ZmDREB1A And ZmDREB2A Regulate The Balance Between Plant Growth And Abiotic Stress Tolerance In Maize

Crop yield is closely associated with plant growth and plant stress tolerance.Understanding the relationship between plant growth and abiotic stress tolerance is important for improving crop yield.DREB1A(DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN 1A)and DREB2A(DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN 2A)are two important transcription factors in response to plant abiotic stresses.Plants over-expressing DREB1 A or DREB2 A enhenced their abiotic stress tolerance,however,the transgenic plants exhibited seriously growth retardation.The molecular mechanism of how DREB1 A and DREB2 A balance plant growth and abiotic stress tolerance remains unclear.The function and expression regulation of ZmDREB1 A and ZmDREB2 A in balancing plant growth and abiotic stress were investigated in this thesis.The results are listed as follows:ZmDREB2 A regulated the balance between IAA stimulated seedling growth and raffinose mediated seed aging tolerance in maize.The unaged seeds of two independent maize mutator(Mu)-interrupted ZmDREB2A(zmdreb2a)lines,with decreased expression of GRETCHEN HAGEN3.2(Zm GH3.2,encoding indole-3-acetic acid(IAA)deactivating enzyme),increased IAA in embryos,and exhibited faster seedling growth than the null segregant(NS)controls.However,the zmdreb2 a seeds,also had a decreased expression of RAFFINOSE SYNTHASE(Zm RAFS)and less raffinose in their embryos,resulting in decreased seed aging tolerance,relative to the NS controls.Overexpression of ZmDREB2 A in maize protoplasts increased the expression of Zm GH3.2,Zm RAFS,and the Rennila LUCIFERASE reporter(Rluc)gene controlled by either the Zm GH3.2-or Zm RAFS-promoter.Electrophoretic mobility shift assays and Ch IP q-PCR showed that ZmDREB2 A directly binds to the DRE motif of the promoters of both Zm GH3.2 and Zm RAFS.Exogenous supplementation of IAA to the unaged,germinating NS seeds increased subsequent seedling growth making them similar to the zmdreb2 a seedlings from unaged seeds.There were one predicted DRE motif and two predicted Aux RE motifs(Aux RE1 and Aux RE2)in Zm GH3.2 promoter.The Aux RE2 motif was partially overlapped with the DRE motif.To identify which ARFs that regulate the expression of Zm GH3.2,Zm ARF7,Zm ARF13,Zm ARF14,Zm ARF16 and Zm ARF39 were cloned and characterized in maize protoplasts.Overexpression of Zm ARF7 decreased the expression of Zm GH3.2,while overexpression of Zm ARF13,Zm ARF14,Zm ARF16 or Zm ARF39 up-regulated the expression of Zm GH3.2.In addition,Zm ARF7,Zm ARF13,Zm ARF14,Zm ARF16 or Zm ARF39 was supressed the effects of ZmDREB2 A regulation of Zm GH3.2 expression.Compared with their NS controls,zmdreb2 a mutants were more sensitive to salt stress.ZmDREB2 A was bound to the DRE motif of the Zm RAFS promoter and up-regulated the expression of Zm RAFS.zmrafs mutants were also more sensitive to salt stress compared to their NS.These data suggest that ZmDREB2 A regulate plant salt stress tolerance through regulation of Zm RAFS expression and raffinose biosynthesis.Overexpression of ZmDREB1 A in maize leaf protoplasts increased ZmDREB1 A amounts which consequently up-regulated the expression of Zm RAFS and the Renilla luciferase(Rluc)which was controlled by the Zm RAFS promoter.Deletion of the single drought responsive element(DRE)in the Zm RAFS promoter abolished ZmDREB1A's influence on Rluc expression while addition of three copies of the DRE in the Zm RAFS promoter dramatically increased Rluc expression when ZmDREB1 A was simultaneously over-expressing.Electrophoretic mobility shift assays and CHIP-q PCR demonstrated that ZmDREB1 A directly binds to the DRE motif in the promoter of Zm RAFS both in vitro and in vivo.Characterization of the maize zmdreb1 a mutant showed that Zm RAFS expression and raffinose content were significantly decreased compared with its null segregant(NS)control under chilling stress.These data demonstrate that Zm RAFS,which was directly regulated by ZmDREB1 A,enhances both raffinose biosynthesis and plant chilling stress tolerance.Two independent zmrafs mutant lines,in which raffinose was completely abolished,were more sensitive to chilling stress and their net photosynthetic product(total soluble sugars and starch)accumulation were significantly decreased compared with their NS controls after chilling stress.The crosstalk between ZmDREB1 A,ZmDREB2 A and IAA signal pathways was investiaged.The genes that are involved in IAA degradation(Zm GH3 s or ZmDAO)and IAA signaling inhibitor(Aux/IAA)were upregulated by ZmDREB1 A,ZmDREB2 A or both.In addition,the genes involved in raffinose synthesis were also regulated by ZmDREB1 A or ZmDREB2 A.IAA(auxin)was also upregulated the genes whose products are involved in IAA dedgradation and IAA signaling transduction.In summary,ZmDREB1 A and ZmDREB2 A regulate the balance between plant growth and abiotic stress tolerance by regulating IAA signaling pathways and raffinose metabolism.These data not only help people to understand the molecular mechanisms about the plant growth and abiotic stress tolerance,but also provide useful information for scientists,or alarming those scientists who are aiming to generate plant abiotic resistant crops using transcription factors DREB1 A or DREB2 A.