Response Of MhSultr3;1a To Low Sulfur And Regulation Of Sulfur Metabolism In Malus Hupehensis

Sulfur is an essential macronutrient for plant growth,development and stress response.However,for a long time,agricultural and forestry production has focused on the input of nitrogen,phosphorus,and potassium,neglecting the neglecting the role and timely supplements of sulfur.This has caused many crops growing under low sulfur or even sulfur deficiency conditions.The production of fruit crops and other crops is often limited by low sulfur.Sulfur required for the fruit crops is mainly taken up from the soil throughout the root via specific sulfate transporters（Sultr）.Rootstocks serve as the root of the cultivated apple tree.Malus hupehensis is widely used as an excellent rootstock.Therefore,our study first analyzed the growth and sulfur metabolism of Malus hupehensis response to different sulfur supply levels.Then,MhSultr3;1a was isolated from Malus hupehensis roots according to the results of genome-wide analysis of Md Sultr genes,and its response to low sulfur treatment was characterized.In addition,we explored the regulation of Mh MYB3 on the expression of MhSultr3;1a under low sulfur conditions.Furthermore,the regulatory mechanisms of abscisic acid（ABA）and the combination of biochar and potassium nitrate（KNO₃）on the sulfur metabolism of plant were also investigated.The main results are as follows:1.Sulfur supply level significantly affected the growth and sulfur metabolism of Malus hupehensis.With the increase of sulfur application rates,the sulfur content in the roots and shoots of Malus hupehensis increased,but the leave photosynthesis and root activity increased first and then dreased,and peak values were observed at 2 m M treatment.Compared to normal sulfur treatment（2 m M）,sulfur deficiency treatments（0 m M and 0.5 m M）promoted the root absorption area by increasing total root length,root surface area,root tip number,and hormones ocntent such as IAA,GA3,SA,JA,ZT,whereas high sulfur treatments（8 m M）had the opposite result.In addition,sulfur deficiency significantly increased the activity of ATP sulfatase（ATPS）and APS reductase（APR）and their relative gene expression in roots and leaves,promoted root H⁺-ATPase activity and induced the expression of Sults,thus promoting the uptake and assimilation of sulfur,and enhancing the transport of sulfur from root to shoot.Furthermore,sulfur deficiency improved antioxidant capacity of Malus hupehensis by increasing the GSH content and GSH/GSSG,thus alleviating sulfur deficiency stress.2.Based on the Malus domestica genome,9 Md Sultrs genes were identified,which can be divided into 2 subfamilies,and mapped onto 7 chromosomes.Md Sultrs were highly conserved and contained Sulfate＿transp domain and STAS domains.Furthermore,various cis-regulatory elements related to abiotic stress and plant hormone responsiveness were found in the promoter regions of Md Sultrs.These Md Sultrs exhibited tissue-specific expression patterns and responded to ABA,IAA,and Me JA.The expression of 9 Md Sultrs was found to be highly upregulated in response to ABA treatment.,IAA treatment downregulated the most Md Sultrs except for Md Sultr3;3a and Md Sultr4;2.Most Md Sultrs were upregulated by Me JA treatment except for Md Sultr3;4.The expression of Md Sultrs showed tissue-specific patterns and exhibited different expression patterns in roots and leaves response to low sulfur treatment.Among them,Md Sultr3;1a showed root-specific expression and was rapidly upregulated by low sulfur treatment.3.MhSultr3;1a was isolated from Malus hupehensis roots,which localized on the plasma membrane and nucleus membrane.Further function characterization revealed that MhSultr3;1a complemented a sulfate transport-deficient yeast mutant CP154-7A.The growth of MhSultr3;1a-overexpressing apple calli and Arabidopsis thaliana were better than that of wild type,and the contents of SO₄^2-,Cys and GSH were higher,while that of Arabidopsis thaliana Sultr3;1 mutant and inhibited expression of apple callus were opposite,indicating that MhSultr3;1a encoded a functional sulfate transporter and improved the tolerance of plants to low sulfur stress.4.Mh MYB3 regulated the expression of MhSultr3;1a to enhance low sulfur tolerance.The expression of Mh MYB3 was upregulated by low sulfur streatment.The expression of MhSultr3;1a was upregulated in Mh MYB3-overexpressing apple calli under low sulfur conditions,with significantly higher fresh weight,SO₄^2-,Cys and GSH contents than the wild type.The results of electrophoretic mobility,yeast monohybrid and dual luciferase reporter assays indicated that Mh MYB3 could directly bind MhSultr3;1a promoter and activate the expression of MhSultr3;1a,thereby improving low sulfur tolerance in apple calli.5.ABA can alleviate the low sulfur stress of Malus hupehensis.ABA treatment significantly reduced the contents of IAA,JA,ZT and SA under low sulfur conditions,reduced total root length,root tip number and root surface area by 56.61%,37.65%and 57.10%,respectively,and increased the root diameter by 38.22%,which weakened the root growth stimulated by low sulfur.ABA significantly increased root activity and H⁺-ATPase activity,as well as the activity of APR in both roots and leaves.The expression of most sulfur transporters and genes encoding key enzymes of sulfur assimilation were also significantly upregulated.The content of Cys in leaves and roots was significantly increased,and GSH,GSSG and GSH/GSSG were changed.It can be seen that ABA can increase root diameter and promote the absorption and assimilation of sulfur,maintain the oxidative balance in the plant,and thus alleviating low sulfur stress.6.Biochar enhances the effect of KNO₃ on sulfur accumulation and distribution in apple trees.KNO₃and biochar application exhibited synergistic effects on improving sulfur accumulation and root growth.Meanwhile,KNO₃ application increased the activities of ATPS,APR,SAT,OASTL and upregulated the expression of ATPS,APR,Sultr3;1,Sultr2;1,Sultr3;4,and Sultr3;5 in both roots and leaves,and the positive effects of KNO₃addition on both genes and enzyme activity were enhanced by wood biochar.Wood biochar amendment alone promoted the activities of enzymes described above,upregulated the expression of ATPS,APR,Sultr3;1,Sultr2;1,Sultr3;4,and Sultr4;2 in leaves,and enhanced sulfur distribution in roots.KNO₃ addition alone decreased sulfur distribution in roots and increased that in stems.In the presence of wood biochar in soil,KNO₃ application decreased sulfur distribution in roots but increased that in both stems and leaves.