Identification Of The Heat Response Cis-acting Elements Of The Zmgols2Gene Promoter

Maize (Zea mays) is the premier food crop in China; it is also the highest yielding graincrop in the world. Maize, like other crops, is a victim of many environmental stresses, such asdrought, heat stress, cold stress and salinity, which can affect it at any growth stage. Theseabiotic stresses drastically reduce corn yield and quality. For the past few years, due to globalwarming, high temperature has become the major abiotic constraint affecting maize yield andquality. The key way to solve this problem is by breeding new corn varieties resistant to hightemperature stress. Raffinose is ubiquitous in the plant kingdom and can function as anosmoprotectant; it can protect plants from abiotic stress by keeping the cell membrane in itsnormal physiological state under high temperature or dehydration stress. Raffinosepresumably plays an important role in cold-, heat-, and/or desiccation-tolerance in plants.Regulating the raffinose biosynthetic pathway may improve maize stress resistance andconstitute a new breeding strategy. Maize GALACTINOL SYNTHASE (GolS) is a keyenzyme in the raffinose biosynthetic pathway. We have previously characterized the maizeGolS2(ZmGolS2) gene as heat shock induced in maize germinating seeds and cultured cells.In the present study, a1.5kb ZmGolS2promoter fragment was initially cloned after analysisof its feature motifs using bioinformatics software. Two putative heat shock responsiveelements (HSEs) were predicted in the5’regulatory region of ZmGolS2. Vectors wereconstructed containing a series of ZmGolS2promoter fragment gradually shortened from the5’end of the1.5kb promoter. Transformation of these constructs into maize callus protoplastusing PEG permeation permitted the measurement of these promoter activities after usinghigh temperature to stress the protoplasts. Simultaneously, the ZmGolS2gene5’UTR in theseconstructs was replaced with the5’UTR of the human beta-globin (Hsbeta-globin) gene. Thisstrategy was used to explicitly examine the relationship between the5’UTR and ZmGolS2gene expression during the heat stress response. Finally, the putative HSE were used to assessmaize nuclear extracts from heat shock stressed or unstressed cells for proteins with affinity tothese motifs. The main results are as follows:We have designed a dual luciferase reporter vector for testing the promoter activity ofDNA fragments in vivo by using the experimental promoter to drive Renilla (Renillareniformis) luciferase (Rluc) and the reference promoter (2×35SCaMV) to drive expression offirefly (Photinus ssp)(Fluc) luciferase. Since experimental or control expression cassettes containing both Rluc and Fluc, both driven by2×35S promoter are present in the same vector.The two expression cassettes are transformed into the cell in the same quantity and DNAquality. Using this method can reduce the experiment error.The1.5kb ZmGolS2promoter fragment and its deletions were tested for promoteractivity by their regulation of the Rluc reporter gene expression in maize protoplasts culturedat either25℃or42℃for24h. The activity of constitutively expressed Fluc gene in thesame vector was used as a reference. One heat shock element (HSE) was identified bycomparing the promoter activity of each fragment under normal and heat shock conditions.Deletion or triplication of this HSE motif abolished or enhanced the heat shock response ofthe ZmGolS2promoter, respectively.When the ZmGolS25’UTR was replaced by Hsbeta-globin5’UTR, using real-time PCR,it was found that the ZmGolS25’UTR did not interfere with the up-regulation of transcriptionby heat stress, but decreased the translational efficiency of this gene under these heat shockconditions.This HSE motif is specifically bound by proteins in the nuclear extracts of heat shockstressed, but not unstressed maize cells as confirmed by DNA-EMSA.