Studies On The Relationship Between Biotin And Carbonate Stress In Arabidopsis Thaliana

Soil salinization is a global environmental problem,which seriously affects crop yield.The saline-alkali soil in the northeast of China suffers from severe alkaline salt stress.The saline-alkali soil is rich in NaHCO3 and Na2CO3 alkaline salts,which leads to the high salinity and high pH of the soil.The stress brought by carbonate stress to plant growth is multiple,and the damage it brings to plant growth is also huge.In recent years,the mechanism of salt resistance in plants has been better understood,but the research on the resistance to carbonate stress is still rare.In order to better understood the mechanism of plant resistance to carbonate,this study mainly took the dicotyledonous model plant Arabidopsis thaliana as the research material.By analysis the transcriptome of the shoot part and root part of A.thaliana,the following results were obtained:(1)In the experiment of NaHCO3 treatment to wild-type A.thaliana,it was found that carbonate stress led to slow growth of Arabidopsis seedlings and yellow leaves.In addition,using trypan blue staining,it was found that carbonate stress caused damage to Arabidopsis leaves.The growth of seedlings on agar medium with different concentrations of NaHCO3 was also found that the growth of A.thaliana was severely inhibited when the final concentration of NaHCO3 was 3 mM and 5 mM.Wild-type A.thaliana was treated with 3 mM NaHCO3 for different times and samples were divided into shoot parts and root parts for high-throughput sequencing.The results showed that the number of differentially expressed genes(DEGs)was the highest at 4 h treatment,and the number of DEGs decreased gradually with the increase of treatment time.However,the number of DEGs in the shoot part was relatively stable at each time point.These results suggest that the roots of A.thaliana may be damaged and respond to carbonate stress before the shoot part.(2)In the GO enrichment analysis of all DEGs,we paid attention to two relatively significant pathways:one is the carboxylic acid biosynthesis pathway,and the other is the carboxylic acid metabolism pathway.The synthesis and metabolism of carboxylic acids are often accompanied by HCO3-as the reactant or reaction product.Some known carboxylases often use HCO3-as the reactant and biotin as the cofactor to carboxylate HCO3-into organic matter.All the up-regulated DEGs in the two pathways were analyzed and 5 DEGs related to biotin were found.The expression trend of the 5 genes was basically the same as that of RNA-seq,which was verified by qRT-PCR.The results of transcriptome indicated that biotin might play a role in the resistance of plants to carbonate stress.(3)To determine the role of biotin in carbonate stress,we studied the seed germination rate and growth status of wild-type A.thaliana treated with different concentrations of carbonates(NaHCO3 and Na2CO3)and biotin.It was found that low concentration of carbonate and biotin can promote the growth and seed germination of A.thaliana,while high concentration of carbonate and biotin can inhibit the growth and seed germination.However,when high concentration of carbonate and biotin were both added,the two substances can restore each other.The content of endogenous biotin was detected under carbonate stress,and it was found that different concentrations and different periods of carbonate stress would lead to the upregulation of the content of endogenous biotin.The activity of antioxidant enzymes increased under carbonate stress,but decreased under the co-treatment of carbonate and biotin compared with the treatment of carbonate or biotin alone.The results confirmed that biotin plays an important role in plant resistance to carbonate stress.(4)The expression level of AtBI02 which is encoding biotin synthase was detected under different concentrations and different periods of carbonate treatment,and it was found that carbonate stress could induce the up-regulation of AtBIO2 gene expression.Carbonate stress on AtBIO2-Pro:GUS plants showed a stronger GUS activity.The expression of AtBIO2 and the content of biotin in the shoot part and the root were detected.It was found that the expression of AtBIO2 was significant up regulated in the shoot part under carbonate stress,and the biotin was mainly accumulated in the root.Phenotypic experiments on AtBIO2 mutants and overexpression showed that AtBIO2 mutants were more sensitive to carbonate stress,while overexpression plants had carbonate stress resistant phenotypes.By DAB and NBT staining and antioxidant enzyme activity determination,it was found that under carbonate stress,the antioxidant enzyme activity of AtBIO2 overexpression plants was enhanced and the accumulation of reactive oxygen in leaves was less,while the antioxidant enzyme activity of mutant plants was lower than that of wild-type plants and the accumulation of reactive oxygen in leaves was more.These results suggest that biotin and biotin synthase AtBIO2 can help Arabidopsis resist carbonate stress.Biotin may help plant to remove reactive oxygen species to reduce the damage caused by carbonate stress.(5)We constructed three biotin carboxylase subunits AtBCCP1,AtBCCP2 and AtMCCA overexpression plants,and identified the mutant plants of AtBCCP1 and AtBCCP2.Phenotypic experiments showed that the overexpression plants of AtBCCP1,AtBCCP2 and AtMCCA showed tolerance to carbonate stress,and the overexpression plants of these three genes could resist the growth inhibition caused by high biotin concentration.In contrast,AtBCCP1 and AtBCCP2 mutant plants showed sensitivity to carbonate stress and were similar to wild-type phenotypes under high biotin concentrations.With the treatment of carbonate stress,the addition of exogenous biotin did not help to resist carbonate stress.These results suggest that AtBCCP1,AtBCCP2 and AtMCCA can help Arabidopsis resist carbonate stress,and the ability to resist carbonate stress is stronger under the condition of sufficient biotin.In conclusion,this study confirmed that biotin is involved in the process of Arabidopsis resistance to carbonate stress and that four biotin-related genes play an important role in Arabidopsis resistance to carbonate stress.These results help to construct the gene network of plant resistance to carbonate stress,and provide a good theoretical basis for plant molecular breeding.