Proteomics Study Of Guard Cells Of Sugar Beet M14 Strain Under Salt Stres

Salt stress is one of the main abiotic stresses in plants,which seriously affects the growth and production of crops.The sugar beet monosomic addition line M14 which has been obtained previously by the laboratory is a salt-tolerant germplasm with excellent traits,and is an good material for studying the salt-resistance mechanism of plants.Stomata strictly regulate the exchange of gas,water and information between the organisms and the external environment,and play an important role for plants in response to abiotic and biological stresses.In order to better explore the salt-resistant molecular mechanism of the sugar beet M14,the differentially expressed proteins of guard cells from the leaves before and after salt stress were studied at the cellular level.The method of observation and enrichment of guard cells in sugar beet M14 leaves was explored through a large number of pre-experiments.It was determined that the stomatal aperture was observed by tearing the epidermis and fixing it under confocal microscope;the guard cells were rapidly enriched by stirring leaf epidermis combined with 4.2% cellulase R10,0.15% eductase R10 and 0.2% pectinase Y23 in20 min digestion for subsequent proteomics studies.Firstly,the methods that measure of stomatal aperture and enrichment of guard cells in sugar beet M14 leaves were optimized.The method of measuring stomatal aperture was determined as follows: the epidermis was tore,then was fixed for 45 sec with Kano’s fixative solution,which was imaged by confocal microscope to measure stomatal aperture by image J software.The method of enriching guard cells was determined as follows: after the leaf epidermis was obtained by stirring method,4.2% cellulase R10,0.15% librase R10 and 0.2% pectinase Y23 were used to digest 20 min.The purity of enriched guard cells was identified by real-time quantitative fluorescence PCR of mesophyll cell specific expression gene ATPase-1.The stomatal apertures and APX activities of guard cells in sugar beet M14 leaves under 200 mM and 400 mM Na Cl stresses were measured at different time points within 60 minutes to determine the optimum time point for guard cells to respond to salt stress.The results showed that the optimal response times of guard cells to salt stress under 200 mM and 400 mM Na Cl stresses were 10 min and 20 min after salt stress,respectively.Subsequently,iTRAQ technique was used to label the guard cell proteins of sugar beet M14 under different salt concentrations(0,200,400 mM Na Cl),and the quantitative proteomics analysis was performed by LC-MS/MS.Finally,90 differentially expressed proteins were identified under 200 mM Na Cl treatment,46 of which were up-regulated and 44 were down-regulated.75 differentially expressed proteins were identified under 400 mM Na Cl treatment,19 of which were up-regulated and 56 were down-regulated.There were shared six differentially expressed proteins under both 200 mM and 400 mM Na Cl treatments.The functional classification of differentially expressed proteins in guard cells under 200 mM and400 mM Na Cl treatments indicated that these proteins were involved in metabolism,transportation,biosynthesis,cell structure,energy,signal transduction,transcription,stress resistance and defense,and photosynthesis.The transcriptional level of coding genes corresponding to the important differentially expressed proteins under 200 mM and 400 mM Na Cl treatments were further verified by Real-Time PCR.The differentially expressed proteins identified in this study laid a foundation for revealing the salt-resistant mechanism of sugar beet guard cells,and were of great significance to further improve the salt-resistant molecular model of sugar beet M14.