| Salvia miltiorrhiza Bunge(called Danshen in China)is a perennial herb of Salvia Linn in Labiatae family.Its dried roots and rhizomes are one of the most common Chinese medicinal materials in China and have important medicinal and economic value.With the continuous development of traditional Chinese medicine industry,the wild resources of S.miltiorrhiza can no longer meet the needs of the society.The planting industry develops rapidly in China,and Shandong province is the most important genuine production area.The yield and quality of S.miltiorrhiza are affected by many factors,among which drought is the most important abiotic stress.In order to elucidate the molecular mechanism of S.miltiorrhiza response to drought stress,this study takes the purple and white flowers of S.miltiorrhiza which originate from Shandong province as experiment material.We combine the application of metabonomics,differential proteomics and protein-modified omics with the investigation of physiological traits to systematically study the physiological and biochemical changes and molecular response mechanism of S.miltiorrhiza seedlings under drought stress,which has important theoretical value and practical significance for selecting and breeding drought-resistant varieties and promoting the sustainable development of S.miltiorrhiza industry.The main results were as follows:1.Physiological and biochemical analysis of S.miltiorrhiza in response to drought stressUnder drought stress,the two varieties of S.miltiorrhiza show the same physiological changes.The relative water content of the leaves will gradually decrease with the increase of the degree of drought,the leaves will become smaller and even turn yellow and wither,at the same time,the roots will accelerate the growth,including increasing the number of lateral roots and root length.With the increase of the degree of drought stress,photosynthetic rate,stomatal conductance and transpiration rate show a trend of decline after rising first,and in moderate drought and severe drought stress has a significant reduction.The content of chlorophyll a and chlorophyll b first rose slightly after declining trend,when under serious drought stress the content of chlorophyll a and b are lower.The concentration of intercellular CO2 decrease firstly and then increase.The activity of SOD,POD,CAT and other antioxidant enzymes in plants increased in the early and middle stages of stress.With the increase of drought,the protective enzymes could not normally remove excessive reactive oxygen species,leading to the accumulation of reactive oxygen species and free radicals,which had a toxic effect on plants and affected the normal growth of S.miltiorrhiza.At the same time,proper drought stress can improve the synthesis and accumulation of many active components in root of S.miltiorrhiza.2.Metabonomics analysis of S.miltiorrhiza in response to drought stressGC-MS metabonomics analysis was used to compare the differentially expression metabolites between control,moderate drought and severe drought stress in leaves and roots of S.miltiorrhiza respectively.149 and 133 different metabolites were identified in leaves and roots most of which belong to the carbohydrates,amino acids and organic acid.In leaves there were 74 different metabolites between the control and severe drought,declined 43 raised 31;63 differential metabolites between the control and moderate drought,among which 17 were down-regulated and 46 were up-regulated.In roots,the number were 58,45,13;56,47,9.The results showed that the metabolites in the leaves and roots of S.miltiorrhiza showed different changes under drought stress.The leaves adapted to drought stress by changing the contents of amino acids and their derivatives,lipid organic acids and carbohydrates,and the contents of most carbohydrates increased significantly.And the drought was significantly decreased in the root of metabolites,including most of carbohydrate content,most major participate in the metabolism of amino acids,starch and sucrose metabolic pathways.In different drought conditions,L-alloisoleucine relative content in leaf and root were significantly increased,and can be used as a key metabolite of S.miltiorrhiza response to drought stress.3.Comparative proteomics analysis of S.miltiorrhiza in response to drought stressThe TMT(Tandem Mass Tag)quantitative proteomics technique was used for comparative proteomics analysis of leaves and roots of S.miltiorrhiza under different drought stress.There were 5689 and 5564 identified quantitable proteins in leaves and roots of which the relative molecular weight was mainly concentrated in 10-70kda,with the highest amount in 30-40kda,and the peptide length ranged from 7 to 16 amino acid residues.There were 309and 1060 differentially expressed proteins under moderate and severe drought stress in leaves,of which 181 and 515 were up-regulated,and 128 and 545 were down-regulated.In roots,the numbers were 355,992;175,454;180,538.The results of subcellular localization showed that the differential proteins were mainly located in the chloroplast(>30%)cytoplasm(>24%)and nucleus(>14%).COG functional analysis found that differential proteins can be classified into 24 classes,mainly distributed in posttranslational modification,protein turnover,chaperones;general function prediction only;carbohydrate transport and metabolism;energy production and conversion;secondary metabolites biosynthesis,transport and catabolism;signal transduction mechanisms and so on.In the GO enrichment results showed that the differential proteins in leaves were mainly enriched in S-adenosylmethionine biosynthetic process and chlorophyll metabolic process;in roots were mainly enriched in isoprenoid biosynthetic process and lipid transport.KEGG analysis showed that the differential proteins in leaves were mainly enriched in glycolysis and glycolytic formation,starch and sucrose metabolism,degradation of valine leucine and isoleucine,protein processing in endoplasmic reticulum,cyan amino acid metabolism and glutathione metabolism.In roots the differential proteins were mainly concentrated in the galactose metabolism,starch and sucrose metabolism,protein processing in endoplasmic reticulum and cyan amino acid metabolism.All of these metabolic pathways are closely related to the drought stress response of S.miltiorrhiza.4.Phosphoproteomics analysis of S.miltiorrhiza in response to drought stressIn order to obtain the phosphorylation information of S.miltiorrhiza in response to drought stress,the total proteins in the leaves and roots treated with different drought treatments were extracted.After enzymatic hydrolysis,TMT marker grading,phosphorylation enrichment and LC-MS/MS mass spectrometry identification were performed.Finally in leaves and roots were identified quantitative 7354 sites on 2904 protein and 6384 sites on2742 proteins.After normalization,respectively for 3569 phosphorylation sites on 1346protein and 3510 phosphorylation sites on 1337 proteins.In GO classification,differential proteins were mainly distributed in biological processes such as metabolic process,cell process and single biological process,as well as molecular functions such as binding activity,catalytic activity and transport activity.Most of them were located in the nuclear,chloroplast and cytoplasm,accounting for more than 75%of the total.To further clarify the metabolic pathways involved in drought response proteins among the comparison groups,the different proteins were enriched by KEGG pathway.The phosphorylated proteins mainly enriched in leaves were significantly down-regulated in photosynthetic and metabolic pathways.Carbon metabolism carbon fixation and energy metabolism related metabolic pathways photosynthetic antenna protein and endoplasmic reticulum protein processing pathways differentially phosphorylated protein expression up-regulated.During root enrichment,the expression of SNARE interactions in vesicular transport,amino acid biosynthesis and carbon metabolism pathways were up-regulated,while the spliceosome and RNA transport pathways were down-regulated.The integration of the two proteome groups revealed that among the proteins that existed in both proteome groups,127 were in the leaves and 131 were in the roots,and the same proteins in the leaves were enriched in the photosynthetic antenna protein and the ether lipid metabolic pathway,and 2 proteins were enriched in each.The same proteins in the root were enriched in the oxidative phosphorylation pathway,and there were three enriched proteins.These proteins may coordinate the metabolic process and physiological and biochemical reactions of S.miltiorrhiza under drought stress through phosphorylation.5.Comprehensive analysis of the mechanism of S.miltiorrhiza in responseto drought stressIn terms of plant morphology,S.miltiorrhiza seedlings could reduce water loss by controlling their growth,including reducing leaf area and abandoning some aged leaves,and at the same time increase root length and number of root strips to enhance the ability to absorb water,so as to ensure the growth point growth and water supply,and to survive in the drought stress environment better.Physiologically,photosynthetic rate and transpiration rate of leaves decreased to different degrees.Under certain extent drought conditions,the activity of protective enzymes in leaves increased and the anti-aging ability of leaves was enhanced.At the metabolic level,drought stress induced changes in metabolites involved in metabolic processes including photosynthesis,TCA cycle,glycolysis/gluconeogenesis,and amino acid metabolism.At the protein level,many functional proteins related to drought tolerance were induced to be expressed,including some proteins involved in carbohydrate metabolism,protein metabolism,redox response and stress response,and some also involved in secondary metabolite anabolism.At the post-translational modification level,some phosphorylated proteins related to the processing and metabolism of photosynthesis,carbon metabolism and energy metabolism,splicose-related proteins were induced to be expressed,which played a role in drought regulation at the phosphorylation level.The adaptation mechanism of S.miltiorrhiza to drought stress is a complex regulatory network and need multiple genes and proteins work together. |
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