Functions Of The NADK Family Genes And Their Mechanisms In Stress Tolerance In Plants

NAD kinase(NADK)is the sole enzyme that generates NADP(H)by phosphorylating NAD(H)and plays vital roles in maintaining the balance of NAD(H)/NADP(H)for NADP(H)-based metabolic pathways in cells.NAD(H)and NADP(H)are coenzymes of a number of enzymes in the cell metabolism in all living organisms.They involve in multiple redox reactions and affect the rate and pathways of oxidative metabolism.However,there are few studies about the expression regulation of NADK gene in plants,especially the crops of rice and wheat,the functions and regulated mechanism were still unclear.In this study,we firstly analyzed the phylogenetic and evolutionary mechanism of NADK gene family in the whole genome level by bioinformatics with examined the expression profiles of the NADK genes of Arabidopsis and rice under different stress conditions.The biological functions of NADKs,especially OsNADK1 and TaNADK4,were further studied by the means of molecular biology,cell biology,plant physiology,etc.The main results are as follows:1.We performed a phylogenetic analysis with the 74 NADK homologous genes from 24species representing the eight major plant lineages within the supergroup Plantae.Phylogenetic and structural analysis classified these NADK genes into four well-conserved subfamilies with considerable variety in the domain organization and gene structure among subfamily members.In addition to the typical NAD_kinase domain,additional domains,such as ADK,PTPc or PTPc/DSPc domains,were found in subfamily II.Whereas the NAD_kinase domain in subfamily III was divided into two parts and exhibited low sequence similarity(~30%)in the kinase domain with the other subfamilies.These observations suggest that gene fusion and exon shuffling may have occurred after gene duplication,leading to specific domain organization seen in subfamilies II and III,respectively.Further analysis of the exon/intron structures showed that single intron loss and gain had occurred,yielding the diversified gene structures,during the process of structural evolution of NADK family genes.Finally,both available global microarray data analysis and qRT-RCR experiments revealed that the NADK genes in Arabidopsis and Oryza sativa show different expression patterns in different developmental stages and under several different abiotic/biotic stresses and hormone treatments,underscoring the functional diversity and functional divergence of the NADK family in plants.2.We cloned the gene of OsNADK1 in rice and studied its functions in stress tolerance.It was found that OsNADK1 is a cytosolic localization protein and expressed in all tissues with increased transcript levels under stresses.Compared with wild-type(WT),the mutant osnadk1 exhibited lower plant height and significantly lower ratios of NADPH/NADP~+,ASA/DHA and GSH/GSSG.Moreover,a number of stress-related genes changed their expression patterns in the mutant under both normal growth and drought stress conditions.In particular,the transcription factors OsDREB1B and OsWRKY21,OsWRKY42,showed a well co-expression pattern with OsNADK1 in both the osnadk1 mutant and transgenic plants.In addition,overexpression of OsNADK1 accumulated proline and enhanced the tolerance to drought in rice,while the plants which loss function of the gene were opposite.These results indicated that OsNADK1 may regulate the intracellular redox balance and enhance the tolerance to drought in rice.3.A total 44 NADK genes were identified from 11 species of the three major cereal crops,rice,maize and wheat.According to the phylogenetic and structural characteristics,these NADK genes can be classfied into 3 subfamilies.Based on the wheat EST data,11NADK genes were predicted in wheat genome to encode the four NADK isoforms,TaNADK1~4.We then cloned 4 genes of wheat which encode the four isoforms,respectively.Analysis of the subcellular location showed that both TaNADK1 and TaNADK2 were located in the cytoplasm,TaNADK3 was in chloroplast and TaNADK4 in peroxisomes,respectively.The expression patterns of these NADKs showed the specific characters in different tissues and under various environmental treatments,showing the diverse functions of the wheat NADK genes in the plant stress tolerance.4.The promoter of TaNADK4 on 5B chromosome was cloned and then used for a yeast one-hybrid experiment.Two transcription factors,HOX1 and HDZipI-1,were screened.They belong to the same subfamily and have self-activating activity.The transcriptional activity analysis showed that the two transcriptional factors can bind to the promoter of TaNADK4.Besides,TaNADK4 can also interact with TaPEX5,a transporter protein of PTS1 signal peptide in wheat,and then can be transported into peroxisomes assisted by TaPEX5.Our further study showed that TaNADK4 can form homodimers or polymers in cells.Meanwhile,we obtained a null AtNADK3(a homologous gene of TaNADK4 in Arabidopsis)mutant atnadk3.In addition,we constructed vectors and then introduced the vectors into Agrobacterium tumefaciens to establish the overexpression transgenitic lines of TaNADK4gene.The calli of rice(from cv.Nipponbare)and unpollinated flowers of Arabidopsis thaliana(WT and atnadk3)were used for the genetic transformation,respectively.Then the resulted transgenic plants were identified by the means of GUS staining,PCR of DNA and RNA,and western blot.These materials will be used for further study to clarify the molecular mechanisms of TaNADK4 in the development and stress tolerance of wheat.In conclusion,NADK genes had structural and functional diversity in plants.The gene fusion and exon shuffling play an important role in the evolution and functional diversity.The cytoplasmic protein OsNADK1 was affected by various environmental factors and involved in the drought resistance by regulating the redox balance in rice.TaNADK4 was located in the peroxisomes and also had response to the environmental stresses.It could be transported to peroxisomes by assisted TaPEX5 and its expression might be regulated by the two transcription factors,TaHOX1 and TaHDZipI-1.