Abstract
Brassica juncea is an important leafy vegetable, and flowering time is a key determinant of its yield and quality. In this study, one significantly up-regulated gene, BjuAGL9-2, was identified from RNA-Seq data. qRT-PCR analysis confirmed that BjuAGL9-2 expression was significantly elevated in reproductive organs and reproductive stages. Further five BjuAGL9-2 over-expression (OE) lines were subsequently generated, which showed an early-flowering and pale-yellow leaf phenotype compared to the wild type. qRT-PCR assays found that the mRNA of core floral integrator genes was changed in Arabidopsis OE lines. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays indicated that BjuAGL9-2 interacted with BjuTUA5, BjuZFP7, BjuGSTU5, and BjuMAPK16 in vivo. Sub-cellular localization assays showed that BjuAGL9-2 localizes in the nucleus, whereas its interacting partners localize in the cytoplasm. qRT-PCR assays further revealed that BjuTUA5 and BjuGSTU5 were up-regulated in flower buds, while BjuZFP7 and BjuMAPK16 were down-regulated. During vegetative stages, all four genes were up-regulated in B. juncea. As for BjuAGL9-2 interaction protein-encoding homolog genes, except AtGSTU5, the other three genes were up-regulated in Arabidopsis OE lines. Additionally, qRT-PCR analysis of chlorophyll biosynthesis-related genes showed that 19 of 27 genes were up-regulated, while 8 genes were down-regulated, in Arabidopsis OE lines. Collectively, these findings suggest that BjuAGL9-2 promotes flowering and contributes to the pale-yellow phenotype by regulating its interacting protein-coding genes, floral integrators, and chlorophyll biosynthesis genes.