Molecular Dissection And Expression Profile Of Rice OsFRO Genes Under Various Abiotic Stresses

The ferric reduction oxidase(FRO)gene family is involved in various biological processes,widely found in plants and may play an essential role in metal homeostasis,tolerance and intricate signaling networks in response to a number of abiotic stresses.While the speedy variations in the oxidative state of iron stimulate cellular function,regulation,electron transport and various other metabolic functions.In contrast the Fe toxicity is highly reactive and hamper normal plant growth and development.Previously,it has been reported that FROs functioning in iron(Fe)homeostasis,translocation and uptake mechanism,but the evolutionary history and core functional site especially in rice are still ambiguous.In this report we initially analyzed the phylogenetic relationship of FROs gene and define the characteristics of the expression of FROs gene in rice plant.Subsequently,we studied the functional validation of OsFRO1 gene in stress tolerance by developing several approaches,like RNAi-based strategy,genetic transformation,and analysis of morphological and physiological determination.The following are the obtained results.1.To explore the phylogenetic relationship of FROs,initially we identified 50 ferric reduction oxidases(FRO)genes in Plantae and 15 ‘FRO like' genes in non-Plantae.These genes were retrieved from 16 different species,representing eight major lineages with in the subgroups Plantae: glaucophytes,rhodophytes,chlorophytes,bryophytes,lycophytes,gymnosperms,monocots,and eudicots.The phylogeny analysis classified all FRO and FROlike genes in seven well conserved groups according to close similarity in biological and functional behavior.It is also speculated that the reason of clustering into different groups may be due to low sequence identity among the homologue's protein.The FRO family associates and their homologs originated from a common ancestor sharing three canonical domains,a heme-containing transmembrane region ferric reductase domain(Pfam: PF01794),and the two C-terminal cytoplasmic FAD-binding(PF08022)and NAD-binding(PF08030)domains.In plant FROs the exon intron structure is less complicated due to small number.while,the numbers of introns and intron phases are considerably conserved in plant FROs.Although the position of introns is distinct among the subgroups.The two conserved motifs,HPFT in FAD-binding domain and GPYG in NAD binding domain were quite conserved in two rice OsFRO1 and OsFRO7 genes,which are associated with co-factor binding.However,the other amino acid sequences were imperfectly conserved in both genes.Therefore,these observations suggest that the functional diversity and divergence of the FRO family proteins in plants may be due to long-term evolution history and subsequently the genes duplication may enhance the functional divergence and possibly improve the functional characteristics of FRO genes family.2.We further analyzed the tissue specific expression pattern of rice FROs as well as the induced expression level in stresses and hormonal treatments.Initially we investigated a set of microarray data in various developmental tissues and stress conditions in GENEVESTIGATOR V3 database,the obtained data were also cross verified by rice eFP browser for further validation and accuracy.Following the qRT-PCR and semi-quantitative RT-PCR analysis revealed that OsFRO1 and OsFRO7 genes exhibit ubiquitous expression pattern,like the expression of OsFRO1 was high in flag leaf,and OsFRO7 gene expression was maximum in leaf blade and flag leaf.Further,we demonstrate the expression pattern of OsFRO1 and OsFRO7 genes under abiotic stresses,hormones and metal treatments.The qRT-PCR analysis,bared that the OsFRO1 gene have strongly regulated by salt and drought stresses,while OsFRO7 have shown transcriptional variation under salt and heat stress,Although,there is no obvious expression in heat stress for both genes.Moreover,the expression of both genes was also substantial under heavy metal stresses.As the OsFRO1 gene transcripts were triggered after 6 h under Zn,Pb,Co and Ni treatments,whereas the OsFRO7 gene expression under Fe,Pb and Ni after 12 h,Zn and Cr after 6h,Mn and Co after 3h treatments have shown up-regulation.These findings suggests the possible involvement of OsFRO1 and OsFRO7 genes under multivariate stresses as well as contribution in the regulation of phytohormones.3.Furthermore,we cloned the OsFRO1 gene in rice and studied its functional characterization and stress tolerance mechanism by using RNAi-based technology.We found that the protein of OsFRO1 was localized in plasma membrane as well as the small signals were detected in internal cellular organelles.The knockdown effect of osfro1 significantly suppressed the seed germination,growth retardation,decrease in lateral and adventitious roots,dwarfness in plant height,stunted internodes,Fe-deficiency in flag leaf,reduction in chlorophyll content and modulation of ROS mechanism in transgenic rice plants.Moreover,a number of changes were induced at transcriptional,physiological and morphological levels under multivariate stresses.The antioxidant enzymes exposed increase in the level of MDA and APX,while decrease in the level of SOD,POD and CAT,thus the RNAi plants showed modulation in ROS activity at lowering or modulation in ROS,suggesting that the RNAi plants may help in stress condition and adaptation.In contrast the root morphology markedly affected by Fe-toxicity,thereby causing the root cellular damage by hyperaccumulation of ROS and thus inhibit the lateral root formation as well as reduction in the biomass of rice seedlings.Additionally,the Chlorophyll content and leaf pigments were also reduced due to Fe-deficiency,which further triggered the leaf senescence and further chlorophyll degradation process in RNAi plants.In conclusion,all these results signifying the role of FROs gene in plant growth and development.The possible speculation of the functional diversity and divergence of the FRO family proteins in plants may be due to long-term evolution history or the gene duplication process.The subcellular localization of FROs protein may help in translocation of Fe from outer membrane into inner membrane and further Fe partition.Both FROs in rice have distinct expression pattern as well as responses to various abiotic stress and hormonal treatments.Therefore,the appropriate regulation and correct function of rice FROs are vital for plant growth and development.The highest expression level of rice FROs in flag leaf indicating that these gene participating in grain filling and size.The loss of function of OsFRO1 induces sensitivity to Fe stress and affect plant growth and development.The knockdown effect of osfro1 have shown association with Fe-deficiency,As the plants were unable to resume the metabolism of chlorophyll synthesis,growth and root ferric chelate reductase(FCR)activity.