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Brassica juncea var. tumida Tsen et Lee (Tumorous stem mustard) is an unique vegetable in China. Its enlarged tumorous stem was used as main raw material to produce pickle (Zhacai). In practice, early-bolting happens around 15% of planting area all year and inhibits its production. Here, about 209 PP2C proteins were identified through HMMER software and divided into 13 sub-families in B. juncea. BjuPP2C52 belongs to E sub-family, was up-regulated at reproductive growth stages and interacts with BjuFKF1, a key protein in regulating plant photoperiod flowering, in vitro and in vivo. To explore interactive proteins, BjuPP2C52 was used as bait, 12 potential interactive proteins were screened from yeast library, and they are BjuCOL3, BjuCOL5, BjuAP2, BjuAP2-1, BjuSVP-1, BjuFLC-2, BjuSKP1f, BjuA014572, BjuA008686, BjuO002119, BjuB036787 and BjuA019268. Further study verified that 10 out of the 12 screened proteins interacted with BjuPP2C52 in vivo. qRT-PCR was conducted to understand the expression pattern of those 10 interactive proteins in different tissues and development stages in B. juncea. The results showed that BjuCOL3, BjuCOL5, BjuB036787 and BjuA019268 were significantly up-regulated, while BjuA008686 and BjuO002119 were down-regulated in flowers compared with other four tissues. In developmental stages, BjuCOL5, BjuAP2, BjuAP2-1, BjuA014572, BjuB036787 and BjuA019268 were significantly up-regulated, while BjuSVP-1, BjuA008686 and BjuO002119 were down-regulated at reproductive stages. Based on the results, BjuCOL5, BjuAP2, BjuAP2-1, BjuSVP-1, BjuA014572, BjuB036787 and BjuA019268 may function in regulating flowering time in B. juncea.

Tumorous stem mustard is well known for its swollen stem from which pickled “Fuling Mustard” is made. The molecular mechanisms governing the formation of the modified swollen stems are still poorly understood. This paper aims to identify candidate genes involved in the developmental regulation of the swollen stems. We sought to map previously published transcriptome datasets for Brassica juncea, including those derived from swollen stems at four different developmental stages and a mutant variety without swollen stems. Using pairwise comparisons of the five datasets, we identified 31 368 differentially expressed genes (DEGs). A total of 55 DEGs related to plant hormone signal transduction and 259 continuously up- or downregulated transcription factors were identified during stem development using Gene Ontology (GO) analysis. Quantitative real-time PCR (qRT-PCR) results showed that the expressions of 12 important candidate DEGs were consistent with RNA-seq results. Our study provided digital gene expression profiling and a dynamic view of the swollen stem development process. Furthermore, we identified candidate genes for further studies on mechanisms of modified stem development in non-model species.

Stem mustard (Brassica juncea var. tumida Tsen et Lee) is one of a unique vegetable in China and the tumor stem is its economic product. With the expansion planting area to high altitude in Fuling, early-bolting has become the most critical issue in its cultivation. Previous studies found that stem mustard early-bolting in high latitude closely related to short waves. FKF1 (FLAVIN BINDING, KELCH REPEAT, F-BOX1) is a plant-specific LOV blue light receptor, mainly regulates photoperiodic flowering and cycle rhythm. We used BjuFKF1 as the bait protein and detected its interaction protein BjuPP2C52. Further, GST pull down assay confirmed the interaction in vitro. Yeast two hybrid assay showed that both the F-box domain and KELCH repeat domain of BjuFKF1 interacted with BjuPP2C52 in vivo. RT-PCR and qRT-PCR was used to analyze gene expression in different tissues and development stages, respectively. RT-PCR results indicated that BjuFKF1 was expressed in tumorous stem at an extremely high level, while BjuPP2C52 was highly expressed in both flowers and tumorous stem. qRT-PCR assays showed that both BjuFKF1 and BjuPP2C52 were up-regulated expressed in the reproductive growth stages. Sub-cellular assays showed that BjuFKF1 and BjuPP2C52 located in the nucleus and plasma membrane, respectively. Taken together, our results demonstrated that BjuFKF1 interacted with BjuPP2C52 in vitro and vivo, BjuFKF1 was expressed at an extremely high level in tumorous stem, BjuPP2C52 was highly expressed in flowers. BjuFKF1 and BjuPP2C52 were up-regulated expressed in reproductive growth stages. Sub-cellular assays showed that BjuFKF1 and BjuPP2C52 located in the nucleus and cell membrane, respectively. It was speculated that BjuFKF1 may through the pathway of “BjuFKF1-BjuPP2C52-BjuAGB1-BjuNH3-Bjuphot1” to regulate stem mustard response to blue light and eventually bolting and flowering time.