| Nostoc flagelliforme is a terrestrial nitrogen-fixing cyanobacterium of the Nostoc in Nostocaceae family,which is distributed in arid and semi-arid steppe of China,and it has important ecological,edible,and medicinal values.N.flagelliforme has evolved a unique drought-resistant mechanism and has a strong drought tolerance because its growing environment often suffers from periodic rehydration and dehydration.Protein malonylation is a widespread and highly conserved post-translational modification,which regulates protein functions in photosynthesis,carbon metabolism,amino acid metabolism,lipid metabolism and other pathways,thus affecting physiological metabolic activities of plants.Metabolites are the final products of cell regulation,which can reflect the changes of metabolic response of plants under adversity stress.In order to explore the molecular mechanism of drought tolerance of N.flagelliforme in response to drought stress,N.flagelliforme with sufficient water absorption was used as the control(MA)and water loss 30%(MB),75%(MC)and 100%(MD)of N.flagelliforme as the drought stress treatment groups.The protein malonylation modification of N.flagelliforme was studied by proteomics method.The characteristics of malonylation and its response mechanisms to drought stress were analyzed by subcellular localization,functional enrichment of differential malonylated proteins,metabolic pathway,and malonylation function verification,combined with the determination results of related enzyme activities and metabolites content in malonylated proteins involved in metabolic pathway.At the same time,metabolomics technology was carried out to identify the metabolic profile of N.flagelliforme under different drought stress,the types of metabolites and their expression patterns were analyzed,and a data analysis model was constructed to obtain differential metabolites.Correlation analysis,cluster analysis,functional enrichment analysis and metabolic pathway analysis of differential metabolites were conducted to reveal the metabolic regulation mechanisms of N.flagelliforme in response to drought stress.In addition,the lipid profile of N.flagelliforme under drought stress was identified,the biomarkers of drought tolerance were screened,and the effects of the abundance changes of different lipid types on the membrane lipids was analyzed to determine the relationship between lipid metabolism and N.flagelliforme response to drought stress.In this study,the mechanism of response to drought stress was revealed on the level of malonylation modification of proteins and metabolism.The results not only laid a theoretical foundation for comprehensively analyzing the drought tolerance mechanism of N.flagelliforme,but also enriched the research on drought tolerance of lower plants,and played a positive role in promoting the protection,development and utilization of special plant resources in arid and semiarid regions.The main results of this study are as follows:1.Malonylated modification of proteins in N.flagelliforme in response to drought stressIn this study,421 malonylated lysine modification sites were found in 236 malonylated proteins.Most of the malonylated proteins(38%)were distributed in cytoplasm and 14%in plasma membrane.GO and KEGG enrichment analysis indicated that malonylated proteins were highly enriched in metabolic pathways,such as carbon metabolism and photosynthesis.In addition,88 differential malonylated proteins in N.flagelliforme had orthologs in other species,accounting for 72.95%of all differential malonylated proteins.The decreased malonylation modification level of N.flagelliforme under drought stress may inhibit the reception and transfer of light energy,reduce the fixation of CO2 in Calvin cycle,and thus reduce photosynthesis.Malonylation modification may respond to drought stress by inhibiting the carbon flux in TCA cycle and activating the gluconeogenetic pathway.Furthermore,malonylated antioxidant enzymes of N.flagelliforme participate in early antioxidant defense under drought stress.Malonylated proteins are also involved in lipid degradation and amino acid biosynthesis in response to drought stress.According to multi-sequence alignment analysis,it was found that the K306 modification site of fructose diphosphate aldolase(FBA,EC4.1.2.13),the K237 site of isocitrate dehydrogenase(IDH,EC 1.1.1.42)and the K86 site of nucleoside diphosphate kinase(NDK,EC2.7.4.6)were highly conserved in cyanobacteria.Further,a mutant without a malonyl modification group was produced by site-specific mutation.It was found that malonylation could affect the activities of FBA,IDH and NDK.2.Metabolome analysis of N.flagelliforme in response to drought stressDynamic changes of metabolites in N.flagelliforme under drought stress were analyzed by nontargeted metabolomics technology.A total of 92 differential metabolites were obtained,most of which belonged to carbohydrates,amino acids,nucleotides and their derivatives and lipids.Compared with the control,A total of 45 different metabolites were identified at 30%water loss of N.flagelliforme,including 35 up-regulated and 10 down-regulated.58 differential metabolites were identified at 75%water loss,including 46 up-regulated and 12 down-regulated.69 differential metabolites were identified at 100%water loss,of which 25 were up-regulated and 44 down-regulated.Correlation and cluster analysis of differential metabolites of N.flagelliforme showed that the change trend of carbohydrate content was consistent with that of amino acid metabolites,while the change trend of both was opposite to that of lipid.The content of metabolites was significantly different in response to drought stress through their synthesis and decomposition during the drought process.KEGG enrichment analysis showed that 18 metabolic pathways were significantly affected by drought stress,including galactose metabolism,starch and sucrose metabolism,alanine,aspartic acid and glutamic acid metabolism,unsaturated fatty acid biosynthesis,fatty acid biosynthesis,pyrimidine metabolism,and purine metabolism.The metabolic network of N.flagelliforme in response to drought stress was established based on metabolite changes.Drought stress mainly changed the carbohydrate metabolism,amino acid metabolism and lipid metabolism of N.flagelliforme,and the metabolites more conducive to drought resistance were synthesized through substrate redistribution.13 genes involved in carbohydrate metabolism and amino acid metabolism of N.flagelliforme under drought stress were analyzed by qRT-PCR.It was found that there was no clear correlation between the changes in transcription and metabolites.3.Lipidome analysis of N.flagelliforme in response to drought stressNon-targeted lipidomics technology was used to identify and analyze the lipid molecule species of N.flagelliforme under different degrees of drought stress.A total of 853 lipid molecules were identified,representing 37 subclasses,and 6 main categories.The main lipid subclasses in N.flagelliforme include 168 triglyceride(TG),100 digalactosyldiacylglycerol(DGDG)and 93 ceramide(Cer).In addition,the number of lipid molecules of diglyceride(DG),monogalactosyldiacylglycerol(MGDG),sulfoquinovosyldiacylglycerol(SQDG),phosphatidylcholine(PC),phosphatidylethanolamine(PE),phosphatidylglycerol(PG)and sitosterol ester(SiE)was also relatively high in the N.flagelliforme.Moreover,DG(29:1),SQDG(17:5/23:2),SQMG(16:0),MGMG(16:0)and MGDG(16:1/18:3)can be used as biomarkers for N.flagelliforme to respond to drought stress.The comparative analysis of differential lipid species in N.flagelliforme showed that there were 64(31 up-regulated,33 downregulated),94(69 up-regulated,25 down-regulated)and 98(75 up-regulated,23 down-regulated)differential lipid molecules between the control sample with water loss 30%,75%and 100%samples,respectively.The amount of glycerolphospholipids decreased accompanied by the increase of lysophospholipids.The membrane lipid composition,lipid acyl unsaturation and membrane fluidity were adjusted by increasing the ratio of DGDG/MGDG of N.flagelliforme in response to drought stress.The enzyme activity in the biosynthesis pathway of DGDG was significantly up-regulated,which contributed to the accumulation of DGDG and enhanced drought resistance.The photosynthesis of N.flagelliforme decreased under drought stress,and the stability of membrane was enhanced by increasing the content of unsaturated fatty acids,thus maintaining the integrity of thylakoid membrane structure.The accumulation of antioxidative lipid Co(Q10)was accumulated significantly under drought stress,which beneficial to reduce the lipid peroxidation of N.flagelliforme,and produce a large amount of energy through lipid degradation,and allocate substrate rationally in response to drought stress.This study also found that increasing the level of odd-numbered fatty acids(OCFA)was also an important strategy for N.flagelliforme response to drought stress.4.Multi-omics joint analysis of N.flagelliforme in response to drought stressThe joint analysis of malonylome and metabolome showed that there were 22 common annotated metabolic pathways between differential malonylated proteins and differential metabolites.Among them,the most annotated differential modified proteins and differential metabolites were ABC transporters pathway.Enrichment analysis of KEGG pathway showed that purine metabolism and alanine,aspartic acid and glutamic acid metabolism were highly active in the malonylome and metabolome,indicating that the response of N.flagelliforme to drought stress in the level of protein malonylation and metabolite changes has common characteristics.The joint analysis of malonylome,metabolome and lipidome showed that there were complex,regulatory relationships among differential malonylated proteins,differential metabolites and differential lipid molecules of N.flagelliforme under drought stress,and they participated in the regulation of photosynthesis,osmotic adjustment and antioxidant defense of N.flagelliforme from different dimensions to respond to drought stress.The above results preliminarily revealed the physiological and molecular regulating mechanism of N.flagelliforme under different drought stress.The response mechanism of N.flagelliforme to drought stress is a complex process,which plays a synergistic role through multi-dimensional regulation.The results of the study are helpful to clarify the drought tolerance and evolution mechanism of terrestrial cyanobacteria and provide a reference for study of drought tolerance mechanism in other plants,especially lower plants.At the same time,it has a positive role in promoting the protection,development and utilization of special plant resources,innovation of drought tolerant germplasm resources,and ecological protection in arid and semi-arid areas. |
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