Functional Analysis Of Arabidopsis Transcriptional Factor AtOFP8 Under Drought Stress

Arabidopsis thaliana Ovate Family Proteins?AtOFPs?,a novel transcription factor,is a family of plant-specific proteins involved in the regulation of Arabidopsis growth and development.It was reported that other OsOFPs were able to participate in stress response in abiotic stress,but there was no report on the function in Arabidopsis.Recent studies had shown that there were 19members of this family in Arabidopsis.AtOFP8 was a member of class II of AtOFPs,but there was no report on its function.Our previous study found that AtOFP8 seems to be involved in drought.Could AtOFP8 participate in abiotic stress responses?To investigate whether AtOFP8 was involved in abiotic stress tolerance in Arabidopsis,the transcriptional level of AtOFP8 gene under stress was tested.The results showed that the transcriptional level of AtOFP8 gene was most obvious under drought stress.Therefore,Col,Atofp8-1 mutant and 35S:HA-AtOFP8overexpression plants were used as materials.The seed germination rate,green cotyledons rate,leaf malondialdehyde content,leaf proline content,leaf antioxidant enzyme activity,leaf soluble protein content were measured under drought stress.Analysis showed that 35S:HA-AtOFP8overexpression had stronger drought resistance under drought stress,indicating that AtOFP8 could indeed enhance drought resistance in Arabidopsis and help Arabidopsis resist drought.This experiment provides important clues for the study of AtOFPs participation in response to stress,and has a good theoretical and practical significance for the study of AtOFPs function.The main test results of this study were as follows:1.The AtOFP8 gene?At5g19650?was located on chromosome 5 of Arabidopsis thaliana.The gene was 954 bp in length with no introns.The CDS region was 666 bp in length and the encoded protein contained 221 amino acids.AtOFP8 had a molecular formula of C₁₁₁₈H₁₇₅₅N₃₁₅O₃₄₈S₉,a molecular weight of 25.466 kDa,a theoretical isoelectric point of 9.05,a fat index of 56.88,a stability coefficient value of 67.78,and it was an unstable protein.There were20 kinds of amino acids that made up the AtOFP8 protein,of which the Ser content was the most,accounting for 16.70%of the total number of amino acids,and the content of Trp was the least,accounting for only 0.90%of the total number of amino acids.there were 37 positively charged amino acid residues?Arg+Lys?in the AtOFP8 protein,31 negatively charged amino acid residues?Asp+Glu?.AtOFP8 protein had strong hydrophilicity and was a non-secretory protein.In the AtOFP8 protein,the content of?-helix and random coils was the highest.2.AtOFP8 protein localized in the nucleus.Subcellular localization of AtOFP8 protein was predicted by SUBA4 subcellular localization prediction site of Arabidopsis thaliana and the SUBAcon value of AtOFP8 protein located in the nucleus was 0.975,mainly located in the nucleus.By constructing the AtOFP8-GFP vector and transforming the onion epidermis with Agrobacterium,the AtOFP8-GFP fusion protein was located in the nucleus of onion epidermal cells.Both bioinformatics predictions and subcellular localization experiments had demonstrated that AtOFP8was localized in the nucleus.3.AtOFP8 gene was expressed in many organs of Arabidopsis thaliana.AtOFP8 gene was expressed in roots,stems,leaves,flowers.Among them,the transcription level was highest in roots.4.AtOFP8 gene responded to adversity stress.Under several stress conditions,AtOFP8 gene expression was affected,indicating that AtOFP8 gene might be involved in the process of abiotic stress.Under drought stress,AtOFP8 gene transcription level increased significantly.At high temperature,the transcription level of AtOFP8 gene also increased.However,the transcriptional level of AtOFP8 gene did not change significantly under low temperature and dark conditions.These results indicated that the AtOFP8 gene might be involved in the adversity stress process,and was particularly sensitive to drought stress and high temperature stress.5.AtOFP8 gene enhanced drought resistance in Arabidopsis.Under normal conditions,there was no significant difference in seed germination rate,green cotyledons rate,leaf malondialdehyde content,leaf antioxidant enzyme activity,leaf proline content,leaf soluble protein content among Col,Atofp8-1 mutant and 35S:HA-AtOFP8 overexpression plant.However,under drought stress,Arabidopsis overexpressed AtOFP8 gene had higher seed germination rate and green cotyledons rate,lower content of malondialdehyde in leaves,higher content of proline and soluble protein in leaves,stronger antioxidant enzyme activities in leaves.35S:HA-AtOFP8 overexpression plant showed stronger drought resistance,while Atofp8-1 mutant had weaker drought resistance.These results indicated that AtOFP8 gene could enhance the drought resistance of Arabidopsis thaliana.6.AtOFP8 promoted the expression of drought-responsive genes.The expression level of ADH1,RD26,RDUF2,ERD7 increased significantly in 35S:HA-AtOFP8 overexpression plant.