Aluminum (Al) toxicity is a primary limiting factor for crop production in acidic soils. The WRKY transcription factors play important roles in regulating plant growth and stress resistance. In this study, we identified and characterized two WRKY transcription factors, SbWRKY22 and SbWRKY65, in sweet sorghum (Sorghum bicolor L.). Al induced the transcription of SbWRKY22 and SbWRKY65 in the root apices of sweet sorghum. These two WRKY proteins were localized in the nucleus and exhibited transcriptional activity. SbWRKY22 showed the significant transcriptional regulation of SbMATE, SbGlu1, SbSTAR1, SbSTAR2a, and SbSTAR2b, which are major known Al tolerance genes in sorghum. Interestingly, SbWRKY65 had almost no effect on the aforementioned genes, but it significantly regulated the transcription of SbWRKY22. Therefore, it is speculated that SbWRKY65 might indirectly regulate Al-tolerance genes mediated by SbWRKY22. The heterologous expression of SbWRKY22 and SbWRKY65 greatly improved the Al tolerance of transgenic plants. The enhanced Al tolerance phenotype of transgenic plants is associated with reduced callose deposition in their roots. These findings suggest the existence of SbWRKY22- and SbWRKY65-mediated Al tolerance regulation pathways in sweet sorghum. This study extends our understanding of the complex regulatory mechanisms of WRKY transcription factors in response to Al toxicity.

WRKY Transcription Factors SbWRKY22 and SbWRKY65 Regulate the Expression of Aluminum Resistance Genes in Sorghum

National Natural Science Foundation of China 31972508