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.

We show that the key flowering regulators encoded by Phalaenopsis aphrodite FLOWERING LOCUS T1 (PaFT1) and PaFD share high sequence homologies to these from long-day flowering Arabidopsis and short-day flowering rice. Interestingly, PaFT1 is specifically up-regulated during flowering inductive cooling treatment but is not subjected to control by photoperiod in P. aphrodite. Phloem or shoot apex-specific expression of PaFT1 restores the late flowering of Arabidopsis ft mutants. Moreover, PaFT1 can suppress the delayed flowering caused by SHORT VEGATATIVE PHASE (SVP) overexpression as well as an active FRIGIDA (FRI) allele, indicating the functional conservation of flowering regulatory circuit in different plant species. PaFT1 promoter:GUS in Arabidopsis showed similar staining pattern to that of Arabidopsis FT in the leaves and guard cells but different in the shoot apex. A genomic clone or heat shock-inducible expression of PaFT1 is sufficient to the partial complementation of the ft mutants. Remarkably, ectopic PaFT1 expression also triggers precocious heading in rice. To further demonstrate the functional conservation of the flowering regulators, we show that PaFD, a bZIP transcription factor involved in flowering promotion, interacts with PaFT1, and PaFD partially complemented Arabidopsis fd mutants. Transgenic rice expressing PaFD also flowered early with increased expression of rice homologues of APETALA1 (AP1). Consistently, PaFT1 knock-down Phalaenopsis plants generated by virus-induced gene silencing exhibit delayed spiking. These studies suggest functional conservation of FT and FD genes, which may have evolved and integrated into distinct regulatory circuits in monopodial orchids, Arabidopsis and rice that promote flowering under their own inductive conditions.

Key flowering genes, FD and FD PARALOGUE (FDP) encoding bZIP transcription factors that interact with a FLOWERING LOCUS T (FT) in Arabidopsis were ectopically expressed in rice since we found AtFD and AtFDP also interact with HEADING DATE 3a (Hd3a) and RICE FLOWERING LOCUS T 1 (RFT1). Transgenic rice plants overexpressing AtFD and AtFDP caused reduction in plant height and spikelet size with decreased expression of genes involved in cell elongation without significant flowering time alteration in spite of increased expression of OsMADS14 and OsMADS15, rice homologues of APETALA1 (AP1) in the leaves. Simultaneous overexpression of AtFD and AtFDP enhanced phenotypes seen with overexpression of either single gene while transgenic rice plants expressing AtFD or AtFDP under the control of phloem-specific Hd3a promoter were indistinguishable from wild-type rice. Candidate genes responsible for the phenotypes were identified by comparison of microarray hybridization and their expression pattern was also examined in WT and transgenic rice plants. It has so far not been reported that AtFD and AtFDP affect cell elongation in plants, and our findings provide novel insight into the possible roles of AtFD and AtFDP in the mesophyll cells of plants, and potential genetic tools for manipulation of crop architecture.