meristem identity (reviewed in Ma, 1994). The genes required for specifying the fate of floral organ primordia include AP1 , AP2 , AGAMOUS ( AG ), PISTILATA ( PI ), and AP3 in Arabidopsis and SQUA , PLENA ( PLE ), GLOBOSA ( GLO ), and DEFICIENS ( DEF ) in Antirrhinum (reviewed in Weigel and Meyerowitz, 1994). Excluding AP2 , these floral homeotic genes encode MADS box proteins that are highly conserved transcription factors in plants, animals, yeast, and fungi and that are regulated by the floral meristem identity gene LFY (Parcy et al., 1998;Wagner et al., 1999).Several other MADS box genes have more subtle functions associated with floral meristem and floral organ identity. Expression of AG-LIKE2 ( AGL2 ), AGL4 , and AGL9 of Arabidopsis begins after the onset of expression of floral meristem identity genes but before the activation of floral organ identity genes (Flanagan and Ma, 1994;Savidge et al., 1995;Mandel and Yanofsky, 1998). DEFH72 and DEFH200 of Antirrhinum appear to function in mediating interactions between the meristem and organ identity genes through direct interaction with PLE (Davies et al., 1996). FLORAL BINDING PROTEIN2 ( FBP2 ) o...

The four-member SPA protein family of Arabidopsis acts in concert with the E3 ubiquitin ligase COP1 to suppress photomorphogenesis in dark-grown seedlings. Here, we demonstrate that SPA proteins are, moreover, essential for photoperiodic flowering. Mutations in SPA1 cause phyA-independent early flowering under short day (SD) but not long day (LD) conditions, and this phenotype is enhanced by additional loss of SPA3 and SPA4 function. These spa1 spa3 spa4 triple mutants flower at the same time in LD and SD, indicating that the SPA gene family is essential for the inhibition of flowering under non-inductive SD. Among the four SPA genes, SPA1 is necessary and sufficient for normal photoperiodic flowering. Early flowering of SD-grown spa mutant correlates with strongly increased FT transcript levels, whereas COtranscript levels are not altered. Epistasis analysis demonstrates that both early flowering and FT induction in spa1 mutants is fully dependent on CO. Consistent with this finding, SPA proteins interact physically with CO in vitro and in vivo, suggesting that SPA proteins regulate CO protein function. Domain mapping shows that the SPA1-CO interaction requires the CCT-domain of CO, but is independent of the B-box type Zn fingers of CO. We further show that spa1 spa3 spa4 mutants exhibit strongly increased CO protein levels, which are not caused by a change in COgene expression. Taken together, our results suggest, that SPA proteins regulate photoperiodic flowering by controlling the stability of the floral inducer CO.

SUMMARYFlowering is controlled by a network of pathways that converge to regulate a small number of floral integrator genes. We studied the interactions in Arabidopsis between three of these integrators, FLOWERING LOCUS T (FT), TWIN SISTER OF FT (TSF) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), as well as their repression by the MADS box transcription factor SHORT VEGETATIVE PHASE (SVP). FT is a mobile signal transmitted from the leaf to the meristem to initiate flowering. Using mRNA null alleles, we show that FT and the closely related TSF are not essential for flowering, but that the double mutant is photoperiod-insensitive. Inactivation of both genes also fully suppresses the early-flowering phenotype caused by over-expression of CONSTANS (CO), a transcriptional regulator in the photoperiod pathway. In addition, we demonstrate that TSF and FT have similar biochemical functions by showing that they interact in yeast with the same bZIP transcription factors. Expression of FT or TSF from promoters specific for phloem companion cells drives early flowering of the double mutant, so no expression of either gene is required in the meristem. Furthermore, TSF, like FT, is repressed by SVP, but the triple mutant svp-41 ft-10 tsf-1 expresses SOC1 in the meristem sooner and flowers earlier than ft-10 tsf-1. Thus we distinguish the functions of SVP in repressing FT and TSF in the leaf and SOC1 in the meristem. In addition, a time course of in situ hybridizations suggested that repression of SVP and activation of SOC1 proceed simultaneously in the meristem. These observations clarify the relationships between these early regulators of the floral transition, and further emphasize the relatedness of mechanisms acting in the leaf and meristem to control flowering time.

Rice atypical HLH protein Oryza sativa BRASSINOSTEROID UPREGULATED 1-LIKE1 (OsBUL1) is preferentially expressed in the lamina joint where it controls cell elongation and positively affects leaf angles. OsBUL1 knockout mutant (osbul1) and transgenic rice for double-stranded RNA interference (dsRNAi) of OsBUL1 produced erect leaves with smaller grains, whereas OsBUL1 overexpressors and an activation tagging line of OsBUL1 exhibited increased lamina inclination and grain size. Moreover, OsBUL1 expression was induced by brassinolide (BL) and osbul1 did not respond to BL treatment. To understand the molecular network of OsBUL1 function in rice, we isolated a novel OsBUL1-interacting protein, LO9-177, an uncharacterized protein containing a KxDL motif, and functionally studied it with respect to the lamina inclination and grain size of rice. OsBUL1 COMPLEX1 (OsBC1) is a basic helix-loop-helix (bHLH) transcriptional activator that interacts with OsBUL1 only in the presence of LO9-177 forming a possible trimeric complex for cell elongation in the lamina joint of rice. Expression of OsBC1 is also upregulated by BL and has a similar pattern to that of OsBUL1 Transgenic rice plants expressing OsBC1 under the control of OsBUL1 promoter showed increased grain size as well as leaf bending, while transgenic lines for dsRNAi and/or expressing a dominant repressor form of OsBC1 displayed reduced plant height and grain size. Together, these results demonstrated that a novel protein complex consisting of OsBUL1, LO9-177, and OsBC1 is associated with the HLH-bHLH system, providing new insight into the molecular functional network based on HLH-bHLH proteins for cell elongation.