Abstract
As the world population continues to increase and climate change simultaneously decreases the amount of land available for agriculture in addition to increasing the severity of abiotic stresses experienced during the growing season, it is increasingly necessary to understand plant tolerance to abiotic stresses at the protein level to aid in the development of more stress-resistant cultivars. The ability of plants to respond to dynamic environmental conditions is largely due to expression of transcription factor proteins that play pivotal roles in the genetic reprogramming that occurs at all stages in plant development and in response to environmental challenge. Previous study of the tomato transcription factor SlNAC4 showed that it was significantly upregulated in response to multiple abiotic stresses including low temperature and drought. Agrobacterium-mediated plant transformation and CRISPR/Cas9-mediated gene editing was used to generate transgenic tomato plants that either overexpress (SlNAC4-OX) or completely lack (SlNAC4-KO) the SlNAC4 gene, and transgenic plants lacking both SlNAC4 and the related SlNAC1 (SlNAC4/SlNAC1-KO). SlNAC4-OX transgenic plants exhibited stunting and abnormal fruiting behavior, but showed increased tolerance to both drought and cold stress, whereas SlNAC4-KO and SlNAC4/SlNAC1-KO plants appeared phenotypically normal but had reduced abiotic stress tolerance. SlNAC4 protein was found to be highly unstable in planta and the development of a novel crossing strategy to restore the WT phenotype to SlNAC4-OX plants was required in order to demonstrate SlNAC4 regulation by the E3 ubiquitin ligase SlSINA4. To this end, SlSINA4-OX, SlSINA4-KO, and SlNAC4/SlSINA4-OX crossed plants were generated and subjected to abiotic stresses. SlNAC4/SlSINA4-OX plants exhibited WT characteristics in both unstressed and stressed conditions, confirming that SlNAC4 may be a substrate of SlSINA4. Because SlNAC4 could be a potential target for manipulation towards increasing crop abiotic stress tolerance, understanding the mechanisms of SlNAC4 influence and regulation greatly benefits the plant science community, and this research provides a simple strategy towards the investigation of highly unstable proteins in planta.