GFP Fluorescence Screening in Transgenic Roots with LUYOR-3415RG

In a study published in Antioxidants (Wang et al., 2023), researchers from Nanjing Agricultural University investigated the role of the BcWRKY22 transcription factor in enhancing thermotolerance in non-heading Chinese cabbage (Brassica campestris ssp. chinensis). To verify the successful generation of transgenic hairy roots overexpressing BcWRKY22, the team used the LUYOR-3415 fluorescent protein excitation lamp for GFP visualization.

Abstract

WRKY transcription factors (TFs) participate in plant defense mechanisms against biologi- cal 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.

Visualizing GFP in NHCC Roots with LUYOR-3415RG Fluorescent Protein Excitation Lamp

Since the expression of BcWRKY22 was at its highest in roots (Figure 1c), we obtained transgenic roots using Agrobacterium rhizogenes in NHCC, and the GFP fluorescence of roots was observed with LUYOR-3415RG Fluorescent Protein Excitation Lamp, to ensure that BcWRKY22-overexpressed roots were obtained (Figure 5a).

An RT-qPCR analysis showed that BcWRKY22 was overexpressed in the three root systems (OE-BcWRKY22-1, OE-BcWRKY22-2, and OE-BcWRKY22-3), and their gene expression levels were higher than that of the WT control (Figure 5b). Then, we treated the WT control and the three over-expressed roots with heat stress (HS). The overexpression of BcWRKY22 in the three root systems did not affect their MDA content values at 24 ◦C, while the values of MDA were significantly higher in the WT control than in the BcWRKY22-overexpressed roots under HS (Figure 5c). Meanwhile, we found that the H2O2 content values of the three transgenic roots were lower than that of the WT control at 24 ◦C, and the increased degree of H2O2 content values in the three transgenic roots was markedly lower than that of the WT control after HS (Figure 5d). This conclusion is compatible with the results of the transient expression and overexpression in Arabidopsis thaliana, indicating that the overexpression of BcWRKY22 can affect the content of H2O2. Furthermore, the CAT activity of the BcWRKY22-overexpressedroot systems was higher at 24 C than that of the WT control. After HS, compared withthe WT control, the increase in CAT activity in the BcWRKY22-overexpressed roots was significantly upregulated (Figure 5e). In addition, the expression of BcCAT2 was significantly increased in the BcWRKY22-overexpressed roots (Figure 5f).

In order to further confirm that the over expression of BcWRKY22 can improve the thermotolerance of roots, we determined the expression levels of the heat response genes BcDREB2A, BCHSFA2, and BcGolS1. Their expression levels increased significantly in transgenic roots. Moreover, after HS, the expression of the three genes increased even more (Figure 5g). In summary, these results suggest that BcWRKY22 overexpression improves the thermo tolerance of transgenic roots. At thes ame time, BcWRKY22 may regulate the expression level of BcCAT2 to improve CAT activityand reduce the accumulation of H2O2.

Photo below: GFP fluorescence of the NHCC roots observed by LUYOR-3415RG Fluorescent Protein Excitation Lamp

Reference

Wang, H., Gao, Z., Chen, X., Li, E., Li, Y., Zhang, C., & Hou, X. BcWRKY22 activates BcCAT2 to enhance catalase (CAT) activity and reduce hydrogen peroxide (H₂O₂) accumulation, promoting thermotolerance in non-heading Chinese cabbage (Brassica campestris ssp. chinensis). Antioxidants 12, 1710 (2023).