Effects Of Ectopic Expression Of ATS1 On Glycerolipid Biosynthesis And Response To Phosphorus Stress In Arabidopsis Thaliana

Phosphorus is a vital nutrient for plant survival.One third of the total cellular organic phosphorus is stored with the formation of phospholipids.Under phosphorus stress,plant cell replaces phospholipids by non-phosphorous galactolipids which demands galactolipids massively.It is poorly understood that whether this lipid remodeling involves de novo phospholipid synthesis.Arabidopsis plastidial glycerol-3-phosphate acyltransferase?ATS1?is a key enzyme catalyzing the first acylation reaction of glycerol-3-phosphate in prokaryotic pathway of glycerolipid synthesis utilizing acyl-ACP in plastid as substrate.Enzymatic analysis shows that ATS1 can also utilize acyl-CoA in cytoplasm as substrate to catalyze the acylation reaction of glycerol-3-phosphate.As ATS1 is a soluble protein with a transit peptide at the N-terminus,it is poorly described that whether ATS1 could participate eukaryotic pathway of glycerolipid synthesis in extraplastidial compartment.This study was designed to investigate the effect of ectopically expressing ATS1 in cytoplasm on extraplastidic glycerolipid synthesis and response to phosphorus stress in Arabidopsis.The main achievements of our research are as following:1.N-terminal sequence of 90 amino acid residues was deduced as the transit peptide of ATS1based on multiple sequence alignment of a number of plastidial glycerol-3-phosphate acyltransferases in different plant species.In an attempt to validate the enzyme activity of ATS1 without 90 amino acids at the N-termius,a novel yeast genetic complementation system was conducted.The result showed that ATS1 without transit peptide could complement the growth defects of yeast owing to lack of glycerol-3-phosphate acyltransferase activity.In addition,ATS1 without transit peptide had a higher enzyme activity than ATS1 with transit peptide in yeast,which means ATS1 without transit peptide is a mature protein.2.ATS1 with and without transit peptide sequence were cloned and introduced into Arabidopsis mutant atgpat1,respectively.Although neither of two kinds of ATS1 rescued atgpat1 mutant phenotype of male sterility,the average increment content of seed oil is 13.5%compared to atgpat1 mutant and the relative fatty acid content of C18:1,C18:3,and C20:1 are significantly increased and C18:2 is significantly decreased in ATS1 without transit peptide sequence transgene compared to atgpat1 mutant,which are similar to those in ATS1 with transit peptide sequence transgene.These results imply that ATS1without transit peptide could still exert glycerol-3-phosphate acyltransferase activity in Arabidopsis.3.The ATS1 with and without transit peptide sequence were subcloned into plant overexpression vector and transformed into Arabidopsis wild-type,individually.6 single-copy transgenic lines?T₃generation?of ATS1 without transit peptide sequence and 5 single-copy transgenic lines?T₃ generation?of ATS1 with transit peptide sequence were obtained by resistance screening and DNA molecular identification.Quantitative real-time PCR analysis showed that the relative expression of ATS1 in all transgenic lines were higher than that in transgenic line of empty vector.4 transgenic lines of ATS1 without transit peptide sequence having high expression level of ATS1 and 3 single-copy transgenic lines of ATS1with transit peptide sequence having high expression level of ATS1 were chosen for further experiments.4.Leaf oil analysis of transgenic plants carrying ATS1 with transit peptide sequence and ATS1without transit peptide sequence was conducted.The result showed that overexpression of ATS1 without transit peptide sequence could significantly decrease the relative fatty acid content of C16:0,C16:3 and increase the relative fatty acid content of C18:0,C18:2,and C18:3 compared to overexpression of ATS1with transit peptide sequence.Besides,the fatty acid composition of MGDG in transgenic line of ATS1without transit peptide sequence had no difference from that in transgenic line of empty vector.These results indicate that ectopic overexpression of ATS1 could affect the de novo synthesis of extraplastidial glycerolipid.5.Transgenic lines of ATS1 with and without transit peptide sequence were subjected to phosphorus stress treatment.The results showed that all transgenic lines had a higher root-shoot ratio and chlorophyll amount than the control group under low phosphorus stress condition,which means enhancing de novo synthesis of glycerolipid could relieve the damage done by low phosphorus stress to Arabidopsis.6.Exogenous application of glycerol could reinforce the formation of glycerol-3-phosphate,leading to a phosphorus stress condition because of consumption of free phosphate within the cell.In order to make a further explanation on the correlation between de novo synthesis of glycerolipid and plant response to phosphorus stress,transgenic lines of ATS1 with and without transit peptide sequence were implemented to glycerol application treatment.When the glycerol application reached 0.01%?v/v?,Transgenic lines of ATS1 without transit peptide sequence had a longer primary root length than transgenic lines of ATS1 with transit peptide sequence,which demonstrates that enhancing de novo synthesis of extraplastidic glycerolipid biosynthesis could improve plant tolerance ability to phosphorus stress.The above research results show that ATS1 devoid 90 amino acid at the N-termius is a muature protein and could enhance de novo synthesis of extraplastidial glycerolipid thus might help to improve plant tolerance ability to phosphorus stress.However,the detailed mechanism of relatioship between de novo synthesis of extraplastidial glycerolipid and plant tolerance to phosphorus stress requires further study.