Functional Study Of Sweet Sorghum (Sorghum Bicolor L.) Transcription Factors SbWRKY22 And SbWRKY65 In Response To Aluminum Stress

Acidic soils account for about 30%of the world’s total land area and up to 50%of the global arable land.When the soil p H is less than 5.5,aluminum（Aluminum,Al）will be free from clay minerals and oxides and other forms,and will be absorbed by plant roots in the form of soluble trivalent Al ions(Al³⁺),which will inhibit the growth of plant roots and impair the normal physiological functions.Al stress has been recognized as one of the most important constraints to agricultural production in acidic soils.WRKY transcription factors are one of the larger families of transcription factors in plants and are an important class of transcription factors in response to environmental stresses in plants.Studies have shown thatWRKY transcription factors have important roles in plant response to a variety of adversity stresses,but there is a lack of research on their functions and molecular mechanisms in response to Al stress.In this study,twoWRKY transcription factors,SbWRKY22 and SbWRKY65,were identified in sweet sorghum and found to be induced by Al at the transcriptional level.The potential downstream regulated genes of SbWRKY22 and SbWRKY65 were verified by transcriptional regulation analysis.On this basis,the Al-resistant phenotypes of the transgenic materials were analyzed by heterologous genetic transformation,and the functions of SbWRKY22 and SbWRKY65 were verified,revealing their mechanisms of action involved in Al stress response.The specific research results are as follows:1.Bioinformatics analysis showed that SbWRKY22 belongs to class IIIWRKY transcription factors,whereas SbWRKY65 belongs to class IIWRKY transcription factors.SbWRKY22 and SbWRKY65 differ in the conformation of theWRKY structural domains,especially the zinc finger motif.The expression of SbWRKY22 and SbWRKY65 at the transcriptional level were both induced by Al,and the expression at the root tip（0-1 cm）was induced by Al to a greater extent.2.Subcellular localization analysis showed that both SbWRKY22 and SbWRKY65 were localized to the nucleus.Further transcriptional activity analysis showed that both SbWRKY22 and SbWRKY65 were transcriptionally active.3.SbWRKY22 had the highest homology to AtWRKY54 in Arabidopsis,and SbWRKY22 restored the Al-sensitive phenotype of the Atwrky54 knockout mutant;overexpression of SbWRKY22 in the wild type significantly improved Al tolerance in transgenic plants.Luciferase activity analysis and yeast one-hybrid activity analysis,together,revealed that SbWRKY22 had a significant transcriptional regulatory effect on the Al-tolerant genes Sb MATE,Sb Glu1,Sb STAR1,Sb STAR2a,and Sb STAR2b.Further studies revealed that SbWRKY22 improves plant Al tolerance in several ways by increasing the expression level of At BG2,a homologous gene of Sb Glu1 in Arabidopsis thaliana,by decreasing callose accumulation in the root system of the plant,and by decreasing the hemicellulose content in the cell wall of the root system of the plant.4.SbWRKY65 was overexpressed in the wild-type background of Arabidopsis thaliana.under Al stress,SbWRKY65 overexpression lines showed significantly higher relative root elongation than that of the wild type and exhibited strong Al tolerance.Although SbWRKY65 had almost no regulatory effect on the above five Al tolerance genes,it had a significant transcriptional regulatory effect on SbWRKY22.It was further found that SbWRKY65 reduced callose accumulation in the root system of plants by regulating the expression of the SbWRKY22 homologous gene AtWRKY54,thus improving plant Al tolerance.5.A protein kinase,SbCIPK11,was identified in sweet sorghum,and the expression of Sb CIPK11 at the transcriptional level was found to be not only induced by Al but also regulated by SbWRKY65.At CIPK11 and Sb CIPK11 have the highest homology in Arabidopsis,and SbCIPK11 restored the Al-sensitive phenotype of the Atcipk11 knockout mutant;overexpression of Sb CIPK11 in the wild type significantly increased the Al tolerance of the transgenic plants.It was further found that Sb CIPK11modulated plant antioxidant responses by increasing the activities of superoxide dismutase（SOD）and peroxidase（POD）in Arabidopsis thaliana and decreasing the contents of H₂O₂ and malondialdehyde（MDA）in the root system of the plants,which in turn improved the Al tolerance of the plants.Yeast two-hybrid interactions analysis and bimolecular fluorescence complementation analysis together verified that Sb CIPK11 interacted with Sb CBL2,Sb CBL3,Sb CBL4,Sb CBL5,and Sb CBL7 all at the plant cell membrane.In summary,this study elucidated the functions and underlying mechanisms of the sorghum transcription factors SbWRKY22 and SbWRKY65 in response to Al stress.It laid the foundation for understanding the mechanisms by which plants respond to Al stress and offered new insights for the identification of aluminum-tolerant genes in sorghum.