Background: Leaf color is an important trait in breeding of leafy vegetables. Y-05, a pakchoi (Brassica rapa ssp. chinensis) cultivar, displays yellow inner (YIN) and green outer leaves (GOU) after cold acclimation. However, the mechanism of this special phenotype remains elusive. Results: We assumed that the yellow leaf phenotype of Y-05 maybe caused by low chlorophyll content. Pigments measurements and transmission electron microscopy (TEM) analysis showed that the yellow phenotype is closely related with decreased chlorophyll content and undeveloped thylakoids in chloroplast. Transcriptomes and metabolomes sequencing were next performed on YIN and GOU. The transcriptomes data showed that 4887 differentially expressed genes (DEGs) between the YIN and GOU leaves were mostly enriched in the chloroplast- and chlorophyll-related categories, indicating that the chlorophyll biosynthesis is mainly affected during cold acclimation. Together with metabolomes data, the inhibition of chlorophyll biosynthesis is contributed by blocked 5-aminolevulinic acid (ALA) synthesis in yellow inner leaves, which is further verified by complementary and inhibitory experiments of ALA. Furthermore, we found that the blocked ALA is closely associated with increased BrFLU expression, which is indirectly altered by cold acclimation. In BrFLU-silenced pakchoi Y-05, cold-acclimated leaves still showed green phenotype and higher chlorophyll content compared with control, meaning silencing of BrFLU can rescue the leaf yellowing induced by cold acclimation. Conclusions: Our findings suggested that cold acclimation can indirectly promote the expression of BrFLU in inner leaves of Y-05 to block ALA synthesis, resulting in decreased chlorophyll content and leaf yellowing. This study revealed the underlying mechanisms of leaves color change in cold-acclimated Y-05.

The transcription factor WRKY33 is a vital regulator of the biological process of the necrotrophic fungus Botrytis cinerea (B. cinerea). However, its specific regulatory mechanism remains to be further investigated. In non-heading Chinese cabbage (NHCC, Brassica campestris (syn. Brassica rapa) ssp. Chinensis), our previous study showed that BcWRKY33A is induced not only by salt stress, but also by B. cinerea infection. Here, we noticed that BcWRKY33A is expressed in trichomes and confer plant defense resistance. Disease symptoms and qRT-PCR analyses revealed that BcWRKY33A-overexpressing and -silencing lines were less and more severely impaired, respectively, than wild type upon B. cinerea treatment. Meanwhile, the transcripts’ abundance of indolic glucosinolates’ (IGSs) biosynthetic genes is consistent with plants’ B. cinerea tolerance. Identification and expression pattern analysis of BcMYB51s showed that BcMYB51-3 has a similar trend to BcWRKY33A upon B. cinerea infection. Moreover, BcWRKY33A directly binds to the BcMYB51-3 promoter, which was jointly confirmed by Y1H, dual-LUC, and EMSA assays. The importance of MYB51, the homolog of BcMYB51-3, in the BcWRKY33A-mediated B. cinerea resistance was also verified using the TRV-based VIGS system. Overall, our data concludes that BcWRKY33A directly activates the expression of BcMYB51-3 and downstream IGSs’ biosynthetic genes, thereby improving the B. cinerea tolerance of NHCC plants.