Mass Spectrometry Analysis Of Cotton Secondary Metabolites Under The Stress Of Verticillium Dahliae

Cotton is one of the most important economic crops in the world,and verticillium wilt caused by Verticillium dahliae is one of the most serious cotton diseases worldwide.The use of resistant cotton cultivars is regarded as a fundamental component in managing this disease;however lack of the disease resistant source materials has tremendously limited the breeding programs.The traditional source of disease resistant materials includes collection of wild cotton strains,distant hybridization and resistant cultivar acclimation,etc.Unfortunately,these practices seem too inef ficient to meet the needs of breeding programs.With the development of molecular biology,molecular cotton breeding has drawn more and more attention from scientists.Molecular breeding improves cotton resistance against verticillium wilt by introducing exogenous genes or regulating gene expressions.Although important advances have been made in molecular cotton breeding,there remains problems before any related technologies can be applied in cotton production.In order to accelerate the cotton molecular breeding,we need to have more understandings on the interaction between cotton and pathogen,pathogenesis pathway,and resistance mechanisms.Secondary metabolites play an important role in all these aspects.In this study,cotton materials with the same genetic background,but with significant difference in resistance to cotton verticillium wilt were determined by field evaluation in an artificial verticillium wilt nursery.Secondary metabolite profiles of 12 groups of cotton seedling tissue samples were detected by UPLC-Q/TOF-MS,and the difference among treatments and among cultivars were analyzed.Fingerprint metabolites(FPMs)and universal pathogenesis-related metabolites(UPRMs)were determined by linkage analysis.Using data retrieved from the open literatures and databases,a dedicated cotton secondary metabolite database was constructed.An approach for characterizing the detected substrate using LC/MS was proposed.The following are the main results:1.Two cotton cultivars with the same genetic background but with a significant difference in disease resistance against cotton verticillium wilt were obtained(90472-4 and 91079-80)and used for consequent secondary metabolite study.Cotton 90472-4 was a transgenic offspring of cotton 91079-80,which was obtained by introducing chitinase and glucanase genes into cotton 91079-80.SSR marker analysis showed that two materials have the same genetic background.After 2 years of field evaluation in an artificial disease nursery,these 2 cotton cultivars showed a very clear resistant difference against verticillium wilt.The transgenic cotton 90472-4 was significantly more resistant than the parent cotton 91079-80 and its resistant efficacy reached more than 70.0%compared to parent cotton with disease index lower than 60.0.2.UPLC-Q/TOF-MS technology can be used in high-throughout separation and detection of secondary metabolites in cotton tissues at seedling stage.There were 4744 and 3370 detected characteristic ions of secondary metabolites in positive and negative ion modes respectively.The mass ratio(m/z)ranged from 101.022312 to 1484.517108 and from 100.040801 to 1483.382150 respectively.3.A dedicated cotton secondary metabolite database was established.It contained 115 metabolites with data on their molecular weights,including 61 terpenoids,28 phenols,8 lipids,6 nitrogen-containing substances,5 simple organic acids,1 aromatic hydrocarbons and 6 saccharides.4.One hundred and thirty-nine characteristic ions of cotton metabolites were mined as fingerprint metabolites(FPMs),which were potential to distinguish cotton cultivars,including 112 ions under positive ion mode and 27 ions under negative ion mode.These ions showed stable relative quantity between the tested cotton cultivars,which was not affected by treatments,indicating they can be used as potential biomarkers.5.Two hundred twenty-eight characteristic ions of metabolites were mined as universal pathogenesis-related metabolite(UPRMs)including 184 ions under positive ion mode and 44 ions under negative ion mode.All of them were stable in the relative quantity among treatments,which was not affected by cotton cultivars,indicating the usefulness as biomarkers.6.The distribution of FPMs and UPRMs was determined to be tissue-specific,and this may be due to their different physiological functions.Most of the FPMs occurring in cotton roots and stems as UPRMs distributed in roots.However,distribution of the FPMs occurring in cotton leaves as UPRMs was varied.They occurred either in cotton roots or stems alone or in both of these tissues,but they never showed up in the leaf itself.It was presumed that although FPMs could be synthesized in the roots,stems and leaves,they were transported and stored in the root and stem first due to the pathogen infection.Root is the most important tissue in the interaction between cotton andthe pathogen.7.F orty-nine characteristic ions of secondary metabolites were determined in cotton.They included 38 ions detected under positive mode,which were matched to 74 cotton metabolites,including 59 terpenoids,1 lipid,1 organic acid,1 saccharide and 12 phenolic compounds.Eleven characteristic ions detected under negative ion mode were matched to 18 cotton metabolites,including 15 terpenoids,1 lipids and 2 organic acids.These results will not only enrich the data of cotton metabolomics,but also provide a scientific basis for further identification of cotton cultivars with resistance against verticillium wilt.It may help build a theoretical foundation for efficient breeding disease resistant cotton cultivars using molecular regulation.