Functional Study Of Sweet Sorghum (Sorghum Bicolor L.)transcription Factor SbHY5 In Response To Aluminum Stress

Over the years,the process of soil acidification is intensifying,accounting for40-50%of the potential arable land in the word.In general,soil acidification is accompanied by aluminum toxicity.As the acidic soil increases,the p H of the soil solution decreases（p H<5.0）.Aluminum（Al）,which originally existed in the solid phase of the soil as insoluble oxides and aluminum silicates,gradually dissolves into the soil solution in the active form of Al³⁺.Micromolar concentrations of Al³⁺can cause toxicity to plant root tips,thereby inhibiting the absorption of water and mineral nutrients by plants.Therefore,Al toxicity has become one of the main factors limiting crop yields on acid soil.Sweet sorghum（Sorghum bicolor L.）is a high-efficient energy crop widely grown in tropics and subtropics of the world,where acid soil is common and Al toxicity is widespread.ELONGATED HYPOCOTYL 5（HY5）is a member of the basic leucine zipper（b ZIP）transcription factor family and an key pivot involved in higher plant photomorphogenesis.Recent studies showed that HY5 plays a critical role in plant response to various stress conditions,such as salinity,drought,low temperature and high temperature.However,there is a lack of research on its functions and molecular mechanisms in response to Al stress.In this study,a novel transcription factor SbHY5 related to Al tolerance was identified in sweet sorghum and found to be induced by both Al stress and increasing light intensity in transcriptional level.The Al-tolerance phenotype of the transgenic materials were analyzed by heterologous genetic transformation.On the basis,further exploration of downstream genes regulated by SbHY5 was conducted through transcriptome analysis,and the function of SbHY5 was verified,revealing its mechanisms in response to Al stress.The specific research results are as follows:1.The stronger the light intensity,the weaker the inhibitory effect of Al treatment on root elongation in the sweet sorghum seedlings.The effect of light on Al tolerance might be mediated via the modulation of SbHY5 transcript levels in roots and shoots.The Al tolerance of Arabidopsis seedlings were also improved by enhancing light intensity,while the hy5-215 mutant did not exhibit such a light intensity dependent Al tolerance improvement,revealing the importance of HY5 in integrating light signal and plant Al stress response.2.SbHY5 clusters closely with Zm HY5,at the identity of 89.8%,and a homology of 70.0%with At HY5.SbHY5 is specifically induced by Al stress at the transcriptional level,and its expression is significantly induced at 6 h.SbHY5 was localized to the nucleus and was transcriptionally active.SbHY5 restored the Al-sensitive phenotype of the hy5-215 mutant;overexpression of SbHY5 in wild-type significantly improved Al tolerance in transgenic plants.3.Transcriptome analysis indicated that SbHY5 affects the organic acid secretion metabolic pathway and transmembrane transport protein activity in transgenic Arabidopsis roots under Al stress,and the expression levels of At MATE and At STOP2 were significantly upregulated.Yeast one-hybrid activity analysis,luciferase activity analysis and EMSA,together,revealed that SbHY5 could directly bind to Sb MATE promoter and positively regulate its transcription.Overexpression of SbHY5 participats in the MATE-dependent citrate secretion pathway to improve plant Al tolerance by increasing root citrate secretion and reducing root tip Al accumulation.4.SbHY5 can directly bind to the promoter of Sb STOP1a-d,inducing its expression,further revealing the important role of SbHY5 in the plant Al stress response network.There is no protein-protein interaction between SbHY5 and Sb STOP1a-ds.Moreover,SbHY5 can directly bind to its own promoter and positively regulate its transcription in response to Al stress.5.Overexpression of SbHY5 has an effect on cell wall components,cell wall modification in transgenic Arabidopsis roots,playing an important role in the process of cell wall xyloglucan metabolism.A Xyloglucan endoglycosyltransferase/hydrolase gene（SbXTH7）was further identified in sweet sorghum.SbHY5 can directly bind to the SbXTH7 promoter and negatively regulate its expression.The expression level of SbXTH7 significantly decreased in the root and shoot of sweet sorghum under Al stress.Overexpression of SbXTH7 increases the hemicellulose content in transgenic Arabidopsis root cell walls,increases Al accumulation on hemicellulose,and reduces plant tolerance to Al stress.In summary,this study revealed the function and mechanism of a sweet sorghum transcription factor,SbHY5,in response to Al stress.It lays the foundation for understanding the mechanisms by which plants respond to aluminum stress and offers new insights for the identification of aluminum-tolerant genes in sorghum.