The Mechanism Of Transcription Factor SbSTOP1 Activates The Transcription Of A ?-1,3-Glucanase Gene SbGlu1 In Sweet Sorghum Under Aluminum Stress

In acid soil,Aluminum?Al?toxicity is the main factor limiting crop yield.When the pH value is lower than 5,Al in the soil changes from solid phase to liquid phase and exists in the rhizosphere soil solution in the form of trivalent Al ion(Al³⁺).Quite small amount of Al³⁺in soil solution can affect the elongation of plant root system in a relatively short time,and then affects the physiological metabolism of plants.It was found that callose deposition in plant roots is a sensitive indicator of Al toxicity and mechanism of Al toxicity.Callose degradation is regulated by?-1,3-glucanase.Sweet sorghum SbGlu1,encoding a?-1,3-glucanase for callose degradation,showed an important role in sorghum Al resistance,yet its regulatory mechanisms remain unclear.Arabidopsis thaliana AtSTOP1 and rice OsART1,two zinc finger transcription factors,can regulate the expression of multiple genes related to Al resistance and Al tolerance,which plays a crucial role in improving the Al tolerance of Arabidopsis thaliana and rice.Their homolog SbSTOP1 in sweet sorghum has been identified and functionally verified.In this study,sweet sorghum?Sorghum bicolor L.,variety:POTCHETSTRM,POT?and Arabidopsis?Arabidopsis thaliana?were used as materials to verify that SbSTOP1 can transcriptionally activate SbGlu1 expression by binding specific regions of SbGlu1 promotor,so that the degradation of callose in plant roots under Al stress is promoted and the Al-resistance of plants is improved,through transcriptional regulation analysis,expression pattern analysis of?-1,3-glucanase and determination of callose content.The results are as follows.1.The SbSTOP1 effector vector and the pSbGlu1 reporter vector were co-transduced into human embryonic kidney cell HEK293T and Arabidopsis protoplasts.By detecting luciferase activity,it was confirmed that SbSTOP1 can regulate the expression of SbGlu1.2.The transcriptional regulation analysis in Arabidopsis protoplast indicated that SbSTOP1 interacts with two 20-bp DNA sequences of the SbGlu1 promoter that do not contain the cis-acting elements binding OsART1.This indicated that there is another transcriptional regulatory mechanism of SbSTOP1 that is not similar with OsART1.Using these two DNA sequences as probes,electrophoretic mobility shift assay was conducted with GST-SbSTOP1^DD protein and the probes.The results showed that these two 20 bp DNA sequences can directly interact with SbSTOP1protein,and the interaction is specific.3.Using the amino acid sequence of SbGlu1 as query,one homologous protein,named AtBG3,was identified in Arabidopsis database.The expression pattern of AtBG3 by real-time PCR showed that the expression level of AtBG3 in the SbSTOP1transgenic complemented lines was significantly higher than that of Atstop1 mutant,indicating that SbSTOP1 regulates the expression of AtBG3.The callose content in SbSTOP1 transgenic complemented lines roots was significantly lower than that of Atstop1 roots.The result indicated that SbSTOP1 can promote the degradation of callose in the roots,and thus improve the Al tolerance of transgenic plants.