Flavonols have been shown to respond to a variety of abiotic stresses in plants, including cold stress. Higher total flavonoid content was found in non-heading Chinese cabbage (NHCC, Brassica campestris (syn. Brassica rapa) ssp. chinensis) after cold stress. A non-targeted metabolome analysis showed a significant increase in flavonol content, including that of quercetin and kaempferol. Here, we found that an R2R3–MYB transcription factor, BcMYB111, may play a role in this process. BcMYB111 was up-regulated in response to cold treatment, with an accompanying accumulation of flavonols. Then, it was found that BcMYB111 could regulate the synthesis of flavonols by directly binding to the promoters of BcF3H and BcFLS1. In the transgenic hairy roots of NHCC or stable transgenic Arabidopsis, overexpression of BcMYB111 increased flavonol synthesis and accumulation, while these were reduced in virus-induced gene silencing lines in NHCC. After cold stress, the higher proline content and lower malondialdehyde (MDA) content showed that there was less damage in transgenic Arabidopsis than in the wild-type (WT). The BcMYB111 transgenic lines performed better in terms of antioxidant capacity because of their lower H₂O₂ content and higher superoxide dismutase (SOD) and peroxidase (POD) enzyme activities. In addition, a key cold signaling gene, BcCBF2, could specifically bind to the DRE element and activate the expression of BcMYB111 in vitro and in vivo. The results suggested that BcMYB111 played a positive role in enhancing the flavonol synthesis and cold tolerance of NHCC. Taken together, these findings reveal that cold stress induces the accumulation of flavonols to increase tolerance via the pathway of BcCBF2–BcMYB111–BcF3H/BcFLS1 in NHCC.

WRKY transcription factors (TFs) participate in plant defense mechanisms against biological and abiotic stresses. However, their regulatory role in heat resistance is still unclear in non-heading Chinese cabbage. Here, we identified the WRKY-IIe gene BcWRKY22(BraC09g001080.1), which is activated under high temperatures and plays an active role in regulating thermal stability, through transcriptome analysis. We further discovered that the BcWRKY22 protein is located in the nucleus and demonstrates transactivation activity in both the yeast and plant. Additionally, our studies showed that the transient overexpression of BcWRKY22 in non-heading Chinese cabbage activates the expression of catalase 2 (BcCAT2), enhances CAT enzyme activity, and reduces Hydrogen Peroxide (H₂O₂) accumulation under heat stress conditions. In addition, compared to its wild-type (WT) counterparts, Arabidopsis thaliana heterologously overexpresses BcWRKY22, improving thermotolerance. When the BcWRKY22 transgenic root was obtained, under heat stress, the accumulation of H₂O₂ was reduced, while the expression of catalase 2 (BcCAT2) was upregulated, thereby enhancing CAT enzyme activity. Further analysis revealed that BcWRKY22 directly activates the expression of BcCAT2 (BraC08g016240.1) by binding to the W-box element distributed within the promoter region of BcCAT2. Collectively, our findings suggest that BcWRKY22 may serve as a novel regulator of the heat stress response in non-heading Chinese cabbage, actively contributing to the establishment of thermal tolerance by upregulating catalase (CAT) activity and downregulating H₂O₂ accumulation via BcCAT2 expression.

A well-developed root system is crucial for the rapid growth, asexual reproduction, and adaptation to the drought environments of the watercress. After analyzing the transcriptome of the watercress root system, we found that a high concentration of auxin is key to its adaptation to dry conditions. For the first time, we obtained DR5::EGFP watercress, which revealed the dynamic distribution of auxin in watercress root development under drought conditions. Via the application of naphthylphthalamic acid (NPA), 4-biphenylboronic acid (BBO), ethylene (ETH), abscisic acid (ABA), and other factors, we confirmed that auxin has a significant impact on the root development of watercress. Finally, we verified the role of auxin in root development using 35S::NoYUC8 watercress and showed that the synthesis of auxin in the root system mainly depends on the tryptophan, phenylalanine, and tyrosine amino acids (TAA) synthesis pathway. After the level of auxin increases, the root system of the watercress develops toward adaptation to dry environments. The formation of root aerenchyma disrupts the concentration gradient of auxin and is a key factor in the differentiation of lateral root primordia and H cells in watercress.

Based on the established efficient regeneration system for watercress in our laboratory, we optimized the processes of pretreatment, co-culture, and differentiation culture. Through GFP fluorescence and PCR identification, we successfully obtained transgenic watercress with the DR5 gene, which allowed us to investigate the distribution details of auxin in the growth process of watercress. Our findings provide an effective method for gene function research and lay the foundation for innovative utilization of germplasm resources of watercress.

Watercress (Nasturtium officinale R.Br.) is a cruciferous aquatic vegetable that possesses significant nutritional value. The NAC family is a transcription factor family specific to plants that play an important role in regulating plant responses to abiotic stress. In order to investigate the response of NAC genes to flooding stress in watercress, we conducted a study on the NoNAC family. In this study, a total of 119 NoNAC genes were obtained through genome-wide identification. Phylogenetic analysis indicated that the NoNAC family members can be categorized into ten subgroups. The results of gene structure analysis revealed that each branch within the subgroups exhibited similar motif composition and gene structure. The heatmap analysis showed that several NoNAC genes demonstrated tissue-specific expression patterns, suggesting their potential as regulators of associated tissue development. As an aquatic plant, watercress serves as a valuable material for investigating plant resistance to flooding stress. This study found that flooding can significantly increase the watercress plant height, which is a typical escape strategy under flooding. The analysis of the expression of NoNAC genes in the stem transcriptome after flooding indicated that only NoNAC36a consistently exhibited significant differential changes and down-regulated expression at the three time points of flooding treatment. This suggests that NoNAC36a may be involved in regulating watercress plant height increases under flooding stress. The utilization of a virus-induced gene silencing assay to investigate the biological function of NoNAC36a revealed that NoNAC36a silencing caused cell elongation and expansion, thus increasing watercress plant height. The yeast one-hybrid and dual luciferase assays demonstrated that NoNAC36a binds the promoter of NoXTH33 and inhibits its expression. Subsequently, the results of yeast two-hybrid, luciferase complementary, and pull-down assays revealed the interaction between NoMOB1A and NoNAC36a in vivo and in vitro. Sequence alignment indicated that NoMOB1A and AtMOB1A share an identical amino acid sequence. RT-qPCR analysis indicated that flooding prompted the expression of NoMOB1A in stems. Thus, it is speculated that NoMOB1A may exhibit functions similar to AtMOB1A and that the up-regulation of NoMOB1A expression in stems may facilitate an increase in plant height under flooding. In summary, the NoNAC family was analyzed, and revealed a regulatory network centered on NoNAC36a that facilitates watercress resistance to flooding stress. This study enhanced the understanding of the NoNAC genes and established a theoretical foundation for investigating plant flooding tolerance.