The effect of CaNAC2csilencing on pepper growth, thermotolerance and response to RSI
To assay the possible role of CaNAC2c in pepper response to HTS or RSI, we assayed the effect of CaNAC2c silencing by VIGS on response of pepper plants to HTS or RSI. The results showed thatCaNAC2c was successfully silenced by the two vectors (Figure 1A and B). It was found that upon HTS, the CaNAC2c silenced plants exhibited decreased basal and acquired thermotolerance, displayed with serious wilt phenotype, high levels of plant mortality from 1 to 42 hpt with or without pretreatment of nonlethal high temperature (Figure 1C and D). The decreased thermotolerance was accompanied with significant downregulation of thermotolerance relatedCaHSP24, CaHSFB2a and CaHSP70 (Ashraf et al., 2018) (Figure1E-G).We also found that ion leakage upon HTS displayed by conductivity in CaNAC2c silenced plants was significantly higher than that of the control plants (Figure 1H and I). The DAB and NBT staining representing H2O2 and ROS accumulation, which were previously found to be negatively related to thermotolerance(Yu et al., 2019; Zhuang et al., 2020), were much darker in the leaves and stems of CaNAC2c -silenced plants than that in the control plants (Figure 1J and K).We also foundCaNAC2c -silenced plants exhibited lower levels of Fv/Fm and actual photochemical efficiency of PSII in the light (фPSII), indicator of thermotolerance and thermostability of the photosynthetic apparatus, respectively(Guan et al., 2018; Wang, Zhang, Goatley, & Ervin, 2014; Yan et al., 2008), immediately after the HTS treatment(Figure 1L and M). All these data indicate that CaNAC2c acts as positive regulator in basal and acquired thermotolerance. By contrast, the CaNAC2csilenced pepper plants were inoculated with R. solanacearum , but no difference was found between the CaNAC2c silenced and the control plants (Data not shown). In addition, the CaNAC2csilenced pepper plants exhibited promotion not only in plant height and size of roots, stems and leaves, but also in leaf and flower number (Figure 2), indicating that CaNAC2c acts negatively in pepper growth and development.