Molecular Mechanisms Of Phosphoethanolamine N-methyltransferase PMT1 Mediated Root Elongation Of Arabidopsis Under Salt Stress

Soil salinization has become a severe issue,which imposes ionic stress,osmotic stress,and a series of secondary effects such as oxidative stress on plants,resulting in growth inhibition of plants and crop yield reduction.Revealing the salt stress tolerance mechanisms of plants can provide a theoretical basis for breeding new salt-tolerant crops.In this study,we identified a salt-sensitive mutant,SALK＿108751,which exhibited severe root elongation inhibition and swollen epidermal cells in the transition and elongation zones after salt stress,and we confirmed that mutation of phosphoethanolamine N-methyltransferase PMT1 responsible for this phenotype via constructing CRISPR/Cas9 mutant lines and complementation lines subsequently.Using genetics in combination with a series of physiological and biochemical experiments,we revealed the molecular mechanism of how PMT1 involved in salt tolerance,and the main findings are as follows:1.q RT-PCR showed that the PMT1 had the highest transcript level among PMTs in the Arabidopsis roots,and the expression of PMT1 was upregulated more obvious than PMT2 by Na Cl treatment.Unexpectedly,Na Cl treatment inhibited the expression of PMT3.Salt sensitivity analysis assay revealed that only pmt1 but not pmt2 or pmt3was sensitive to salt stress.Among double mutant,pmt1 pmt2 was similar to pmt1 under salt stress,pmt2 pmt3 was not sensitive to salt treatment,and pmt1 pmt3 was developmental defect,which indicated that different PMTs are not functional redundancy in response to salt stress,and PMT1 plays a significant role in regulating primary root elongation under salt stress in Arabidopsis.2.We introduced CYCB1;1:GUS reporter to detect cell division activity and DR5:GUS reporter to observe auxin response in both pmt1 and WT,which revealed that the lack of PMT reduced the cell division of root meristem zone,and altered the auxin response of root tip.ABA treatment could mimic this phenomenon.Suggesting that the regulation of primary root development by Na Cl as well as ABA is dependent on the maintenance of PMT1-mediated meristematic zone activity.3.Na⁺and K⁺content analysis showed that salt stress blocked the root elongation of pmt1 was not caused by the change of K⁺/Na⁺ratio.We measured glycerolipids content subsequently.Statistics indicated that only phosphorylethanolamine abundance in the root of pmt1 was higher than WT without Na Cl treatment.phosphoethanolamine N-methyltransferase and monogalactosyldiacylglycerol content in the roots of pmt1were all lower than those of the WT,but the triacylglycerol and phosphatidic acid content were higher than that of the WT after Na Cl treatment,indicating that salt stress altered the metabolism of glycerolipids of pmt1 roots.Exogenous adding lecithin reverted the short root phenotype of the pmt1 under salt stress.It can be seen that the primary root elongation of pmt1 is associated with PMT1-dependent metabolism of glycerolipids.4.By the analysis of JASPAR database online,we found that the ABA response elements（ABREs）existed in the promoter of PMT1.Besides,exogenous ABA was able to induce the expression of PMT1.However,in aba2-1 and pyl112458-T mutant,this regulation was attenuated or even completely disappeared,indicating that Na Cl regulation of PMT1 expression is dependent on the ABA signaling pathway.5.The primary roots of the pmt1 are sensitive to exogenous ABA,and knocking out ABA2 in the background of pmt1 alleviated the inhibition of pmt1 root elongation and the swollen epidermal cells after salt stress effectively.It was shown that the inhibition of root elongation and swelling of epidermal cells in pmt1 under salt stress were associated with ABA.6.NBT and DAB staining showed that Na Cl treatment caused more accumulation of H₂O₂and O₂^-in the root stele of pmt1 compared with the WT,and the area of H₂O₂in the pmt1 was reduced.Exogenous addition of GSH did not significantly alleviate the restrain of the root elongation of pmt1 under salt stress,indicating that the change of ROS distribution in the root tip of pmt1 was more like the cause of the impaired primary root development.The accumulation and distribution of ROS in pmt1-2 aba2-1 after salt treatment is more like WT or aba2-1 than pmt1-2.So,we support that ABA affects root development of pmt1 by regulating ROS homeostasis.In conclusion,salt stress depends on ABA signaling to regulate PMT1 transcription,and PMT1 regulates root development under salt stress by affecting cell division,auxin distribution,and glycerolipids metabolism.PMT1-dependent lipid synthesis can alleviate ABA-induced reactive oxygen species burst and maintain cell membrane integrity and primary root elongation under salt stress.