Functional Analysis Of LuPDH-E1β1 From Linum Usitatissimum On Seed Fatty Acid Accumulation

Flax(Linum usitatissimum L.,2n=30),an autogamous annual crop,is grown worldwide primarily for its seed oil(oilseed flax)and stem fiber(fiber flax).Flax seed oil is rich in polyunsaturated fatty acids(PUFAs),particularly linolenic acid,which is converted from linoleic acid.Pyruvate dehydrogenase complex(PDC)is a key rate-limiting enzyme in the process of glycolysis,catalyzing the conversion of glucose into pyruvate and acetyl-CoA.However,the function of PDC,especially the E1 component,in seed oil accumulation and response to abiotic stress in flax is not well understood.Therefore,this experiment systematically analyzed the physicochemical properties of AtPDH-E1β1 and LuPDHE1β1proteins and its relationship with other crops.The full-length coding domain sequence of LuPDH-E1β1 was obtained from "Longya 10".We preliminarily investigated the function of LuPDH-E1β1 in seed oil accumulation and response to abiotic stress in flax,and analyzed its regulatory mechanism.The main results of this paper are as follows:(1)We investigated AtPDH-E1β1 expression in various tissues of wild-type plants by using quantitative qRT-PCR and found that AtPDH-E1β1 was expressed in various tissues including roots,stems,rosette leaves,stem leaves,flowers and siliques.During seed development,AtPDH-E1β1 expression increased initially and then decreased during seed development,with the highest expression level observed on the 12th day after pollination.(2)Phenotypic analysis showed that the mutant seeds had significantly lower size and weight than the wild-type.Furthermore,the seed total FAs and major seed FAs were significantly lower in the mutant seeds than in the wild-type.Additionally,during the critical period of seed oil accumulation in Arabidopsis,the expression levels of oil accumulationrelated genes,AtBCCPl,AtMCAT,AtKASl,AtKAS2,AtFAB2,AtFAD2,AtFAD3,AtKCS17,and AtPDAT2,were significantly lower in the mutant seeds compared to the wild type.These results indicate that A tPDH-E1β1 positively regulates seed oil accumulation in Arabidopsis.(3)There was only one paralog(LuPDH-E1β1)of AtPDH-E1β1 in flax.The results of multiple sequence alignment showed that the amino acid sequence of LuPDH-E1β1 was 82.15%similar to AtPDH-E1β1,which had a conserved domain.Phylogenetic tree analysis showed that AtPDH-E1β1 and LuPDH-E1β1 were closely related.Bioinformatic analysis shows that both AtPDH-E1β1 and LuPDH-E1β1 are stable hydrophilic proteins without signal peptides or transmembrane domains,and are non-secretory proteins located outside the membrane.The secondary and tertiary structures of the two proteins are mainly composed of α-helices and disordered loops,with similar structures and phosphorylation and N-terminal glycosylation modification sites.(4)Overexpression of flax LuPDH-E1β1 in the background of Arabidopsis thaliana mutant can restore the phenotype of low seed size,seed weight,seed total FAs and major seed FAs of the mutant,and restore the expression of genes related to seed oil accumulation to the level of wild-type.Therefore,Arabidopsis AtPDH-E1β1 and flax LuPDH-E1β1 have similar functions in regulating seed oil accumulation,and both can positively regulate seed oil accumulation.(5)Under the stress of 150 mM NaCl or 300 mM mannitol,the germination rate of Arabidopsis mutants pdh-e1β1-1 and pdh-e1β1-2 significantly decreased,and the seed germination rate of transgenic line pdh-e1β1-2 35S:LuPDH-E1β1-6HA returned to the level of wild type.At the same time,under stress conditions,the expression levels of ABA synthesis and signaling response genes,such asAtNCED3、AtABI3、AtABI5、AtEM1、AtEM6、AtRD29A,were significantly higher in the seeds of the Arabidopsis mutant than in the wild type.The expression levels of these genes in the transgenic line pdh-e1β1-2 35S:LuPDH-E1β1-6HA were not significantly different from those in the wild type.These results demonstrated that both Arabidopsis AtPDH-E1β1 and flax LuPDH-E1β1 have similar functions in plant responses to abiotic stress,and can improve plant tolerance to salt and osmotic stress.In summary,this study investigated the functions of LuPDH-E1β1 and AtPDH-E1β1 in seed oil accumulation and response to abiotic stress,and provided insights into their molecular regulatory mechanisms.The results not only contribute to the construction of a molecular regulatory network for crop oil metabolism and response to abiotic stress,but also provide valuable genetic resources for improving fatty acid composition of oil crops and breeding new resistant crop varieties with high levels of α-linolenic acid.