Phosphoproteomic Analysis Of Tamba Black Soybean Under Aluminum Stress And Molecular Mechanisms Of GmMYB183 In Aluminum Tolerance

Approximately 40%～50%arable land in the word is acidic soil,which severely limits the yeileds of global food crops and affects the healthy development of world agriculture.Aluminum stress is one of the major constraints restricting the normal growth of plants in acidic soil.When the soil p H is less than 5.0,the aluminum in the soil mainly in the form of Al³⁺and Al（OH）²⁺,of which Al³⁺is extremely harmful to plants.The inhibition of root elongation is the earliest and easiest phenotype to be observed when plants suffered from aluminum toxicity,which further affects the absorption of water and nutrients,sequentially inhibiting the growth of the whole plant by damaging plant cell nuclei,reducing cell mitotic activity,affecting enzyme activity,and destroying ion channels.Al³⁺acts as a signal molecule to stimulate the signal transmission in the plant,activate the expression of related genes,and initiate a variety of physiological mechanisms to deal with aluminum poisoning.The signal transduction of aluminum signal in plants depends on protein phosphorylation.Protein phosphorylation modification is especially crucial in post-translational modification,which serves as participating in the whole life of plants and playing an essential role in response to abiotic stress.The secretion of citric acid under aluminum stress,regulated by protein phosphorylation,is one of its significant mechanisms to alleviate aluminum toxicity in soybean.Over the past decade,the research on the responsiveness of plants to aluminum stress had mostly focused on the transcription level and protein level.Using a combinationoftandemmasstag（TMT）labeling,immobilized metal affinity chromatography（IMAC）enrichment and liquid chromatography-tandem mass spectrometry（LC-MS/MS）,Al³⁺-induced phosphoproteomic changes in roots of Tamba black soybean（TBS）were investigated in this study.GmMYB183,an important transcription factor from the phosphoproteomic,was taken for in-depth study.Using plant transgenic technology and molecular biology methods,to clarify the GmMYB183 transcription factor in Tamba black soybean from the phenotypic,physiological,biochemical and molecular levels.The mechanism of regulating the secretion of citric acid in aluminum tolerance provides a scientific basis for the use of genetic engineering technology to improve the aluminum resistance of plants.The main findings were as follows:（1）Differentially phosphorylated proteins（DPPs）regulating citric acid anabolism and secretion under aluminum stressUnder aluminum stress,3245 sites on 2697 proteins with quantitative information were detected,of which 189 proteins were significantly changed（fold change>1.2 or<0.83,p<0.05）,with 88 upregulated,96 downregulated and 5 up-/downregulated,among which 22 differentially phosphorylated proteins were involved in the signal cascade,20 in transcription,translation and post-translational modification,10 in carbohydrate metabolism,and 30 in DNA and RNA processing and modification,15 in the cytoskeleton,37 in other function,8 transporters and 39 unknown functional proteins.Enrichment functional analyses revealed that DPPs were associated with citric acid synthesis and secretion under the Al³⁺treatment.A transcription factor GmMYB183,which was selected from the phosphorylated proteomic data for the largest phosphorylation diversity（hyperphosphorylated by 1.377 fold at Ser36）,was taken for in-depth study.（2）GmMYB183,in response to aluminum stress by post-translational protein phosphorylation modification as a transcriptional activatorGmMYB183,cloned from Tamba black soybean,encodes a protein which belongs to the typical R1-MYB type located in the nucleus.Yeast two-hybrid test results showed that GmMYB183 is a transcriptional activator.The overexpression vector of GmMYB183 and its Ser36 mutation GmMYB183-S36A was constructed,and was transformed into Arabidopsis and soybean hairy roots.Under aluminum stress,the relative root growth and the citric acid secretion of Arabidopsis overexpressed GmMYB183 were significantly higher than those of wild type,while the relative root growth and the citric acid secretion of Arabidopsis overexpressed GmMYB183-S36A were significantly lower than those of wild type.Hematoxylin staining results showed that root tips of Arabidopsis overexpressed GmMYB183 were less stained than wild type Arabidopsis,while the root tips of Arabidopsis overexpressed GmMYB183-S36A were more stained heavily than wild type.The citric acid secretion of soybean hairy root overexpressed GmMYB183 and hematoxylin staining results were consistent with those of Arabidopsis overexpressed GmMYB183.The results indicated that GmMYB183responded to aluminum stress due to its main dependence on its Ser36 phosphorylation.（3）The GmMYB183 transcription factor regulating the secretion of citric acid by directly modulating the expression of Gm MATE75Our previous study found that the expression of citric acid transporter gene Gm MATE75 was up-regulated by more than 200 times under aluminum stress in Tamba black soybean,which promoted the secretion of citric acid to alleviate aluminum toxicity.In this study,the secretion of citric acid in soybean hairy roots of overexpressed GmMYB183 and the relative expression of Gm MATE75 were both significantly higher than the control under aluminum stress.Transient dual-LUC analysis was showing GmMYB183 can activate Gm MATE75 promoter in N.benthamiana leaves,while can’t activate Gm MATE75 promoter with single mutation or double mutations.Meanwhile,GmMYB183-S36A can’t activate Gm MATE75 promoter.The results of dual luciferase reporter system and yeast one-hybrid experiments show that GmMYB183 directly binds to the promoter of Gm MATE75 to regulate the expression of Gm MATE75,and it depends on the phosphorylation of Ser36.In addition,GmMYB183 regulates Gm MATE75 and requires simultaneous binding to the two binding sites.（4）The phosphorylation of Ser36 of GmMYB183 transcription factor regulating the growth and development of Tamba black soybean.Under normal growth condition,the root elongation of GmMYB183 and GmMYB183-S36A transgenic Arabidopsis was significantly lower than that of the wild type,and the root elongation of GmMYB183-S36A transgenic Arabidopsis was significantly lower than that of GmMYB183 transgenic Arabidopsis.Similar results were also found in soybean hairy roots overexpressed GmMYB183 or GmMYB183-S36A.The auxin content of the GmMYB183-S36A transgenic hairy roots was significantly higher than that of the control.The relative expression of auxin synthesis and transport-related genes of Arabidopsis and soybean hairy roots overexpressed GmMYB183-S36A of were significantly different from those of Arabidopsis and soybean hairy roots overexpressed GmMYB183.It is speculated that the phosphorylation of GmMYB183 plays an vital role in regulating root growth.In conclusion,protein phosphorylation plays a vital role in regulating the anabolism and the secretion of citric acid in Tambo black soybean in response to aluminum stress.The transcription factor GmMYB183 responds to aluminum stress and regulates the root growth by depending on phosphorylation at Ser36.