Functions Of NOX Gene Family In Wheat And Their Molecular Mechanism Participating In Plant Growth Regulation And Stress Tolerance

NADPH oxidases(NOXs),also known as respiratory burst oxidase homologues(RBOHs)in plants,are key enzymes producing intracellular reactive oxygen species(ROS).NOXs have diverse biological functions in plants,involving in a number of signal transduction processes and have become the new research focus of plant biology.Recent years,more and more NOX homologues have been identified in plants and their biological functions and involved signal pathways have been gradually revealed.However,almost all the results obtained now are just based on the model plants,few field crops have been considered for deep research.As is known to all,increasing yield and improving quality are the foundation of agriculture development and also are the final aim of scientific researches.Wheat(Triticum aestivum)is one of the main food crops all over the world.Therefore,defining the developmental characteristics of wheat and analyzing the mechanism of the plant responding to biotic and abiotic stresses will provide a solid theoretical basis for improving agricultural production.It has been known that the ROS homeostasis is not only important for plant normal development,but also is a commol/Lon mechanism for plants responding to a number of external stresses.However,as the main producers of ROS,the composition and functions of NOXs/RBOHs family members in wheat are still barely reported.In the present study,we firstly identified the NOX family members at the genome-wide level in wheat to systematically understand the member composition,phylogenetic relationships and transcriptional expression profiles of this gene family.Then,we focused on the calarification of molecular mechanism of TaNOX7 and TaNOX10 in wheat development,fertility,and drought tolerance.The main results obtained are as follows:(1)Identification of wheat NOX gene family members and their phylogenetic relationships.A total of 46 NOXs family members including 10 primitive NOXs(TaFROs),4NOX likes(TaNOX-like)and 32 typical NOXs(TaNOXs)were identified at genome-wide in wheat using a seriels of bioinformatics methods.According to the gene and protein structures,phylogenetic relationship,and chromosome localization of the NOX family members,it can be concluded that the wheat NOX family undergoes a complicated evolution history.The TaFROs on Chr 2 might be the more ancient forms of NOXs in wheat.The TaNOXs may be derived from TaFROs by acquiring a NADPH＿Ox domain,while the new members TaNOX-likes may be derived from the typical TaNOX by lossing some gene sequences during the long evolutionary process.(2)Tissue-specific and stimulus-inducible transcriptional expression profiles of wheat NOX family genes and their coexpression networks.Using a series of molecular biology techniques and bioinformatics methods,the tissue-specific and environmental stress-inducible expression profiles as well as the co-expression patterns of NOX gene family members in wheat were investigatied comprehensively and systematically.Meanwhile,the cis-acting elements existing in the promoters of TaNOXs were predicted and mapped.Then,7promoters of TaNOX family genes were selected and cloned for further investigated the transcriptional expression characteristics of TaNOXs genes by performed a dual luciferase reporter assay.The results showed that the expression of wheat NOX genes has obvious tissue and/or stress specificity and sensitivity,revealing their functional diversity and specificity as well as the complexity of transcriptional regulation.The large-scale expression and/or coexpression analysis demonstrated that TaNOXs can be divided into four functional groups with different expression patterns under a broad range of environmental stresses.The four functional groups include Group Ⅰ(TaNOX1/2/6/7/8/12/13/15/TaNOX-like1/4);Group Ⅱ(TaFRO1 and TaNOX4);Group Ⅲ(TaNOX5 and TaFRO3/4);Group Ⅳ(TaNOX3/9/10/11/14 and TaFRO2)。Different TaNOXs are coexpressed with different sets of other genes,which widely participate in several important intracellular processes such as cell wall biosynthesis,defence response,and signal transduction,suggesting their vital but diversity of roles in plant growth regulation and stress responses of wheat.(3)Cloning of TaNOX7 gene and its functional mechanism in wheat development,fertility,and drought stress tolerance.TaNOX7 and its promoter(TaNOX7pro)were successfully cloned,and then a series of heterogenous transgenic lines of Arabidopsis thaliana and rice including overexpression lines(OE),co-surpression lines(CS),complementary lines(CO),were generated,which laid a well genetic foundation for further study of the function and transcriptional regulation of TaNOX7.By employing a series of physiological,biochemical,and immol/Lunohistochemical experiments,we found that TaNOX7 plays important role in regulation of vegetative and reproductive growth of wheat by regulating intracellular ROS homeostasis.Overexpression of TaNOX7 promoted the development of plant seedlings,shortened the growth period,prolonged the reproductive growth cycle,improved the fertility of plants,and enhanced the tolerance of plants to drought stress,indicating the crucial roles of TaNOX7 in the plant development,fertility and stress tolerance.(4)Co-expression and protein interaction of TaNOX7 with TaCDPK2/4/13.In this study,the co-expression profiles between TaNOX7 and three calcium-dependent protein kinases(CDPKs)genes TaCDPK2/4/13 were clarified by q RT-PCR.The results showed that TaNOX7 has well coexpression relationship with TaCDPK2/4/13 in different tissues or organs of wheat.TaCDPK2 is mainly expressed in the leaves of plants at seedling and jointing stages,TaCDPK4 is more in pistil,and TaCDPK13 dominantly in panicles and floral organs,while,TaNOX7 is expressed in whole plant with higher levels in panicles and floral organs.Moreover,both the multiple in vivo protein interaction assays(including LCI、Bi FC、Su Y2H、Co-IP)and histochemistry staining(DAB)verified that TaCDPK2/4/13 can directly interact with TaNOX7 for ROS production.These results suggested that the interaction of TaCDPKs-TaNOX7 might play important roles in wheat development and stress tolerance.The interaction between TaNOX7 and TaCDPK2 may be involved in the vegetative growth of wheat and therefore mainly contributes to the plant stress tolerance such as drought,whereas,the interaction of TaNOX7 with TaCDPK4 may participate in pistil development and therefore contributes to seed development.The interaction of TaNOX7 with TaCDPK13,by contrast,may function in the development of young panicles and floral organs and contributes to the plant fertility of wheat.(5)Cloning of TaNOX10 and its biological function.Firstly,we cloned the fullength c DNA sequence of TaNOX10 gene and its promotor,then a series of transgenic rice and Arabidopsis thaliana lines were created,providing abundant genetic materials for further studies on the functions of this gene.Next,the tissue-specific expression profiles and co-expression relationship of TaNOX10 and several TaCDPKs genes were systematically studied by q RT-PCR,displaying that TaNOX10 has very similar expression pattern with TaCDPK2/17/21,with all the domainant transcripts in the leaves of wheat plants at the seedling,jointing and heading stages.In addition,both LCI and Su Y2 H assays showed that TaNOX10 can directly interact with TaCDPK2,indicating that TaNOX10 may work together with TaCDPK2 to regulate the growth and development of wheat plants.In addition,the transcriptional regulation mode of TaNOX10 was also analyzed by measuring the GUS activity in the TaNOX10pro-GUS genetic transformation lines of Arabidopsis thaliana.The results obtained suggested that TaNOX10 may mainly function in growth and development of plants at seedling stage and jointing stage,as well as in spike development and response to high temperature stress.As a whole,wheat NOX is a multi-gene family with numerous members,complex structure and diverse functions,which not only participates in the response of plants to drought and other adversity,but also involves in the regulation of normal development of wheat.TaNOX7 and other members of NOX play crucial roles in the regulating drought resistance,fertility and other important life processes by interacting with the members of CDPKs(for instance,TaCDPK2/4/13)for regulating the ROS level in plants.However,the functional mechanisms of the TaNOXs in plant growth regulation and environmental stress tolerance still need to further study.The detailed description of the functions and stress tolerance mechanism of the NOX family members in wheat,will provide new target genes for the subsequent improvement of new wheat germplasm by molecular biological technique,which has important biological significances and great potential application values.