Functional Diversity of CYCLOIDEA-like TCP Genes in the Control of Zygomorphic Flower Development in Lotus japonicus

CYCLOIDEA （CYC）-like TCP genes play key roles in dorsoventral differentiation of zygomorphic flowers in Papilionoideae legumes. In this study, we analyzed the kew mutants whose flowers lost lateral identity, and investigated the diverse functions of three LjCYC genes during zygomorphic flower development in the model legume Lotus japonicus. We showed that kew1 and kew3 are allelic mutants of LjCYC3, a CYC-like TCP gene. Through transgenic experiments, it was shown that LjCYC1 possesses dorsal activity similar to LjCYC2, and that LjCYC3 alone is sufficient to confer lateral activity, and an epistatic effect between dorsal and lateral activities was identified. Sequence analysis revealed a striking alteration at the 3 end of the LjCYC3 open reading frame （ORF） in comparison with those of LjCYC1 and LjCYC2 ORFs. Furthermore, it was found that LjCYC proteins could interact with each other and possess different activities by means of a transcriptional activity assay. Our data demonstrate that the sequence variation and the subsequent alteration of protein property play important roles in the functional diversity of different LjCYC genes in controlling zygomorphic flower development in Lotus japonicus.

Transcriptional and Post-transcriptional Modulation of SQU and KEW Activities in the Control of Dorsal-Ventral Asymmetric Flower Development in Lotus japonicus

In Papilionoideae legume, Lotusjaponicus, the development of dorsal-ventral （DV） asymmetric flowers is mainly controlled by two TB1/CYCLOIDEA/PCF （TCP） genes, SQUARED STANDARD （SQU） and KEELED WINGS IN LOTUS （KEW）, which determine dorsal and lateral identities, respectively. However, the molecular basis of how these two highly homologous genes orchestrate their diverse functions remains unclear. Here, we analyzed their expression levels, and investigated the transcriptional activities of SQUand KEW. We demonstrated that SQU possesses both activation and repression activities, while KEW acts only as an activator. They form homo- and heterodimers, and then collaboraUvely regulate their expression at the transcription level. Furthermore, we identified two types of post-transcriptional modifications, phosphor- ylation and ATP/GTP binding, both of which could affect their transcriptional activities. Mutations in ATP/ GTP binding motifs of SQU and KEW lead to failure of phosphorylation, and transgenic plants bearing the mutant proteins display defective DV asymmetric flower development, indicating that the two conjugate modifications are essential for their diverse functions. Altogether, SQU and KEW activities are precisely modulated at both transcription and post-transcription levels, which might link DV asymmetric flower development to different physiological status and/or signaling pathways.

Petal Development in Lotus japonicus

Previous studies have demonstrated that petal shape and size in legume flowers are determined by two separate mechanisms, dorsoventral （DV） and organ internal （IN） asymmetric mechanisms, respectively. However, little is known about the molecular mechanisms controlling petal development in legumes. To address this question, we investigated petal development along the floral DV axis in Lotus japonicus with respect to cell and developmental biology by comparing wild-type legumes to mutants. Based on morphological markers, the entire course of petal development, from initiation to maturity, was grouped to define 3 phases or 13 stages. In terms of epidermal micromorphology from adaxial surface, mature petals were divided into several distinct domains, and characteristic epidermal cells of each petal differentiated at stage 9, while epidermal cells of all domains were observed until stage 12. TCP and MIXTA-like genes were found to be differentially expressed in various domains of petals at stages 9 and 12. Our results suggest that DV and IN mechanisms interplay at different stages of petal development, and their interaction at the cellular and molecular level guides the elaboration of domains within petals to achieve their ideal shape, and further suggest that TCP genes determine petal identity along the DV axis by regulatincl MIXTA-like clene expression.