Functional Characterization of Cassava A20/AN1 Genes (Metip4, Metip8, and Metip11) in Multiple Abiotic Stresses

Climate/environmental changes have posed significant challenges to crop production. Cassava (Manihot esculenta) is an important starchy root crop of a great significance in food security, energy production, and various bio-industrial applications, as well as a model for studying stress tolerance. Improving stress tolerance can help further increase the starch yield of this crop and expand its planting regions. A20/AN1 domain-containing family genes are master regulators in abiotic stress tolerance, but functions for most cassava A20/AN1 genes (Metip) are unknown. In this study, three Metip genes (Metip4, Metip8, and Metip11) were functionally characterized by prediction, yeast two-hybrid, subcellular localization in rice protoplasts, transgene in Arabidopsis, and virus-induced gene silencing (VIGS) and transcriptome sequencing in cassava. As a result, these genes were intron-free and positively regulated the tolerance of plants to drought, salt, high (32°C) and low (10°C) temperatures, and Mn, but differed in regulating resistance to Cd and Cu, which paralleled changes in plants in contents of proline and relative water, reactive oxygen species, malondialdehyde, endogenous abscisic acid, and/or catalase activity. Metip4, Metip8, and Metip11 proteins were nucleus-localized, had no direct interactions between them, and displayed variations in amino acids within A20/AN1 domains. Moreover, 280 differentially expressed genes (DEG), 4 differentially regulated pathways, and 9 DEG-encoded protein interactions were found to be common in VIGS-treated cassava potted under drought. The results not only clue the formation of multiple functions of A20/AN1 family genes but also strongly suggest that Metip4, Metip8, and Metip11 genes have potentialities in gene engineering abiotic stress-tolerant crops.