Study On The Mechanism Of Root Aerenchyma Formation And Waterlogging Tolerance Of Maize

Achieving grain supply security with limited arable land is a major challenge in the twenty first century,owing to the changing climate and increasing global population.Maize(Zea mays)plays an increasingly vital role in global grain production.Maize is currently the second most plentiful crop globally and the largest crop produced in China,as a result of both larger planting area and greater yield.Oxygen deficiency stress,which is induced by abiotic factors such as flooding(waterlogging or submergence)and soil compaction,negatively affects the growth of plants and ultimately their yields,especially for upland crops such as maize.Zea nicaraguensis is a wild relative of Zea mays subsp.mays(maize)that has high waterlogging tolerance.One of its traits is constitutive aerenchyma formation(CA)in roots and this may be one of the reasons for the tolerance,but it has not yet been proven by comparing plants that differ only in CA in the same genetic background.We therefore produced an introgression line AE24-50-44-91(AE91)possessing four quantitative trait loci(QTLs)for CA from Z.nicaraguensis in the background of maize(inbred line Mi29).The AE91 genome has ~14% of the Z.nicaraguensis genome,including the four CA QTLs,and ~86% of the maize genome.In this study,Z.nicaraguensis,maize Mi29 and its introgression line AE91 were used as materials.Measurement items include plant morphology,physiology and root anatomy of observed parameters under waterlogging stress and normal conditions.Therefore,waterlogging tolerance of these three lines was preliminarily evaluated and the physiological mechanism of CA on waterlogging tolerance was studied.Moreover,root cortex cells at different developmental stages will be isolated by laser microdissection(LM)technology for transcriptome analysis,screening key genes controlling the formation of constitutive aerenchyma.In addition,the protocol for LM-based proteomics was studied.Different tissue fixation methods,protein extraction methods and protein identification methods were used to explore the best way for the identification of LM-based proteome.Then,the established protocol for LM-based proteomics was used for comparative proteomics analysis of cortex of maize primary root under waterlogging conditions and under normal conditions.The main results of this study are as follows:1.QTLs for constitutive aerenchyma from Zea nicaraguensis improve tolerance of maize to rootzone oxygen deficiencyZ.nicaraguensis,Mi29 and AE91 were used as materials.The result of anatomical observations of roots showed that the degree of root CA in AE91 was intermediate between that of Mi29 and Z.nicaraguensis,as Z.nicaraguensis > AE91 > Mi29.The results of hydroponic experiments and folding in the field showed that the dry weight,plant height and leaf SPAD were in the order of Z.nicaraguensis > AE91 > Mi29.These results indicate that seedlings of AE91 grown aerobically were more tolerant to transfer to oxygen-deficient conditions than were Mi29 seedlings.On day 2 of oxygen deficiency,the root extension rate and viability of root-tip cells in AE91 were ~2.7 and ~1.3 times greater,respectively,than they were in Mi29.On day 4,the area of aerenchyma at 80 mm from the root tips was ~1.5 times greater in AE91 and radial oxygen loss from the apical parts of roots was ~3.4 times higher than in Mi29.These results demonstrate that CA reduces the stress from low external oxygen levels caused by soil waterlogging.2.Transcriptome analysis of CA in the adventitious roots of Z.nicaraguensis,Mi29 and AE91Under aerobic conditions,CA can be clearly observed in 45 mm from the root tip in the adventitious root of Z.nicaraguensis.In AE91,CA was found to start at 55 mm from the root tip in the adventitious root.However,CA can be barely observed in the adventitious root of Mi29.Thus,the segments of 30 mm and 40 mm of Z.nicaraguensis,40 mm and 50 mm of Mi29 and AE91 from the root tip were collected and used for LM and RNAseq.The result showed that some differentially expressed genes were more highly expressed in the root of Z.nicaraguensis and AE91 than in Mi29,including genes related to cell wall loosening and degradation(e.g.xyloglucan endotransglucosylase/hydrolase),auxin related genes(e.g.Aux/IAA-transcription factor)and some transcription factor(e.g.transcription factor b HLH60).There is a hypothesis that auxin may regulate CA in maize root cortex through the combined action of transcription factors b HLH60 and Aux/IAA-transcription factor 4,and transcription factor NAC may play a negative regulatory role here,which need further study.3.The optimization of the protocol for laser microdissection(LM)-based proteomics analysisMicroproteomics refers to proteomic analysis at the cellular level.In contrast to transcript profiling that can be performed from very small sample amounts due to efficient amplification strategies,there is as yet no amplification procedure for proteins available.In the current study,we compared different tissue preparation techniques prior to LM with respect to their suitability for protein retrieval.Also,we compared different protein extraction methods and protein identification methods for maize cortical cells isolated by LM.Root tissues fixed by formalin(FAA)solution was identified as the best compromise between tissue morphology and effective protein extraction.Liquid tissue MS Protein Pre Kit is used for the isolation of proteins and peptides for formalin fixed tissue in preparation for mass spectrometry analysis.The full protocol includes FAA fixation,slide preparation with 20 μm thickness of slice and 200 mm2 cutting area by LM,using Liquid tissue MS Protein Pre Kit for protein extraction,and high sensitivity instrument in HPLC-MS/MS for protein identification.4.Proteomic analysis(iTRAQ)of cortical cells of maize primary rootsAccurate isolation and sampling of root cortex cells at different development stages,and effective extraction of proteins are the key steps to identify the differential proteins related to aerenchyma formation analyzed through microproteomics.For now,studies related to proteomics on waterlogging stress in maize root cortex have not been carried out yet.In the current study,primary roots of maize B73 under normal and waterlogging conditions were used as materials.Root cortical cells of maize root was isolated by LM.According to protocol established above,we carried out the comparative proteomics analysis of cortex of maize primary root under waterlogging conditions and under normal conditions.The results showed that proteins related to inducible aerenchyma formation(IA)were identified,including cysteine protease and ethylene pathway related proteins,which may involve in the process of programmed cell death during IA.In summary,there are different regulatory pathways and factors involved in the formation of CA and IA in maize root.Among them,ethylene dependent signaling pathway and auxin pathway may participate in the formation of CA in maize roots.PCD initiated by cysteine protease may promote the formation of CA in maize roots.Ethylene signaling pathway is involved in the formation of IA in maize roots under waterlogging stress,while PCD initiated by cysteine proteases may not be the dominant factor in the formation of IA in maize roots under waterlogging stress.In addition,Ca2+ signal related genes / proteins,ROS mediated signal transduction related genes / proteins and very-long-chain fatty acid metabolism may participate in the signal transduction process in the formation of CA and IA in maize roots,affect the changes of enzymes and their coding genes related to cell wall loosening and degradation,lead to cell wall loosening /degradation,and finally form aerenchyma.These results will provide new information for understanding the molecular mechanism of adventitious roots induced CA and IA in maize,and thus the results are expected to be of great significance for the genetic improvement of waterlogging tolerance and the cultivation of tolerance varieties in maize.