| Maize(Zea mays L.)is an important food crop in China,and its growth and yield are severely affected by chilling stress,especially in the seedling stage.Various physiological methods have been established and applied to study the effects of chilling stress on the growth and development of maize seedlings.However,the detailed molecular biological mechanism of how chilling stress affects the growth and development of maize seedlings are still unclear,especially in the protein and metabolism levels.Protein in another word,is the ultimate undertaker of life activities,while metabolites is known as the ultimate embodiment of macromolecular functions.The expression level of protein and metabolites displays the best-match consistency to the phenotype of individual plant.Therefore,analyzing the molecular mechanism of maize resistance to chilling stress at seedling stage from protein and metabolic level proffers new insight into cultivating new maize varieties with chilling tolerance by means of genetic improvement,which is of great and deep importance to the development of agriculture.In this study,chilling-tolerant inbred line KR701 and chilling-sensitive inbred line hei8834 screened in the early stage of our laboratory were used as experimental materials to simulate the situation that maize seedlings suffered from chilling stress at 4°C under laboratory conditions.At the same time,two different seedling stages,the first leaf stage and third leaf stage,were added in this study.The changes of endogenous hormone ABA content through comprehensive physiological,comparative proteomics and specific hormonal analyses,trying to identify the similarities and differences of different inbred lines in different stages of seedling growth and development.This study is aiming to explore the key proteins and key pathways in response to chilling stress in maize seedling stage,and to provide valuable resources for analyzing the molecular mechanism of response to chilling stress in maize seedling stage by combining the verification of related biochemical levels and functional verification of related proteins.The main research results are as follows:(1)Chilling stress at different stages of seedling(the first leaf stage and third leaf stage)would cause damage and inhibition to chilling tolerant line KR701 and chilling sensitive line hei8834 in different degrees.The morphology were mainly shown as inhibition of plant height and root length,obvious dwarfism and slow growth of leaves.On the physiological level,malondialdehyde(MDA)content and relative conductivity of membrane system,superoxide dismutase(SOD)and peroxidase(POD)content of antioxidant system were increased,while chlorophyll content(Chl)and chlorophyll fluorescence parameters(F_v/F_m)of photosynthetic system shown the decreasing trend.(2)Under the same chilling stress condition,the damage change patterns of chilling tolerant line KR701 and chillingsensitive line hei8834 were similar at the first leaf stage and third leaf stage with dissimilar change degree,which showed that compared with the change degree of related indexes at the first leaf stage,the related indexes at the third leaf stage showed the same change pattern,but the change degree was obviously smaller,indicating that under the same chilling stress condition,the first leaf stage was more sensitive to chilling stress than the third leaf stage.At the same time,we also found that the difference of phenotypic between chilling tolerant line KR701 and chilling sensitive line hei8834 were obvious at the first leaf stage and all the indexes were different,but the difference of phenotypic between them became obviously smaller at the third leaf stage.(3)Quantitative proteomics analysis of chilling stress showed that there were 394 proteins responding to chilling stress in chilling tolerant line KR701 and 404 proteins responding to chilling sensitive line hei8834,respectively.At the third leaf stage,the number of responsive proteins decreased significantly,and the number of responsive proteins in chilling tolerant line KR701 and chilling sensitive line hei8834 were 132 and 46,respectively.The functional enrichment analysis of differentially expressed proteins and specific expressed proteins classified by WGCNA showed that at the first leaf stage,214 CRPs were specifically linked to the chilling response in KR701 lines,mainly functioning in photosynthesis and various hormone-mediated signalling pathways(especially the ABA signalling pathway),and a small number of them were related to cell wall organization and catabolic pathways.225 CRPs specifically responding in hei8834 were mainly focused on cell wall organization,biogenesis and catabolic processes,ATP biosynthesis-related processes and hormone(only auxin)transport pathways.At the third leaf stage,the biological processes of most CRPs in KR701were defence response and cell wall organization&metabolic pathways,which is similar to the scenario of hei8834-specific CRPs at the first leaf stage.In contrast,the CRPs were reduced significantly,and only 35 CRPs were specific to hei8834 at the third leaf stage compared to those at the first leaf stage,and these CRPs were involved in the photosynthesis pathway.(4)Metabonomic analysis under chilling stress showed that there were 17 different metabolites in chilling tolerant line KR701 and 25 different metabolites in chilling sensitive line hei8834 under chilling stress at the first leaf stage,respectively.At the third leaf stage,there were 15 different metabolites in chilling tolerant line KR701 and 22 in chilling sensitive line hei8834,respectively.Meanwhile,metabonomic found that the endogenous ABA content of chilling tolerant line KR701 at the first leaf stage increased 32-fold after chilling stress.(5)At the first leaf stage,moderate exogenous ABA pre-treatment significantly increased the plant height and root length of chilling sensitive line hei8834,the change of relative conductivity decreased significantly.For chilling tolerant line KR701,moderate exogenous ABA pre-treatment only slightly increased the root length,but had no effect on plant height and relative conductivity.At the third leaf stage,Appropriate exogenous ABA pre-treatment only slightly increased the plant height of chilling sensitive line hei8834.For chilling tolerant line KR701,appropriate exogenous ABA pre-treatment had no effects on the changes of plant height,root length and relative conductivity.The results showed that appropriate exogenous ABA pre-treatment only affected the first leaf stage.At the same time,it is important that exogenous ABA pre-treatment has an obvious effect on the chilling sensitive line hei8834 at the first leaf stage,which significantly improves the chilling resistant ability of the chilling sensitive line hei8834,and has almost no effect on the chilling tolerant line KR701.(6)The key transcription factor ZmOCP3 was found in the hormone signaling pathway of chilling tolerant line KR701 at the first leaf stage,and subcellular localization analysis showed that ZmOCP3 was located in the nucleus.Meanwhile,through the analysis of ZmOCP3expression pattern in maize and Arabidopsis thaliana,it was found that the expression of ZmOCP3 was significantly induced by chilling stress.Phenotypic analysis showed that under chilling stress,compared with wild-type Col4,ZmOCP3 overexpressing lines had significantly enhanced chilling resistance,and ZmOCP3 overexpressing lines were ABA-insensitive phenotype compared with wild-type Col4.(7)IP-MS analysis found that 1,010 non-specific binding proteins were found in ACGFP,and 1,674 potential interacting proteins were found in ACGFP-ZmOCP3.We removed the non-specific interacting proteins and obtained 805 potential ZmOCP3 interacting proteins. |
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