| Drought stress is one of the mainly abiotic stresses affecting maize production.Previous studies have demonstrated that the external signals stimulated by drought stress are amplified and transmitted to the nucleus to activate the transcription factors,and the downstream drought resistance genes are activated to response the drought stress.LBD gene family is a plant specific transcription factor.The class I members play an important role in plant growth and development and biological stress resistance,but the function of class II members is not clear.Therefore,the study of class II members in growth and development and drought stress is conducted to expand the understanding of the function of LBD gene family and enrich the gene resources of drought resistance molecular breeding.Combining with physiological and biochemical experiment,this study investigated the biological functions and regulatory mechanisms of ZmLBD5 and ZmLBD33 under drought stress in ZmLBD5 and ZmLBD33 overexpressed Arabidopsis and maize as well as maize knockout material.The main results can be concluded as follows:1.The basic character of ZmLBD5 and ZmLBD33.The expression of ZmLBD5 and ZmLBD33 were detected in different tissues at different stages and the expression level differed in each tissue.The expression levels of ZmLBD5 and ZmLBD33 increased significantly under PEG6000 and ABA treatment.GA3,JA and low nitrogen also affected the expression levels of ZmLBD5 and ZmLBD33.Given ZmLBD5 and ZmLBD33 proteins had no or incomplete GAS and leucine zipper domains,they can form homologous or heterologous dimers.Subcellular localization in tobacco leaves and maize protoplasts showed that ZmLBD5 and ZmLBD33 were localized in the nucleus,belonging to typical transcription factors.These results suggested that ZmLBD5 and ZmLBD33 responded to drought stress and the proteins played a role in the nucleus.2.ZmLBD5 and ZmLBD33 affected plant development and enhanced drought sensitivity.Overexpression of ZmLBD5 resulted in the larger leaf area,biomass accumulation and promoted growth in Arabidopsis and maize,while the phenotype of lbd5 mutant wascompletely opposite to that of overexpressed maize under normal conditions.Overexpression of ZmLBD33 inhibited the growth of Arabidopsis and maize.Analysis of root traits in hydroponic showed that primary root length,total root length,root surface area and root volume of ZmLBD5 overexpressed maize were significantly bigger than those of wild type,while the root traits of ZmLBD33 overexpressed maize and lbd5 mutant were opposite to those of the ZmLBD5 overexpressed maize.The cotyledons greening rate and survival rate of overexpressing ZmLBD5 and ZmLBD33 were significantly lower than those of wild type under osmotic stress(mannitol)and drought stress.ZmLBD5 and ZmLBD33 may down-regulate the expression of drought tolerant genes At RD20,At RD26,At RD29 A and At RD29 B to response drought stress in Arabidopsis.Compared to the wild type,both ZmLBD5 and ZmLBD33 overexpressing maize displayed severer leaf withering,and this effect was more remarkable in ZmLBD5 overexpressing maize than in ZmLBD33 overexpressing maize.The biomass penalty and survival rate of ZmLBD5 and ZmLBD33 overexpressing maize under drought stress were lower than those of wild type.Importantly,the plant height and the development of cob and tassel were significantly inhibited after drought stress while the ear weight,grain weight and hundred-grain weight obviously decreased.The above results showed that ZmLBD5 promoted the plant growth and development while ZmLBD33 showed the opposite effect.Furthermore,both of ZmLBD5 and ZmLBD33 weakened drought resistance.3.Mechanisms of ZmLBD5 and ZmLBD33 affected plant growth and drought resistance.ZmLBD5 and ZmLBD33 increased stomatal density by up-regulating the expression level of Zm MUTE,Zm SPCH,Zm FAMA and Zm SCRAM,which were positive regulators of stomatal development.In addition,ZmLBD5 and ZmLBD33 reduced the stomatal sensitivity to ABA and H2O2,keeping stomatal opened,resulting in the water loss rate of detached leaves significantly higher than that of wild type.So ZmLBD5 and ZmLBD33 regulated drought stress by increasing stomatal density and aperture.Go and KEGG analysis of differential expressed genes in ZmLBD5 and ZmLBD33 overexpressing maize showed that ZmLBD5 and ZmLBD33 were involved in terpene/terpenoid biosynthesis and phenylpropanoid biosynthesis.A total of 15 DEGsannotated in terpene/terpenoid biosynthesis were mainly involved in the TPS-KSGA2 ox metabolic pathway,which was the key enzyme genes upstream of GA and ABA biosynthesis.Phenylpropanoid biosynthesis was the main pathway of lignin synthesis.Y1 H and double luciferase report assays indicated that ZmLBD5 and ZmLBD33 directly regulated the expression of TPS-KS-GA2 ox module gene and reduced the content of ABA,while ZmLBD5 and ZmLBD33 had slight differences in regulating GA synthesis.ZmLBD5 and ZmLBD33 negatively regulated the content of GA3 and GA4,while ZmLBD5 positively regulated the synthesis of GA1.Exogenous GA1 and GA3 can restore the growth inhibition phenotype of ZmLBD33 overexpressing maize and lbd5 mutant,indicating that ZmLBD5 and ZmLBD33 affected plant growth by regulating the biosynthesis of GAs.4.ZmLBD5 and ZmLBD33 may weaken plant drought resistance by reducing lignin deposition.Y2 H,Bi FC and Co-IP assays showed that ZmLBD33 interacted with Zm EXPB7,which is a cell wall loosen gene.The expression of Zm EXPB7 was induced by PEG6000.The cotyledons greening rate and survival rate of Zm EXPB7 overexpressing Arabidopsis were significantly increased.Zm EXPB7 overexpressing Arabidopsis displayed drought tolerance by increasing H2O2 content,stomata closed,lignin deposition and decreasing detached leaves water loss rate.The survival rate and drought tolerance of expb7 mutant were significantly decreased because of the reduced H2O2 content,the open stomata,the decreased lignin deposition and the increased detached leaves water loss rate.Thus,Zm EXPB7 and ZmLBD33 may regulate stomatal aperture and lignin deposition to response the drought stress. |
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