The Phylogenetic Development Of NOX Gene Family In Plants And Their Function In Stress Response

Plants frequently encounter diverse environmental stresses which restrict their growth and development.Water stress is one of the adverse stimuli that affect plant production.The production of reactive oxygen species?ROS?is a common feature in plant response to adversity,and the plasma membrane NADPH oxidases?NOXs?are key producers of reactive oxygen species under both normal and stress conditions in plants.It has been found that NOXs can form different functional subfamilies and these subfamilies indicated considerable evolutionary selection.But the function of different NOXs and their evolutionary relationship in plants are still under investigation.In our present study,we first analyzed the phylogenetic development and evolutionary relationship of NOXs in plants on the whole genome level,and preliminary defined the characteristics of the expression of NOX genes and their co-expression network in plants.Then,we performed a further study on the function of OsNOX1 in stress tolerance by employing a series of approaches including genetic transformation,resistance analysis,and physiological and biochemical determination.The main results of this study are as follows:?1?The study of phylogenetic development and evolutionary relationship of NOXs in plants.We identified 50 ferric reduction oxidases?FRO?and 77 NOX gene homologs from 20 species representing the eight major plant lineages,and then phylogenetic and structural analysis classified these FRO and NOX genes into four well-conserved groups represented as NOX,FROI,FROII and FROIII,every subfamilys contain a Ferric_reduct domain,FROI contained only Ferric_reduct domain and then obtained FAD_binding₈,NAD_binding₆ and NADPH_Ox domains by gene fusion and duplication and clustered into NOX subfamilies.Further analysis of exon/intron structures in FROII and NOXs indicated that single intron loss and gain had occurred,yielding the diversified of gene structures,and FROII and NOXs were classified into three and four conserved subfamilies which are represented as FROIIa,FROIIb and FROIIc and Sub.I,Sub.II,Sub.III and Sub.IV,respectively.Considerable diversity in the number and the length of introns in the FRO and NOX family genes had found,but they shared similar gene structure in terms of intron numbers and intron phases within the same subfamily.FROI perceived as ancient NOX led to the expansion of NOX family and the functional diversity and divergence by gene fusion and duplication.?2?Analysis of the characteristics of the expression of NOX genes under various stresses and their co-expression network.Both available global microarray data and qRT-PCR experiments analysed the expression of NOX genes in different developmental stages,various stresses and co-expression network in Arabidopsis and rice.We found that there were ten AtNOXs?AtRBOHA^J?and nine OsNOXs?OsNOX1⁹?in Arabidopsis and rice,respectively.and they expressed in all tissues during the whole development stages but with strong tissue specificity.The promoters of the most AtNOXs and OsNOXs contain anaerobic-,heat stress,drought?MYB binding sites?,ABA and MeJA response elements,and the expressions of AtNOX and OsNOX genes were induced to varying degrees by different abiotic stress including heat,cold,drought?PEG?,salt?NaCl?and oxidative?methyl viologen,MV?stresses and hormones such as ABA,MEJA,salicylic acid?SA?,Zeatin,gibberellin?GA?,Indole-3-acetic acid?IAA?,kinetin?KT?and cytokinin?6-BA?.The expression profiles of these AtNOX and OsNOX genes shown no distinct subfamily characteristics in response to these abiotic stresses and hormone treatments.In the frameworks of the co-expression networks,AtNOXs are mainly involved in plant-pathogen interaction,plant hormone signal transduction,secondary motabolites and phenylpropanoid biosynthesis,and the OsNOXs are mainly co-expressed with the genes functioning in plant-pathogen interaction,endocytosis,fatty acid and unsaturated fatty acid biosynthesis and many other metabolism progresses.All these results suggest that NOXs show crucial roles in plant metabolism progresses and defense system and different NOXs can be involved in different stimuli but also show a complicated and cross-talk signaling network.?3?The study of the functional of OsNOX1 in drought response.We first cloned the OsNOX1 and its promoter,then obtained over expression?OE?and ProOsNOX1-GUS transgenic lines,and a TOS17 insertion mutant which knockout OsNOX1 were purchased from japan.In our study,we analyzed the functional mechanism of OsNOX1 gene in stress response by morphological,physiological and biochemical and resistance analysis,and we found that OsNOX1 encoded a membrane-localized protein,and the expression of OsNOX1 was found in all tissues throughout development,and it was upregulated by PEG,NaCl,ABA,MeJA and SA.Compared with WT and OE,disruption of OsNOX1 in osnox1 showed lower germination rate,organ size,ROS production,ABA content and expression of stress related genes,but enhanced stomatal aperture opening and sensitivity to environmental.Additionally,the activities and the expressions of Ascorbate peroxidase?APX?and Catalase?CAT?and the content of Malondialdehyde?MDA?and proline were significantly elevated after drought stress.These results suggest that osnox1 keep a lower level of the ROS production after drought stress and enhance the sensitivity to drought.Taken together,FROI,the ancient form of NOX,yield plant NOXs by repeatedly gene fusion and duplication.Single intron loss or gain likely occurred in NOXs and it led to the expansion of NOX family and the functional diversity and divergence.Different NOXs show a different expression profiles in response to various abiotic stress and hormone treatments.NOXs in plants play a crucial role in many metabolic and resistance progresses and different NOXs which belong to different subfamilies can be involved in different stimuli but also show a complicated and cross-talk signaling network.OsNOX1 shows a positive regulation in drought tolerance by control of the OsNOX1-dependent ROS production interaction with ABA signaling pathway.