Vernalization can regulate flowering time through microRNA mechanism in Brassica rapa.

Vernalization is an important process that regulates the floral transition in plants. MicroRNAs (miRNAs) are endogenous non-coding small RNA (sRNA) molecules that function in plant growth and development. Despite that miRNAs related to flowering have previously been characterized, their roles in response to vernalization in pak-choi (Brassica rapa ssp. chinensis) has never been studied. Here, two sRNA libraries from B. rapa leaves (vernalized and non-vernalized plants) were constructed and sequenced. Two hundred eight known and 535 novel miRNAs were obtained, of which 20 known and 66 new miRNAs were significantly differentially expressed and considered as vernalization-related miRNAs. The corresponding targets were predicted on the basic of sequence homology search. In addition, 11 miRNAs and eight targets were selected for real-time quantitative PCR to confirm their expression profiles. Functional annotation of targets using gene ontology and Kyoto encyclopedia of genes and genomes results suggested that most targets were significantly enriched in the hormone signaling pathway. Moreover, a decreased indole-3-acetic acid (IAA) and an increased GA3 hormone were detected after vernalization, indicating that the IAA and GA3 might response to vernalization. These results indicated that vernalization regulates flowering through microRNA mechanism by affecting endogenous hormone level in B. rapa. This study provides useful insights of promising miRNAs candidates involved in vernalization in B. rapa, and facilitates further investigation of the miRNA-mediated molecular mechanisms of vernalization in B. rapa.

Role of vernalization-mediated demethylation in the floral transition of Brassica rapa.

MAIN CONCLUSION: Vernalization-mediated demethylation of BrCKA2 (casein kinase II α-subunit) and BrCKB4 (casein kinase II ß-subunit) shorten the period of the clock gene BrCCA1 (circadian clock associated 1) in Brassica rapa. Photoperiod and vernalization are two environmental cues involved in the regulation of floral transition, but the ways in which they interact remain unclear. DNA methylation is one of the main mechanisms involved in controlling the functional state of chromatin and gene expression in response to environmental signals. To study the interaction between photoperiod and vernalization in floral transition, we carried out a comparative genomic analysis of genome-wide DNA methylation profiles in normal (CK) and vernalized (CA) leaves from Brassica rapa using methylated-DNA immunoprecipitation sequencing (MeDIP-seq). Two subunits of casein kinase II (CK2), BrCKA2 (catalytic α-subunit of CK2) and BrCKB4 (regulatory ß-subunit of CK2), exhibited gradual DNA demethylation and increased expression in vernalized B. rapa. DNA methylation-defective plants demonstrated the causal link between DNA demethylation changes and changes in gene expression. Virus-induced gene silencing (VIGS) of BrCKA2 and BrCKB4 in B. rapa resulted in no change to the period of BrCCA1 (circadian clock associated 1) and a 1-week late flowering time. Finally, we demonstrated that increased levels of BrCKA2 and BrCKB4 in vernalized B. rapa confer elevated CK2 activity, resulting in a shortened period of the clock gene BrCCA1, which plays an important role in perceiving photoperiod in plants. Thus, our results suggest that there is a direct interaction between photoperiod and vernalization through DNA methylation mechanisms.