Molecular Cloning And Characterization Of NADPH Oxidase Gene From Zea Mays Seedlings

NADPH oxidases, termed RBOHs (for respiratory burst oxidase homolog) in plants, are enzymes whose biological function is electron transport and the generation of reactive oxygen species (ROS). A wide range of plant responses have been found to be triggered by ROS that are generated in a genetically controlled manner by NADPH oxidases. Several reports show that plant RBOH isoforms have different functions and participate in multiple distinct signaling pathways. In order to obtain more information on the function and regulatory patterns of rbohs, it is necessary to isolate and characterize more rboh genes from different species. During the past ten years, the molecular and biochemical characterization of rboh genes in plants has been studied extensively, but, most of studies are carried out in dicot plants such as Arobidopsis, tobacco and potato. Maize (Zea mays L.) is a model plant and economically important species. The rbohs, however, have not been reported yet. On the other hand, although the phytohormone abscisic acid (ABA) regulates many important aspects of plant growth and development, its main function is to regulate plant adaptive responses to various adverse environmental conditions. ABA can cause the generation of ROS in various plant cells or tissues, and enhance the capacity of antioxidant defense systems, including enzymatic and non-enzymatic constituents. The diphenylene iodonium (DPI) is widely used as an inhibitor of flavoenzymes, particularly NADPH oxidase. DPI-sensitive ROS are an important intermediate component in the ABA-induced antioxidant defense. However, the detailed mechanism about how ABA induces antioxidant defense in plant cells remains to be determined.In the present study, a series of studies based on the materials of maize seedlings have been conducted on the gene isolation, sequence analysis, expression analysis and the further study was carried on the relationship between rbohs and the ABA-induced antioxidant defense. The results as follows:According to the homologous sequences from other plants, degenerate primers were designed to amplify partial sequences of putative Zmrbohs. Then, a combination of an in silico comparative cloning with a PCR-based strategy was developed to obtain new Zmrboh genes. Four novel maize rbohs were identified, designated ZmrbohA, ZmrbohB, ZmrbohC and ZmrbohD. Sequence analysis showed that their deduced amino acid sequences were highly similar to each other and many their structural features were characteristics of plant RBOHs, such as FAD and NADPH binding sites, Ca2+ binding EF-hand motifs, six transmembrane -spanning domains (TMD1-6) and so on. The result of phylogentic analysis of the rboh gene family suggested that ZmrbohB and ZmrbohC were duplicated genes originated from one ancestor. Expression analysis showed that these genes can be detected in all of the organs examined, however, their relative levels in each organ varied.Alternative splicing is an important posttranscriptional regulatory mechanism that can increase protein diversity and affect mRNA stability. In this study, using RT-PCR assay, we identifyied two novel maize rboh splicing variants designated ZmrbohB-βand ZmrbohD-β. Spliced transcript ZmrbohB-βretains an unspliced intron 11 that carries a premature termination codon and probably leads to nonsense-mediated mRNA decay. This variant was coexpressed with ZmrbohB-a in all organs analyzed, but the major product was ZmrbohB-a. In addition, several abiotic stimuli also affected the alternative splicing pattern of ZmrbohB and led to a net increase in the functional isoform, ZmrbohB-α. We also showed here that a splicing variant of ZmrbohD which lacks the six transmembrane-spanning domains, due to alternative splicing in exon 4, was localized to the cytoplasm of cells. Our results suggest that alternative splicing might serve as a regulatory mechanism of ZmRBOH activity.Reactive oxygen species that are generated by NADPH oxidases have been suggested to be a primary event responsible for triggering the cascade of responses in ABA signaling. However, the molecular mechanisms are not well known. Using pharmacological and quantitative real-time PCR approaches, the signaling pathways between hydrogen peroxide (H2O2), MAPK and NADPH oxidese isoforms transcription pattern in ABA signaling were investigated in leaves of maize plants. Treatments with ABA, a biphasic response in the expression of ZmrbohA-D was observed. Pretreatment with MAPK kinase inhibitors (PD98059 and U0126) or reactive oxygen species scavengers (DMTU and CAT) substantially arrested ABA-induced gene expression after 1 h of ABA treatment, but did not affect the transcription within 1 h of ABA treatment. On the other hand, treatment with H2O2(10 mM) also resulted in the activation of the Zmrbohs expression in leaves of maize plants. Meanwhile, pretreatments with two MAPK kinase inhibitors (PD98059 and U0126) also suppressed the H2O2-induced increases in the transcript levels of these Zmrboh genes. These results clearly suggest that Zmrbohs transcription is involved in the ABA signaling, and a cross talk between Zmrbohs transcription, H2O2 production and MAPK activation plays a pivotal role in the ABA signaling.Using eight maize lines as target genotypes, a highly efficient transformation system was developed based on the study of factors influencing the Agrobacterium-mediated maize transformation. The results showed that the immature embryos of 1.0-2.0 mm in length in DH11 maize were optimal transformation explants. We inserted ZmrbohB gene-specific sequences into the binary vector pGSA1285 in the sense and antisense orientations (named pGZMBi) on the principle of RNA interference (RNAi). Using the optimized Agrobacterium-mediated transformation procedure, the expression vector pGZMBi was transformed into hybrid maize line DH11. The result of PCR showed that the targeted segment had been intergrated into the maize genome and the ZmrbohB transcription was partial inhibited. The ABA-induced H2O2 production and the increase in the activities of antioxidant enzymes in leaves of ZmrbohB RNAi variants and untrasformed plants were examined. Compared with untransformed plants, ABA-induced H2O2 accumulation was partial arrested in the leaves of variants. Meanwhile, the ABA-induced activities of antioxidant enzymes were partial blocked in the leaves of variants, but exogenously-applied H2O2 restored the increase in the activities of antioxidant enzymes. These results indicate that ZmrbohB gene is an important component of ABA signaling in the leaves of maize plants.