The Molecula Mechanism Of Lipid-anchored MfNACsa Transcription Factor In Response To Drought Stress

Studying and exploring the drought stress signal perception,transduction and response are the emphasis and difficulty of drought-resistant mechanism.The transcription factors play an important role in drought signal transduction,which transfer the drought signal into cell response.Although a majority of transcription factors are exclusively localized to the nucleus to activate responses,some identified integral membrane transcription factors,must be activated for nuclear translocation upon exposure to environmental or developmental cues through post-transcriptional or post-translational regulation.Notably,no studies are available on the nuclear translocation mechanism of lipid-anchored transcription factors without transmembrane domains.Medicago falcata,closely related to the model legume,Medicago truncatula,grows in adverse environmental regions.'PI502449',a diploid variation of M.falcata,is confirmed more tolerant against drought and cold stresses.The research on the regulatory mechanisms underlying drought stress in M.falcata 'P1502449' can provide guidance for the breeding of drought resistance and variety improvement.In the study,we screened a dehydration responsive MJNACsa gene that was previously identified from Medicago falcata RNA-seq data,and the research work focused on the regulation mechanism of MfNACsa transcription factor in response to the drought stress.In our research,the subcellular fraction analysis and FM4-64 staining experiments confirmed that MfNACsa was a lipid anchored protein,and the distribution of MfNACsa protein was dependent on the S-palmitoylation/depalmitoylation regulation.The deleted form of the CRKCAGQ motif or the inhibitor(2-bromopalmitate)of S-acyl transferase treatment compromised MfNACsa protein association with lipid bilayer biomembrane,and the palmitoylation reaction of MfNACsa in vitro further confirmed that the S-acylation(also known as palmitoylation)modification of MfNACsa was responsible for biomembrane location.To identify that the translocation of MfNACsa protein resulted from depalmitoylation,the transgenic M.truncatula roots transiently expressing the 35S::MfNACsa-GFP fusion protein were treated with hydroxylamine(NH2OH),which was used to detect protein depalmitoylation through the cleavage of thioester linkages between Cysteine residues and palmitate chains.The confocal image and cell fractionation experiments showed that the enrichment of MfNACsa-GFP fusion protein in the nucleus during NH2OH treatment.The result suggested that the MfNACsa translocated to the nucleus upon depalmitoylation in response to the drought stress.The reversal of S-palmitoylation in vivo is catalyzed through thioesterases that cleave the thioester bond between the S-palmitoylated protein and the palmitate group.Increasing evidence suggests APT1 and APT2 in the animal system are the major APTs(Acyl-protein thioesterases)that mediate the depalmitoylation of diverse cellular substrates.In our research,we showed that the newly characterized M.truncatula acyl-protein thioesterase 1(MtAPT1)regulated MfNACsa translocation.Firstly,we searched the possible acyl lipid thioesterases based on Medicago JCVI database,which was conducted a cluster analysis of identified thioesterase-like proteins(ALT1-4 from Arabidopsis thaliana,MKS2 from the wild tomato species solaum habrochaitis subspecies glabratum,and YBGC from Escherichia coli),revealing that the MtAPT1 exhibited 45%bootstrap value with AtALT family members and belonged to a single Hotdog fold acyl lipid thioesterase,the active site residues(aspartate,glycine,and valine in the DXXGXV motif)were conserved in the MtAPT1 protein.MtAPTl protein was localized in the cytoplasm.The colocalization and cell fractionation experiments confirmed that MfNACsa-GFP was able to be detected in the nucleus,whereas inactived form of MtAPT1 and other pollen thioesterase overexpression did not affect the translocation of MfNACsa,suggesting that MtAPT1 translocated MfNACsa protein into nucleus through the activity of MtAPT1 induced by unkown mechanism in response to drought stress.In the present study,we deduced that MfNACsa is a positive regulator in response to drought stress.The ectopic expression of MJNACsa in M.truncatula remarkably enhanced drought tolerance,which exhibited remarkably more blade number of aerial parts and lateral root number under PEG simulated drought stress,whereas two Tntl knockout mutants of the homologous MfNACsa gene in M.truncatula were more sensitive to PEG simulated drought stress.The soil drought stress experiment also confirmed that an increased survival rate and reduced electrical conductivity were detected in MfNACsa ectopic expression lines compared with wildtype(WT,R108)plants,whereas nacsa mutants were more sensitive to soil drought stress.To identify the target genes of nuclear relocated MfNACsa under drought stress,the RNA-seq analysis of MfNACsa ectopic expression lines and WT plants under 50%PEG-8000 treatment for 4 h was carried to identify the transcriptional response regulated through MfNACsa.The gene ontology(GO)annotation revealed that oxidation-reduction-,lipid transport-and lipid localization-related genes were significantly differentially expressed compared with WT.The Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis revealed that the differentially expressed are involved in starch and sucrose metabolism,phenylpropanoid biosynthesis,glycolysis/gluconeogenesis,and fatty acid metabolism.Among which MtGly I(lactoylglutathione lyase),a key gene of the methylglyoxal detoxification system was significantly upregulated,while the relative expression of MtGly 1 was not significant different between the two nacsa mutants and WT plants under 50%PEG-8000 treatment.The results of electrophoretic mobility shift(EMSA)and chromatin immunoprecipitation(ChIP)assays confirmed that MfNACsa directly binds to the MtGly I gene promoter to active transcription under drought stress.It had previously been reported that elevated glyoxalase I(Gly I)activity could maintain glutathione homeostasis.In our research,The ratio of reduced(GSH)and oxidized(GSSG)glutathione in the seedlings of the MfNACsa ectopic expression plants was remarkably increased compared with WT plants,whereas the GSH/GSSG ratio in Tntl knockout mutants was remarkably decreased compared with WT plants.The result indicated MfNACsa involved in the regulation of GSH homeostasis under drought stress.These results demonstrated that MfNACsa protein associates with the biomembrane through S-acylation.Under drought stress,MfNACsa relocates to the nucleus through depalmitoylation catalyzed by MtAPT1.The processed MfNACsa protein directly binds to the MtGly I gene promoter,thereby involved in the GSH homeostasis under drought stress.Furthermore,we demonstrate that the conserved CRKCAXX motif of ATAF subfamily members in Glycine max is vital for biomembrane association,thus providing a general mechanism for the nuclear relocation of lipid-anchored ATAF transcription factors,suggesting that the lipidation regulation plays important role in plant responses to drought stress rapidly.