The Role Of Leucine-Rich Repeats Receptor-Like Kinases In Antarctic Moss Pohlia Nutans To Adapt Harsh Environment

Antarctic composing of two parts of the terrestrial and marine is the extremely environmental areas with drastic temperature,permanent ice sheets,strong wind and paradoxically low levels of liquid water availability.The unique geographical configuration and the extreme climate of Antarctic coastal region makes the crytogams(mosses and lichens)have the abundant species diversity and unique charactieristics,which are fundamentally different from those of other regions in the world.They have unique adaptability to extremely low temperature resistance,high salt tolerance and anti-ultraviolet radiation.Mosses are the dominate Antarctic vegetation.Howerver,the cellular and molecular mechanism of moss adapting to environmental stress remains unknown.The LRR-RLKs(leucine-rich repeat receptor-like kinases,the largest superfamily of receptor-like kinases)as the primary receptor in perception and processing of extracellular stimuli,may play key roles in response to harsh environment of the Antarctic.In this study,the transcriptome annalysis of the Antarctic moss Pohlia nutans was performed using the Illumina high-throughput sequencing technology after salt treatment.We discovered the important stress-related genes leucine-rich repeat receptor-like kinases and analyzed their transcriptional pattern in response to salt stress.From which,we screened three receptor-like kinases genes(PnLRR-RLK19,PnLRR-RLK44 and PnLRR-RLK27),ascertained their roles in resistance to abitotic stresses through overexpression in Arabidopsis and Physcomitrella patens,and uncovered the molecular mechanisms of these genes in adaption to abiotic stress in plants.These results will contribute to clarify the molecular mechanism of Antarctic moss in their physiological progress of acclimatizing and adapting the harsh Antarctic environments,and accelerate practical exploitation of Antarctic moss gene resources in improving the stress resistance in economic crops.1 Screening transcription patterns of PnLRR-RLK geneHight-throughtput illumina sequencing was used to analyze the gene transcription pattern of P.nutans under salt treatment(200 mM NaCl,1 h).72922 unigenes with mean length of 704 bp were obtained;23747 transcripts were longer than 500 bp.Moreover,a total of 56 putative LRR-RLK genes were screened out from the Antarctic moss P.nutans transcriptome,named as PnLRR-RLK1-56.The phylogenetic tree showed that these PnLRR-RLKs could be classified into 11 major subgroups(LRR I-XIII)according to the kinase domains sequence of the Arabidopsis homologues.According to the differential expression analysis of the Antarctic moss P.nutans transcriptome,4 LRR-RLK genes were upregulated and 10 were downregulated after salt treatment.Three genes PnLRR-RLK19/27/44 with significantly upregulation were screened out for bioinformational analysis.Multiple sequence alignment revealed that PnLRR-RLK19/27/44 contained conserved protein kinase domain and had 45%-60%identity with with other species LRR-RLKs family proteins(eg P.patens subsp.patens,Arabidopsis and Oryza sativa).PnLRR-RLK19 had 43.7%and 16.9%identity with PnLRR-RLK27 and PnLRR-RLK44.PnLRR-RLK27 had 19.6%identity with PnLRR-RLK44.Phylogenetic analysis showed that PnLRR-RLK19 and PnLRR-RLK27 protein belong to the LRR II subgroup,PnLRR-RLK44 belongs to the LRR XI subgroup.Real-time qPCR analysis showed that LRR-RLK19/27/44 were significantly upregulated after salt,cold,20%PEG6000,UV and ABA treatment.And the functional characteristics of them will be further analyzed Genome-wide characterization of the Antarctic moss Pohlia nutans transcriptome.2 Function analysis of PnLRR-RLK19/27/44 inabiotic stress toleranceWe cloned these PnLRR-RLK genes,identified their subcellular localization,uncovered their phenotypic characteristic and molecular mechenism through overexpression in Arabidopsis and Physcomitrella patens.The results were as follows:2.1 PnLRR-RLK27 positively regulates salinity and oxidation-stress toleranceThe GFP and PnLRR-RLK27:GFP was transformed into the protoplasts isolated from P.patens protonema or Arabidopsis Col-0 and incubated in the dark for a period of time.GFP signal was observed with a confocal laser scanning microscopy.The results showed that in the control pBI221 vector transformed protoplasts,GFP proteins distributed evenly in the cell membrane,the cytoplasm and nucleus.However,the PnLRR-RLK27:GFP were localized on the plasma membrane.Consistently,the PnLRR-RLK27:GFP proteins colocalized with both plasma membrane marker H+-ATPase-RFP protein(the marker protein of the plasma membrane with emiting red fluorescence)and the lypophilic dye N-(3-triethylammoniumpropyl)-4-(6-(4-(diethyl amino)phenyl)hexatrienyl)pyridiniu m dibromide(FM4-64)in the plasma membrane.These results suggested that the the green signal of PnLRR-RLK27:GFP proteins colocalized with both the red signal of the lypophilic dye FM4-64 and plasma membrane marker H+-ATPase-RFP protein,and emited yellow signal.These results indicated that the PnLRR-RLK27 is a plasma membrane protein.The function research of PnLRR-RLK2 7 in Physcomitrella:PnLRR-RLK27/pTFH15.3 was constructed using homologous recombination and transformation of P.patens protoplasts to generate the transgenic moss.The 3 mm size stem tips of control and transgenic gametophytes were placed on BCD medium containing 0 mM or 125 mM NaCl for 7 weeks.On standard BCD medium,the overexpression of PnLRR-RLK27 did not affect the growth of P.patens.On 125 mM NaCl medium,the overexpression of PnLRR-RLK27 in Physcomitrella significantly increased the salinity tolerance in their gametophyte growth;the clone size of the WT plants was 4.7 mm,while three transgenic gametophytes were 10.2-11.4 mm.At the BCD solid medium containing 10 ?M and 15 ?M ABA,the gametophyte sizes of the transgenic P.patens plants was 1.6-fold and 1.8-fold larger than that of the WT plants,respectively.These results indicated that the overexpression of PnLRR-RLK27 in Physcomitrella significantly enhanced the salinity tolerance and reduced ABA sensitivity in their gametophyte growth.Meanwhile,the transcription levels of salt-related ion chanal protein genes PpENA2,PpSHPl and PpSHP2,ABA-related genes PpABI3a,PpAB13b,and stress-responsible genes PpDBFl,PpCOR47 and PpCORTMC-AP3 in transgenic P.patens were also significantly increased in transgenic plants.The function research of PnLRR-RLK27 in Arabidopsis:PnLRR-RLK27/pROK2 vector was constructed and transformed into Arabidopsis Col-0 plants by floral-dip method,and finally generated PnLRR-RLK27 heterologous expression.After NaCl treatment,the germination rates of the two transgenic plants were 1.5-fold higher than that of WT plants,the two transgenic plants formed 1.3-fold longer primary roots in compared to WT plants.After ABA treatment,the germination rates of two transgenic plants were 2-fold compared to WT plants.After 0.5 ?M and 0.75 p.M ABA treatment,the root length of the transgenic plants was 2,2-fold and 10-fold longer than that of the WT plants.The root length of the transgenic plants was averagely 1.6-fold longer than 1.4-fold longer at 1.0?M H2O2,and the lateral root numbers in the transgenic Arabidopsis plants were averagely 1.6-fold more than the WT plants.The results suggested that overexpression of the PnLRR-RLK27 in Arabidopsis enhanced plant tolerance to salinity and oxidation stresses,reduced the sensitivety to ABA.PnLRR-RLK27 overproduction reduced the levels of malondialdehyde(MDA)and ROS Meanwhile,overexpression of PnLRR-RLK27 increased the activities of reactive oxygen species(ROS)scavenging enzymes superoxide dismutase,peroxidase and catalase,and enhanced the expression levels of antioxidantive genes AtAPX1,AtAPX2,AtCAT2 and AtZAT10,and improved the contents of scavenger proline.The real-time qPCR showed that after 2 h treatment with 200 mM NaCl,the transcription levels of salt tolerance genes AtHKT1 and AtSOS3,stress-/ABA-responsible genes AtMYB2,AtABF3,AtDREB2A,AtRD22,AtRD29A,AtRD29B,AtKINl and AtCOR47 in transgenic Arabidopsis were all significantly higher in transgenic plants.Taken together,these results suggested that PnLRR-RLK2 7 as a conserved signaling regulator conferred salt tolerance by associated with the regulation of the antioxidative system and the stress-/ABA-mediated signaling network.2.2 PnLRR-RLK19 functions in salinity and drought stress adaptationSubcellular localization analysis revealed that it was a plasma membrane protein.To elucidate the role of PnLRR-RLK19 in response to abiotic stress,it was transformed into Arabidopsis Col-0.On 1/2 MS agar medium,there was no obvious phenotypic difference between the transgenic plants.After NaCl treatment,the germination rates of three overexpressing plants were 25.0%higher than the control plants,three overexpressing plants showed 2-fold longer primary roots than the control plants.The germination rates of three overexpressing plants were more than 11.5%in compared to the control plants at 0.5 ?M ABA.The root length of the overexpressing plants was 1.5-fold longer than that of the control plants at 7.5 ?M ABA.In the presence of H2O2 and mannitol,three overexpressing lines showed 1.5-fold and 2-4-fold more lateral roots than the WT plants,respectively.We observed the growth statues of soil-grown plants in response to salt and drought stress conditions.After NaCl treatment,the overexpressing plants showed less etiolated and yellowing leaves than the control plants with some died.In addition,assays of drought stress showed that the overexpressing plants lost water much more slowly than the control plants.Compared with control plants,the stress-/ABA-reponsive genes(AtMYB2,AtDREB2A,AtRD22,AtRD29A,AtRD29B and AtKIN1),sodium channel protein genes(AtP5CS1 and AtSOS3)and ABA-responsive genes(AtAREBl,AtAREB2 and AtABF3)all had higher transcript levels in transgenic plants after salt or drought treatment.These results speculated that PnLRR-RLK19 not only increased plants tolerance to salt stress and insensitivity to ABA,but also enhanced plant drought and H2O2 tolerance by positively controled the expression of stress-/ABA-related gene.2.3 PnLRR-RLK44 confers salinity and ABA stress toleranceSubcellular localization revealed that PnLRR-RLK44 was also mainly localized on plasma membrane.Two ransgenic Arabidopsis plants constitutively overexpressing the PnLRR-RLK44 were generated to further analyze the function by Agrobacterium-mediated floral-dip method.On 100 mM NaCl medium,the germination rates of two over-expressing lines were 17.6%more than the WT plants;the root length of two over-expressing lines were 28.1%and 12.6%longer than that in the WT plants.On 0.5 ?M ABA medium,the germination rates of two over-expressing lines were 20.0%higher than the WT plants,the root length of two over-expressing lines were more than 28.9%in compared with the WT plants.Therefore,the real-time qPCR was used to test the expression levels of ABA signaling genes and salt-related genes.And qRT-PCR analysis conformed that the expression levels of ABA negatively regulating gene AtABI1 and sodium channel protein genes AtHKTl and AtSOS3,salt stress-reponsive genes AtP5CS1 and AtADH1 were significantly increased in transgenic plants.Meanwhile,the expression levels of ABA biosynthesis genes AtNCED3,AtABA1 and AtAAO3,and ABA early response genes AtMYB2,AtRD22,AtRD29A and AtDREB2 were lower in transgenic Arabidopsis after salt stress treatment.Therefore,these results suggested that PnLRR-RLK44 might involve in regulating salt stress-related and interacting with negative regulator of ABA signaling pathway.Taken together,PnLRR-RLK27 conferred salt tolerance by associated with the up-regulation of ROS antioxidative system and the stress-/ABA-mediated signaling network.PnLRR-RLK19 not only increased plants tolerance to salt stress and insensitivity to ABA,but also enhanced plant drought and H2O2 tolerance by positively controled the expression of stress-/ABA-related gene.PnLRR-RLK44 might involve in regulating salt stress-related and interacting with negative regulator of ABA signaling pathway,which was different with PnLRR-RLK19 and PnLRR-RLK27,thereby contribute to the salinity tolerance of the Antarctic moss P.nutans.The results suggested PnLRR-RLKl9/27/44 not only has certain redunancy,but also has specificites in role of abiotic stress response,suggesting their functional conservation and differentiation during plants evolution.3 Function analysis of PnE3 in abiotic stress toleranceE3 ubiquitin ligases poly-ubiquitinate a target protein for its degradation or post-translational modification.And they play vital role in plants development and regulating plants responses to abiotic stress.However,the role of ubiquitin ligase in the Antarctic mosses are still unclear.The full-length of PnE3 was obtained from the mRNA isolated from the Antarctic moss,and its cDNA contained a 2097 bp open reading frame(ORF)and encoded a protein of 698 amino acids with a calculated molecular weight of 76.2 kDa and a pI of 6.79.Amino acid sequence analysis showed that PnE3 contained a U-box domain(amino acids 288-351)and four ARM domain(amino acids 418-625).PnE3 was induced by cold,high salinity,drought and ABA,indicating that PnE3 might involved in plants abiotic stress signaling pathways.The GFP and PnE3:GFP was transformed into the protoplasts isolated from Arabidopsis Col-0.The results showed that PnE3 were localized in cytoplasm.The function research of PnE3 in Physcomitrella:After NaCl treatment,the gametophyte sizes of the WT plants was 1.4-fold larger than that of overexpressing P.patens plants.After ABA treatment,the gametophyte sizes of the WT plants was 1.6-fold larger than that of overexpressing P.patens plants.Meanwhile,the transcription levels of ABA-responsible genes PpABI3A,PpABI3B and PpABI3C in overexpressing P.patens were also significantly increased in overexpressing plants after salt stress treatment.The function research of PnE3 in Arabidopsis:on NaCl medium,the germination rates of the WT plants were more than 40%compared to overexpressing plants,the roots length of the WT plants was 2.4-fold longer than overexpressing plants.On ABA medium,the germination rates of the WT plants were more than 25.6%compared to overexpressing plants,the roots length of the WT plants was 3.7-fold longer than overexpressing plants.The expression levels of abitic stress related genes(AtABI3,AtABF3,AtDREB2A and AtRD29A)were significantly decreased in overexpressing Arabidopsis plants.Therefore,these results suggested that PnE3 enhanced plant tolerance to abitic stress possibly through interacting with negative gene of the ABA signaling pathways.