Bioinformatics Analyses Of Alkaline-Salt Stress Response Mechanism In Rice(Oryza Sativa L.)

Rice(Oryza sativa L.)is a very important cereal because it is feeding half of the world population.During the life of a rice plant,it faces different kinds of biotic and abiotic stresses.Among these stresses,alkaline-salt is a most dangerous and causes severe losses in rice production.Different molecular as well as conventional techniques have been used to improve the final grain yield.Different gene families have been recognized for their potential role in the development of stress tolerance.Cytokinin is crucial in order to protect plants against excessive light exposure and a certain type of abiotic stress brought on by a changed photoperiod.Secondly,it demonstrates multi-stress resilience mechanisms in response to fluctuating environmental conditions.Through the cytokinin signaling,Type-B authentic response regulators(ARR-Bs)respond positively against the environmental stimuli.ARR-Bs are involved in abiotic stress tolerance and plant development.During previous studies,gigantic amount of transcriptomic data was generated and deposited into data banks,which intensify requirements for the more accurate data classification,prediction,and analysis.Computational techniques and Bioinformatics approaches have been under consideration in several ways for data processing,visualization,and prediction.However,their widespread use in RNA editing sites prediction is still rare.Therefore,this study was designed aiming to evaluate the role of RNA editing sites and ARR-B genes in stress tolerance.Furthermore,long non-coding RNA(lnc RNA)based Competitive Endogenous RNA(ce RNA)network constructed in rice under alkaline-salt stress conditions to understand it’s functional mechanism.RNA editing(RE)technique in rice has provided a quick and better understanding of in-depth gene expression,revealing functional variants(insertions,deletions,and substitutions).RNA editing site(RES)is a powerful and cost-effective functional genomics approach for underlying novel variations and their expression patterns in rice responding to alkaline stress.However,RES in rice genome is still not well understood,and in particular,there is little known about alkaline stress regulatory network.In the current study,comparative transcriptomic analysis has been used to recognize the genes and molecular mechanisms associated with the alkaline stress tolerance in rice at the seedling stage.For this purpose,total RNA from root tissues of 20-days-old seedlings of two rice genotypes Caidao(alkaline-sensitive)and WD20342(alkaline-tolerant)were extracted in control and alkaline stress conditions.The rice genotypes were treated under 0.5m Sodium carbonate(NA2CO3)stress for 36 hrs.The transcriptomic data further revealed 3336 RNA editing sites in both rice genomes including345 RESs in chloroplast and 2991 RESs in mitochondria,with an average of ~55% RES efficiency.The majority of editing events were annotated as non-synonymous codon variations tending to be hydrophobic amino acids.The RNA editing events among alkaline treated and normal rice samples were mainly distributed into four different substitutions A-G(A-I),C-T(C-U),G-A,and T-C.Overall,RNA editing efficiency under alkaline treated samples was significantly enriched.Although,the clustering of differentially expressed genes(DEGs)through utilizing the Gene Ontology(GO)analysis revealed that most of the DEGs were frequently annotated for alkaline related cellular,biological,and molecular functions.Moreover,the expression profile of PPR(pentatricopeptide repeat),OZ1(organelle zincfinger),and MORF/RIP(RNA editing factor interacting proteins)genes in alkaline-treated and control rice cultivars were validated by quantitative real-time PCR(q RT-PCR)using genespecific primers.Among these selected genes,PPR and OZ1 genes in alkaline treated samples were found up-regulated for pentatricopeptide repeat and organelle zinc-finger.Furthermore,genome-wide characterization of Os ARR-B genes under alkaline stress mined 24 genes that categorized significantly into four subgroups using the phylogenetic analysis.These genes were non-randomly scattered among all chromosomes of rice genome except chromosome 8 and 10.Functional characterization of Os ARR-B genes for gene duplications,gene structure,cis-elements,protein-protein interactions,and mi RNA were also studied.All the ARR-B members contain a conserve domain ARR-B domain(PF00072).The GO functional enrichment analysis of Os ARR-B genes,among which most enriched terms were involved in plant development through the regulation of molecular functions(MFs),biological processes(BPs),and cellular components(CCs).Total of 235 mi RNAs target the 22 Os ARRB genes.Os ARR-B8 and Os ARR-B10 were not targeted by any mi RNA.osa-mi R414 and osami R164 target the rice genes and have their role in adaptation against abiotic stress and plant growth.Our reference protein Os ARR-B4 interacts with the HK3,HK4,HK6,WOX11,and CKX4 proteins.These proteins are cytokinin histidine kinase receptor and involved in developmental processes and stress tolerance.Moreover,a total of 117 and 192 RESs were discovered in the chloroplast and mitochondrial genes,encoding proteins for Os ARR-B genes,respectively.Among the chloroplast and mitochondrial genes,six(A-V,T-I,H-Y,P-S,P-L,and R-C)and nine(A-V,T-I,H-Y,P-S,P-L,R-C,Q-X,R-X,and X-R)types of different amino acid variations were caused by RNA editing.In alkaline stress condition,Os ARR-B5,Os ARRB7,Os ARR-B9,Os ARR-B10,Os ARR-B16,Os ARR-B22,and Os ARR-B23 showed higher transcript levels as compared to susceptible ones.q RT-PCR showed up-regulation of these gene expressions under alkaline stress.In conclusion,this study explored the gene expression pattern along with RESs in alkaline tolerant and sensitive rice genotypes,which will help to understand the molecular mechanisms of alkaline-stress tolerance in rice.Long non-coding RNAs(lnc RNAs)are endogenous RNA found in plants that engage in a variety of cellular functions and stress responses.The regulation mechanism of lnc RNAs as ce RNAs in alkaline-salt stress response is yet to be investigated.During this study,we identified alkaline-responsive lnc RNAs in rice using transcriptome-wide analysis.Thereby,we built a lnc RNA-mediated ce RNA network by investigating competitive connections between m RNAs and lnc RNAs.An alkaline stress ce RNA network were 321 and 217 lnc RNA-mi RNA-m RNA triplets were discovered in CD vs.CDT,and WD vs.WDT with lnc RNAs(32 and 22),mi RNAs(121 and 93),and m RNAs(111 and 77),respectively.Network analysis demonstrated that NONOSAT000455-osa_mi R5809b-LOC_Os11g01210 triplet with highest degree is a hub node in alkaline stress response.Our findings emphasized on the regulatory roles of lnc RNAs as ce RNAs in rice alkaline-stress tolerance mechanism.Overall,these findings pave the ways for better development of alkaline-salt stress tolerant rice genotypes,and provision of basics of RES prediction in other crops.Moreover,future understanding of computational techniques will be easier to explore the RNA editing modifications.