The BrPPR Of Brassica Rapa L.Ssp.Pekinensis Conferring Enhanced Multiple Abiotic Stress Tolerances In Arabidopsis

Soil salinization is an important factor limiting plant growth and yield formation,and is one of the main obstacles to improve the efficiency of cultivated land production in China.The worsening alkalized soils are dengerious for plant growth and development,which cause ion toxicity,osmotic,oxidative and high pH stress.To select new salt-alkalized tolerant crop germplasm and avoid yield loss is one of the main strategies in todays' China.Chinese cabbage(Brassica rapa L.ssp.pekinensis)is the highest yield vegetable per unit area,rich in vitamins,is the most popular food during autumn and winter.Isolation and identification of the stress-resistant genes in Chinese cabbage is the main purpose of effectively utilizing saline soil for its breeding in the future.Several studies have shown that PPR in plants can respond to salt,ABA and drought stress in many plants,but there is still no report on the stress resistance function of PPR gene in Chinese cabbage.In this paper,the BrPPR gene derived from Chinese cabbage was studied.The function and salt-alkali tolerance mechanism of BrPPR gene were explored by using wild type(Col-0)and the transgenic lines of Arabidopsis thaliana as experimental materials.The results are as follows:1.Functional analysis of BrPPR geneThe BrPPR gene was cloned from RNA-SEQ of Chinese cabbage pol CMS in our laboratory.Transient expression analysis showed that BrPPR gene localizes to the mitochondria by using the subcellular localization assay.According to the information of NCBI,The full length of BrPPR gene is 1950 bp which encodes 650 amino acids with no introns in it.The BrPPR is composed of 15 tandem motifs and belongs to the subclass P-type protein.Evolutionally,BrPPR gene has the highest homology with PPR gene in rapeseed.Promoter analysis also showed that the HSE,LTR,MBS,and TGA elements of the BrPPR gene promoter region responded to heat,low temperature,drought,and auxin stress,respectively.Stress expression pattern analysis showed that BrPPR gene could be induced by alkali and hydrogen peroxide stress.With the prolongation of treatment time,the expression level of BrPPR gene increased first and then decreased after treatment under two kinds of stress.The overexpression line of this gene showed resistance to high pH,NaHCO3 and H2O2 stress,such as seed germination in advance,germination rate increased,cotyledon pre-expanded,aerial part increased,root length increased,lateral root number increased,etc.There was no significant difference between the two in the control.The survival rate of over-expressing line grown in 50 mM NaHCO3 soil was significantly higher than that in wild type.ROS staining experiments showed that the overexpressing lines accumulated less ROS under the stress of alkali and H2O2,and the content of H2O2 and oxidative stress product MDA in the cells was also significantly lower than that in the wild type,indicating that the oxidative stress in the overexpressing lines was weakened.Further analysis revealed that the expression levels of genes involved in ROS synthesis,clearance and signal transduction pathways in overexpression lines were also lower than those in wild type;the activities of antioxidant enzymes related to ROS clearance were also lower than those of wild type,such as SOD,POD,CAT and APX.The addition of GST treatment can eliminate the difference of ROS between the overexpressing lines and the wild type,whether it is self-accumulation or newly generated ROS under various stresses,and its phenotype tends to be consistent.So it deduced that the BrPPR gene is involved in the regulation of redox balance and mediates response to salt-alkaline stress by reducing ROS production and accumulation.2.Mechanism of BrPPRMost nuclear-encoded PPR protein are predominantly targeted to mitochondria and produced reactive oxygen species(ROS)in the main parts of it,the electron transport chain complex I(NADH dehydrogenase complex I).The transcripts and splicing of subunits of complex I were determined by RT-RCR and qRT-PCR.The splicing efficiency of the fourth intron of nad2 subunit and the fourth intron of nad7 were all reduced under treating by NaHCO3 and high pH in wild-type Arabidopsis,however,there were no significant difference between them in untreated group.The activity of NADH dehydrogenase in complex I showed that the activity of NADH dehydrogenase in overexpression lines after stress treatment was significantly higher than that in wild Arabidopsis.If the electron transport based on the cytochrome C pathway is hindered,the alternate oxidase pathway will be supplemented,so the expression level of the alternate oxidase gene was measured.We determined that the expression of AOX gene in the overexpressing line under alkaline stress was significantly weaker than that in the wild type,indicating that the electron transport pathway of the cytochrome C pathway was more efficient than the wild type;the damage of the wild type electron transport chain caused by alkali stress more severe,thereby allowing it to passively upregulate the activity of the A OX gene.In conclusion,the BrPPR gene may maintain normal mitochondrial electron transport by promoting the normal splicing of the electron transport chain complex I,thereby reducing ROS production and exhibiting resistance to alkaline stress.