Gene Expression Analysis in Response to Vernalization in Chinese Cabbage (<i>Brassica rapa</i> L.)

Abstract

Chinese cabbage (Brassica rapa L. var. pekinensis) is an economically and agriculturally significant leafy vegetable that is extensively cultivated throughout the world. Vernalization is exposure to prolonged cold that alters gene expression and accelerates a transition from the vegetative to reproductive phase. Premature bolting caused by exposure to cold inhibits head production and reduces the yield of Chinese cabbage, and developing a late bolting line is important for breeding. Therefore, it is critical to identify the genes showing differential expression patterns during cold treatment in Chinese cabbage. However, there are few studies on the transcriptome profiling of different durations of cold treatments in Chinese cabbage. Here, we analyzed the gene expression profiles in a Chinese cabbage inbred line, RJKB-T24, given different durations of cold treatments using RNA sequencing. Differentially expressed genes (DEGs) between non-vernalized and vernalized samples tended to be downregulated, and some genes involved in the flowering pathway (including BrFLC and BrMAF genes) were downregulated following cold treatment. Functional enrichment analysis indicated that some DEGs were involved in the stress response and hormone signaling pathways. For genes involved in the FRI-containing complex, a known activator of FLC in Arabidopsis thaliana, only BrFRL1 showed changes in expression. In contrast to A. thaliana, BrVIP and BrVRN genes showed different expression patterns between paralogs during cold treatment, suggesting that Chinese cabbage's flowering pathway is somehow different and more complex than in A. thaliana. These outcomes provide significant insights into the genetic control of bolting and flowering that occur during the vernalization of Chinese cabbage.

Genome Triplication Leads to Transcriptional Divergence of FLOWERING LOCUS C Genes During Vernalization in the Genus Brassica

“…In B. rapa , H3K4me3 accumulation was found in the nucleation region of all four BrFLC paralogs before exposure to cold ( Kawanabe et al, 2016 ). During vernalization, accumulation of H3K27me3 within the nucleation region of all four BrFLC paralogs with decreased expression was observed ( Akter et al, 2020 ; Figure 1 ). After return to warm conditions, H3K27me3 accumulation spreads across the four BrFLC paralogs maintaining silencing ( Kawanabe et al, 2016 ; Akter et al, 2019 ; Figure 1 ).…”

Multiplex CRISPR/Cas9 Mutagenesis of BrVRN1 Delays Flowering Time in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

“…The sequence similarity between the paralogs ranged between 89.1% and 90.7%. A previous study showed that under cold treatment, BrVRN1a (Bra022376) is upregulated, while its paralogs BrVRN1b (Bra037544) and BrVRN1c (Bra001729) are downregulated [25,41]. We expected that knocking out any one paralog in Chinese cabbage would give comparable phenotypic results and therefore focused on BrVRN1a.…”

“…Sequences of the Chinese cabbage VRN1 genes (BrVRN1a, Bra022375, BrVRN1b, Bra037544, and BrVRN1c, Bra001729) were compared to find a region with matching sequences (Figures 1a and S1 and Table S1). Then, four sgRNAs were designed in the corresponding region to target the BrVRN1a (Bra022375) gene [25]. To construct a plasmid vector carrying four sgRNAs and the Cas9 cassette, we designed a multiplex CRISPR/Cas9 platform that allows the co-expression of four sgRNA modules targeting different sites of BrVRN1 in one binary vector (pAGM4723) using Golden Gate assembly (Figure 1b) [31].…”

“…In Chinese cabbage, the expression of BrVRN1 genes is altered by cold treatment, according to patterns that vary among the three paralogous genes: two BrVRN1 genes are downregulated during cold treatment, and one is upregulated. It is therefore possible that the suppression of BrFLC expression is dependent on the upregulation of BrVRN1 by vernalization in B. rapa [25].…”

“…Recently, several studies have set out to directly control flowering time via CRISPR/Cas9-mediated genome editing [28,29]. In this study, we used the CRISPR/Cas9 system for targeted mutagenesis of BrVRN1 homologs (BrVRN1s) to introduce the late-flowering trait into B. rapa [25]. CRISPR/Cas9-mediated multi-target mutagenesis of BrVRN1s gave new insights into flowering time regulation and demonstrated that genome editing can be used to improve agricultural traits in B. rapa.…”