Functional Characterization Of Stress-related NAC Family Genes In Rice

Rice(Oryza sativa L.)is one of the most important staple cereal crops worldwide.Agricultural science and technology researchers have always directed their efforts to improve rice quality and production.Drought,high salinity,extreme temperature and other abiotic stresses have become major constraints to agricultural development in many areas since they frequently result in large-scale reduction of rice production.To cope with the increasingly harsh natural conditions today,exploring the biological function and regulation mechanism of stress-related genes in improving stress resistence of rice has become a crucial project of rice functional genomics research.NAC(NAM,ATAF,CUC)transcription factor family play vital roles in the process of stress response and development in rice.Isolation and functional characterization of the stress-related NAC genes has great significance in genetic improvement of rice for abiotic stress tolerance.In this study,bioinformatics approaches were applied to systematically analyse NAC transcription factor family in rice.Expression profiles of a series of genes with a stress-inducible expression pattern in microarray data were confirmed using quantitative PCR method.Twenty-four stress-responsive NAC genes were subsequently chosen as our candidates for further investigation via integrated approaches including overexpression,RNAi and mutant identification.Based on abiotic stress phenotype screening of the overexpression transgenic plants,the stress-responsive NAC gene SN1 and the miR164-targeted NAC genes(OMTN)were chosen for in-depth fuctional characterization.The main results are summarized as follows:The predicted NAC proteins in rice were identified by homology search and checked with profile Hidden Molkov Model(pHMM)to remove false sequences.A total of 138 gene loci were predicted to encode putative NAC or NAC-like transcriptional factors.The conserved NAC domains were extracted to perform the phylogenetic analysis.The result suggested that the rice NAC family can be classified into five groups(Ⅰ-Ⅴ).Quite a few reported members involving the development regulation belong to group I,while a majority of the published stress-related NAC genes fell into group Ⅲ(designated SNAC).Motif composition analysis revealed that the NAC family of rice can be classified into 15 types(A-O).Most NAC proteins contain a conserved N-terminal DNA-binding domain and a highly diversified C-terminal regulation domain.Yeast one-hybrid assay indicated that rice NAC proteins with different motif compositions possess DNA binding activity in yeast.A significant portion of rice NAC genes showed induction ratios higher than 2.0 under abiotic stresses in our microaaray data,and their stress-responsive expression profile were verified by real-time quantitative PCR.In silico cis-element analysis indicated that 17 cis-elements were enriched in the promoter regions of rice NAC family genes which were known to be responsive to environmental stimulus including phytohormones,light,temperature as well as related to tissue/organ-specific expression pattern.SN1 was expressed ubiquitously.Expression level of SN1 gene was increased under drought,high temperature,salinity stress and ABA treatment.Overexpression(OE)of SN1 in rice resulted in enhanced tolerance of the transgenic rice to high temperature,drought and MV(methyl viologen)oxidative stress,whereas inhibition of SN1 by RNAi resulted in increased sensitivity to high temperature,mannitol simulated drought stress and MV oxidative stress.Quantitative PCR experiment showed that expression of a large number of ROS-scavenging enzyme genes were dramatically increased in the SN1-OE plants but significantly decreased in the SN1-suppressed transgenic plants.DAB staining analysis indicated no significant difference in H2O2 accumulation between the SN1-OE and wild-type plants under normal conditions.However,lower level of H2O2 accumulation was detected in the SN1-OE transgenic plants compared with the wild-type plants under heat condition.We also found that the MDA content and the relative electrolyte leakage of the SN1-OE plant were significantly lower compared to the wild-type under heat stress.These results demonstrated that SN1 was likely involved in heat tolerance regulation by adjusting the redox homeostasis state via controlling the expression of ROS-scavenging enzyme genes.A total of five ROS-scavenging enzyme genes which were up-regulated in SN1-OE plants showed co-expression pattern with SN1 gene.Interaction of SN1 protein and the promoters of three ROS-scavenging enzyme genes(R1,R11 and R45)sharing co-expression pattern with SN1 were verified by yeast one-hybrid assay,indicating that these three genes might be direct downstream targets of SN1.MiR164-targeted genes in Arabidopsis were five NAC genes.Our study indicated that six NAC genes(OMTN1-OMTN6)are putative targets of miR164 in rice.The RLM-RACE(RNA Ligase Mediated-RACE)result confirmed that miR164b guided cleavage of OMTN1 and OMTN2 mRNAs,and the cleavage sites were identical to the position reported in Arabidopsis.OMTN genes were responsive to a variety of abiotic stresses,and they showed diverse tempo-spatial expression patterns in rice.OMTN proteins have the characteristics of typical NAC transcriptional factors and their transactivation domains are located in the C-terminal of the proteins.The miR164 recognition sites of OMTN genes were highly conserved among 158 rice varieties selected from a mini-core-collection of germplasm resources.Both site-directed mutagenesis and deletion of miR164 recognition sites did not affect the DNA binding activity of OMTN proteins in yeast assay.Site-directed mutagenesis of miR164 recognition sites showed no effect on the transactivation activity of OMTN proteins.Nevertheless,deletion of miR164 recognition sites deprived the transactivation activity of OMTN proteins,indicating that miR164 recognition sites played a crucial role in maintaining the transactivation ability of OMTN proteins.Overexpressing OMTN2,OMTN3,OMTN4 and OMTN6 in rice led to visible negaitive effects on drought tolerance at reproductive stage.Expression of numerous stress-responsive genes,development-related genes,regulatory genes and metabolism-related genes altered in the OMTN2,OMTN3,OMTN4 and OMTN6-overexpressing plants.Suppression of OMTN4 and OMTN6 by RNAi caused abnormal developmental phenotypes such as organ fusion,twisted leaf blade,which were very similar to the phenotypes observed in the miR164-overexpressing transgenic plants,suggesting that OMTN genes participate not only in the modulation of abiotic stress response,but also in developmental regulation.