The Study On The Differential Water Stress Responses Of Leaves Of Different Ages And The Role Of Leaf Starch Degradation By ZmBAM8 In Maize Drought Tolerance

With the increasing frequency and duration of drought worldwide,maize growth and yield have been severely affected.As the major organ of photosynthesis,leaves are very sensitive to various abiotic stresses,especially drought.To date,the studies on the stress response mechanism of maize leaves have been carried out by physiological,biochemical or single omics analyses of single leaf or mixed leaf samples.The molecular mechanism of leaf age-related stress response remains unclear,especially in cereal crops such as maize.Recently,our laboratory found that the drought-induced differential genes in the leaves of Chang 7-2drought tolerant mutant（C7-2t）were significantly enriched in the starch metabolism pathway,and the expression level ofβ-amylase ZmBAM8 was highly enhanced.However,it remains unclear whether ZmBAM8 is the key enzyme regulating the degradation of leaf starch（also known as transitory starch）and its role in maize drought tolerance.In this thesis,C7-2t was used to systematically study the mechanism of leaf age-related differential response under water stress by physiology,biochemistry,cytology and multi-omics techniques.Meanwhile,through molecular techniques,enzymatic experiments,especially the knockout and overexpression studies of ZmBAM8,the key enzymes mediating leaf starch degradation and the impact of leaf starch degradation on maize drought tolerance have been demonstrated.The main research findings of this thesis are as follows:（1）Revealing the molecular basis of age-dependent differential stress responses among leaves.After 10-d-old C7-2t seedlings（with 3 visible leaves）were subjected to short-term osmotic stress（0.3 M mannitol,4 h）,fully developed leaves（L1）were more seriously affected by osmotic stress than newly developed leaves（L2）and developing leaves（L3）.Particularly,water loss and the decrease in photosynthetic performance were most significant in L1.Moreover,the changes in transcriptome,proteome and hormones were more drastic in L1 than in L2 and L3.A total of 3195,2673 and 1653 differentially expressed genes（DEGs）were identified,and 1315,633 and 365 DEGs were leaf age-dependent,in L1,L2 and L3,respectively.These DEGs were mainly involved in hormone signal transduction,MAPK signaling pathway,phenylpropane biosynthesis,starch and sucrose metabolism.A total of 143,86 and 58 differentially abundant proteins（DAPs）were identified,and 124,69 and 41 DAPs were associated with leaf age,in L1,L2 and L3,respectively.These DAPs were mainly concentrated in translation,carbohydrate metabolism,amino acid metabolism,energy metabolism and etc.Surprisedly,only one DAP was common to all leaves,i.e.,galactose-sucrose galactosyltransferase,whose accumulation under stress was independent of leaf age.The transcriptome changed more dramatically than the proteome under osmotic stress,and their correlation was poor,especially in L1.Under osmotic stress,leaf ABA levels increased significantly and were positively correlated with leaf age,especially in L1.Under osmotic stress,the hormone composition of leaves was shifted from the dominating growth-related hormones（e.g.auxin and Zeatin）to the dominating stress and aging-related hormones（e.g.ABA）,thus initiating a series of stress responses in a leaf-age dependent way.（2）The early onset of aging-related molecular processes in L1 exacerbates the difference in stress response among leaves under water stress.Under short-term osmotic stress,signaling pathways and metabolic processes related to aging were activated in mature leaves,especially in L1.ABA and ethylene are considered stress hormones or aging hormones.Under osmotic stress,the increased ABA and the upregulated ABA responsive genes were most significant in L1.The number of genes involved in ethylene synthesis and response was the largest in L1,including transcription factor AP2/ERF,ACO（involved in ethylene synthesis）,and ERP（ethylene responsive protein）.Many signaling pathways such as MAPK were activated,leading to a series of aging-like molecular events in L1,especially ubiquitin mediated protein degradation.Many aging-related genes such as SAG12,SAG20,DIN1,sensory regulators,luminal binding protein,and sensory inducible chloroplast stay green proteins（SGR1,SGR2）were highly expressed in L1,leading to premature leaf senescence,whereas the aging-inducible factors in L3 were not activated.Moreover,old leaf and young leaf adopted different strategies to cope with water deficit.Under osmotic stress,old leaf significantly accumulated stress proteins（e.g.dehydrin,aquaporin,and heat shock proteins）,while young leaf exhibited higher proline content and antioxidant enzymes（e.g.CAT,SOD）activities,especially through osmotic adjustment involving transitory starch degradation and proline accumulation.（3）Leaf starch andβ-amylase play important roles in drought stress response.Transmission electron microscopy showed that the chloroplast structure between maize mesophyll and vascular bundle sheath cells differed significantly.Transitory starch mainly existed in the chloroplast of vascular bundle sheath,and also in the stomatal guard cells.Under drought stress,leaf starch in C7-2t significantly decreased under light,soluble sugar content increased,and leaf sheath transitory starch was also degraded,whereas leaf starch in C7-2 did not change significantly.Genome-wide analysis showed that there existed 13β-amylase genes（ZmBAM）in maize,and their promoter regions contained cis-acting elements responsible for abiotic stress（e.g.drought）,hormone（e.g.ABA,auxin）and light signal responses.The expression analysis of ZmBAM revealed that ZmBAM8 was significantly regulated by water stress,ABA and SA.The up-regulated Log₂FC of ZmBAM8 in leaves under osmotic stress was 6.8～8.9,and ZmBAM8 was also significantly up-regulated in leaf sheath.In addition,ZmBAM8 expression was affected by circadian rhythm,which was consistent with the circadian rhythm of leaf starch turnover.These results implied that leaf starch degradation and ZmBAM8 played important roles in drought stress response.（4）The leaf starch degradation mediated by ZmBAM8 under drought stress enhanced maize drought tolerance.In situ hybridization revealed that ZmBAM8 was mainly expressed in the vascular bundle sheath of leaf blade and leaf sheath,consistent with the presence site of transitory starch.Subcellular localization demonstrated that ZmBAM8 was localized in chloroplasts.The recombinant ZmBAM8 protein produced by prokaryotic expression exhibitedβ-amylase activity and was able to degrade leaf starch in vitro.By creating ZmBAM8 knockout and overexpression maize plants,the effects of changes in ZmBAM8 expression levels on transitory starch turnover in vivo and drought tolerance were studied.Under drought stress,the degradation of transitory starch in ZmBAM8 overexpressing plants was almost complete even under light,which was significantly different from the transitory starch degradation that only occurred at night.However,the transitory starch of ZmBAM8 knocked out plants was not degraded under light.After 30 min in the dark,both overexpression and knockout of ZmBAM8 displayed a decrease in leaf starch,indicating that ZmBAM8 does not affect transitory starch degradation normally occurred at night.Importantly,the drought tolerance of the ZmBAM8 overexpressing plants was significantly higher than that of the ZmBAM8 knockout plants.These results provided direct evidence that ZmBAM8-mediated leaf starch degradation enhanced maize drought tolerance.This thesis also showed that ZmBAM8 had no significant effect on plant phenotype and seed traits,but significantly altered the accumulation of starch in root cap.In summary,this thesis systematically revealed the changes of maize leaves of different ages under osmotic stress by using multi-omics approaches,and clarified the molecular basis of age-related differential stress responses.Particularly,the early onset of aging-related molecular events in old leaf exacerbated the differential responses among leaves.The identified DEGs and DAPs here provided new clues for identification of tolerance-related genes and gene function research.By comparison of the performance of C7-2,C7-2t and ZmBAM8 knockout and overexpression plants,it demonstrated that ZmBAM8 was the key enzyme responsible for leaf starch degradation,and ZmBAM8-mediated leaf starch degradation enhanced drought tolerance in maize.The results would contribute to the understanding of the molecular mechanism of drought tolerance in plants,and provide new clues for breeding drought tolerant maize cultivars.