Transcriptomic Analysis Of Wheat Guard Cell Response To Abscisic Acid

Stomata is surrounded by guard cells located on the surface of the plant,control water loss and gas exchange,and closely relate with plant biological processes such as stress response and photosynthesis.Kidney-shaped guard cells were often used for stomatal biological function research,while guard cells of gramineous crops are dumbbell-shaped and function distinguishingly from kidney-shaped guard cells.To clarify the signal transduction mechanism,dumbbell-shaped guard cells need to be isolated though this is extremely difficult considering the dumbbell-shaped guard cells have similar size and morphological features to the adjacent subsidiary cells.Therefore,the related research is currently stagnant.In this study,we isolated the wheat guard cells with high purity and physiological activity by enzymatic hydrolysis.A lot of genes related to ABA signal response in wheat guard cells were explored by transcriptomic analysis,which laid a good foundation for analyzing the mechanism of wheat stomata.The main results list as follows.Microscopic manipulation and enzymatic hydrolysis had been conducted to isolate wheat guard cells.The results show that the micromanipulation method is not suitable for the isolation of wheat guard cells due to complicated operatation,greatly reduced cell activity,and time consuming.The different types of cell protoplasts could be detached by enzymatic hydrolysis successively from wheat epidermis.The protoplasts of long cells and subsidiary cells released quickly,and the protoplasts of guard cells and trichomes were difficult to be free.The guard cells with high purity and activity were successfully isolated from the epidermis of wheat special organs without trichomes,and collected for the follow-up study.Enzymatic hydrolysis of the cells will cause stress responses mainly by high temperature and injury,leading to transcriptomic changes.Therefore,we applied transcription inhibitors to the reaction system before the enzyme reaction in order to reduce the adverse effects of stress and inhibit stress-induced transcriptional changes.The results showed that the transcription-related metabolic pathways were inhibited after transcription inhibitor treatment,and the expressions of glutathione-related genesand heat stress-related genes were significantly down-regulated.These results indicated that the transcription inhibitors can significantly reduce the expression of stress genes during guard cell isolation,and improve the data quality of the guard cells transcriptome.Using the optimized separation procedure,the ABA-treated wheat guard cells were separated for RNA extraction,amplification and next-gereration sequencing.Then the transcriptomic data were obtained and analyzed by bioinformatic analysis.The results revealed that 11415 genes were up-regulated and 7938 genes were down-regulated.GO enrichment and KEGG analysis of differentially expressed genes showed that many stress-related genes were up-regulated.The expression patterns of ABA signaling pathway gene were similar to those in Arabidopsis.In this study,a large number of key candidate genes were identified,and the gene function identification would be carried out based on this study.Hence,this study will further deepen our understanding of the mechanism of wheat stomatal functions.