Identification And Function Characterization Of SnRK2 Family In Marchantia Polymorpha

Green plants are the most important producers in the biosphere.They convert solar energy into biological energy through photosynthesis,thereby providing energy for other life on the earth.Transition from aquatic to terrestrial is the most important direction in the plant evolution.Plant landing has affected the evolution of life on earth and laid the foundation for biodiversity.One of the main problems faced by pre-land plants during the landing process is how to absorb and transport water and control transpiration.For this reason,plants have evolved a series of structures and signal transduction systems.Abscisic acid(ABA)signal directly affects water absorption and transpiration by controlling plant root development and regulating stomata opening and closing,thus becoming the main signal pathway for higher plants to respond to drought stress,salt stress and other abiotic stresses.At present,the ABA signal transduction pathway of angiosperms has been resolved,and its core components include: ABA receptor(Pyrabactin Resistance/PYR1-Like/Regulatory Component of ABA Receptor,PYR / PYL / RCAR),protein phosphatase 2C family(protein phosphatases 2 C,PP2C),Sucrose non-fermenting 1-related protein kinases 2,Sn RK2,ABA responsive element binding protein/ABRE binding factors,AREB / ABF.As an early landing plant,bryophyte do not have real roots or typical stomatal apparatus composed of guard cells.However,recent studies have shown that there is a conservative ABA signaling pathway between the mosses Physcomitrella patens and the angiosperm Arabidopsis thaliana,and its Sn RK2 family member Pp OST1 has been shown to regulate opening and closing of the Arabidopsis’ guard cells.In Marchantia polymorpha,researchers found that the homologous genes of the key elements of ABA signal transduction in angiosperms,including receptors,phosphatases and kinases,can participate in ABA signal transduction.Studies based on gene phylogeny also proved that genes related to ABA signal transduction had been differentiated and formed before the differentiation of bryophytes.Although it has been identified in previous studies that both the ABA receptor and phosphatase PP2 C of Marchantia polymorpha are involved in ABA signal transduction,little is known about the evolution and function of MpSnRK2.In order to study the function and evolution of the members of the Sn KR2 family of Marchantia polymorpha,we found that there were only two members of the Sn RK2 family in Marchantia polymorpha,which were named MpSnRK2.1 and MpSnRK2.2,respectively.A series of bioinformatics,molecular biology,biochemistry and genetic modification studies have been carried out,and the following research results have been obtained:1.Cloning and bioinformatics analysis of MpSnRK2.1 and MpSnRK2.2Bioinformatics analysis revealed that there are only two members of the Sn RK2 family in geocache,which were named MpSnRK2.1 and MpSnRK2.2.The coding region of the MpSnRK2.1 gene is 1041 bp,encoding 347 amino acids,with a predicted molecular weight of 58.68 k Da.The coding region of the MpSnRK2.2 gene is 1059 bp,encoding 353 amino acids,with a predicted molecular weight of 58.65 k Da.Phylogenetic analysis showed that both MpSnRK2.1 and MpSnRK2.2 are located in the clade III of plant Sn RK2,which is the oldest Sn RK2 subfamily in plants.Motif analysis revealed that the motif of Sn RK2 in terrestrial plants is highly conserved.Genetic structure analysis showed that the gene structures of MpSnRK2.1 and MpSnRK2.2 were different,with only one exon in MpSnRK2.1 and nine exons in MpSnRK2.2.The amino acid sequence multiple alignment showed that the amino acid sequences of MpSnRK2.1and MpSnRK2.2 are highly conserved with known Sn RK2 s protein sequences.Both of them contained a conserved kinase catalytic domain and a relatively diverse C-terminal structural domain and the MpSnRK2.2 was more similar to Arabidopsis Sn RK2 s than MpSnRK2.1.The above analysis indicated that MpSnRK2.1 and MpSnRK2.2 are in the clade III of plant Sn RK2,which is the oldest clade and the major clade involved in ABA signaling.Moreover,the MpSnRK2.1 and MpSnRK2.2 are more ancient than the Sn RK2 s of the moss plant,Physcomitrella patens.MpSnRK2.2 was more similar to Arabidopsis Sn RK2.2/3/6 than MpSnRK2.1 in both gene structure and amino acid sequence similarity.2.The expression of MpSnRK2.1 and MpSnRK2.2 is induced by ABATo analysis the relative expression of MpSnRK2.1 and MpSnRK2.2 under the treatment of ABA in different concentrations,fluorescent quantitative PCR was employed.The relative expression of MpSnRK2.1 did not change significantly under the treatment of 0.25 μM and 0.5 μM ABA,and was induced only when the ABA concentration increased to 1 μM;whereas the expression of MpSnRK2.2 showed a significant increase when 0.25 μM ABA was used,and had the greatest increase at 0.5μM ABA.The above results indicated that the expression of MpSnRK2.1 and MpSnRK2.2 was induced by ABA with difference sensitivity,and MpSnRK2.2 is more sensitive to ABA compared with MpSnRK2.1.3.MpSnRK2.1 localizes to the nucleus while MpSnRK2.2 localizes to the nucleus and cytoplasmMpSnRK2.1 and MpSnRK2.2 fusion yellow fluorescent protein(YFP)subcellular localization vectors were constructed and transiently expressed in the tobacco leaves for subcellular localization analysis.Strong YFP fluorescence was detected in the nucleus of the tobacco cells expressing MpSnRK2.1-YFP,while strong YFP fluorescence was detected in the nucleus and cytoplasm when MpSnRK2.2-YFP was expressed.Subcellular localization analysis showed that the intracellular distribution of MpSnRK2.1 and MpSnRK2.2 was obviously different.4.Both MpSnRK2.1 and MpSnRK2.2 have the ability to autophosphorylateTo verify the autophosphorylation ability of MpSnRK2 s,recombinant proteins of MpSnRK2.1 and MpSnRK2.2 were obtained in this study using an E.coli expression system.Using a Phos-tag-based protein phosphorylation validation method,it was found that MpSnRK2.1 and MpSnRK2.2 migrated slower on SDS-PAGE gel with the addition of Phos-tag,while MpSnRK2.1 and MpSnRK2.2 migrated faster after treatment with alkaline phosphatase because of some of the phosphate groups on the proteins were dephosphorylated.The migration rates of MpSnRK2.1 and MpSnRK2.2on SDS-PAGE gel without the Phos-tag did not change whether the ATP or alkaline phosphatase exist.The above results indicate that both MpSnRK2.1 and MpSnRK2.2have the ability of autophosphorylation.5.MpSnRK2.1 interacts with Mp ABI1 but not with At ABI1/At ABI2PP2C is a core component in the ABA signaling pathway.In ABA signaling,PP2 Cs bind to Sn RK2 and regulate its activity.While At ABI1 and At ABI2 are the major PP2 Cs involved in ABA signaling in Arabidopsis,there is only one homolog of ABI1 in the liverwort genome,Mp ABI1,which has been reported to be involved in ABA signaling as well.To investigate the function of MpSnRK2.1,we analyzed its interaction with Mp ABI1,At ABI1 and At ABI2 using yeast two-hybrid.The yeast two-hybrid(Y2H)results showed that yeast harbouring MpSnRK2.1 and Mp ABI1 were able to grow normally on amino acid-deficient screening medium,while the control was unable to grow on it.Yeast harbouring MpSnRK2.1 and At ABI1 or At ABI2 could not grow normally on amino acid-deficient screening medium.The above results showed that MpSnRK2.1 interacted with Mp ABI1 but not with At ABI1 or At ABI2.6.MpSnRK2.2 interacts with both Mp ABI1,At ABI1 and At ABI2Yeast two-hybrid(Y2H)results showed that yeast expressing both MpSnRK2.2and Mp ABI1,both MpSnRK2.2 and At ABI1,and yeast expressing both MpSnRK2.2and At ABI2 grew normally on it,while the control could not.It showed that MpSnRK2.2 interacted with Mp ABI1,At ABI1 and At ABI2.7.Overexpression of MpSnRK2.2 in Arabidopsis causes transgenic Arabidopsis to be more sensitive to ABASince MpSnRK2.2 interacts with both At ABI1 and At ABI2 in Arabidopsis,the MpSnRK2.2 was overexpressed in Arabidopsis,revealed that the MpSnRK2.2overexpression lines were more sensitive to ABA compared with wild type.The growth of MpSnRK2.2 overexpressed Arabidopsis was not significantly different from that of the wild type on MS medium without the addition of ABA,whereas the root length of overexpressed MpSnRK2.2 Arabidopsis on MS medium with the addition of ABA was significantly shorter than that of the wild type control,only 37.7% to 54.3% of the control.In summary,two members of the liverwort Sn RK2 family,MpSnRK2.1 and MpSnRK2.2,were analyzed and cloned in this study.The phylogenetic analysis showed that both of them are located in the clade III of Sn RK2.Both MpSnRK2.1 and MpSnRK2.2 have the ability of autophosphorylation,but differ in gene structure,sequence similarity,degree of response to ABA,subcellular localization,and interaction pattern with ABI1.Compared with MpSnRK2.1,MpSnRK2.2 not only has a more similar gene structure and amino acid sequence to the clade III members of Sn RK2 in Arabidopsis,but also interacts with At ABI1 and At ABI2 in Arabidopsis.Overexpression of MpSnRK2.2 in Arabidopsis led to more sensitivity to ABA in transgenic Arabidopsis.Those experimental results indicated that the functional diversity occurred in Sn RK2 family of liverwort,and MpSnRK2.2 is closer to Sn RK2 s in angiosperm compared with MpSnRK2.1.This study provides a preliminary exploration of the evolution and functional differentiation of the Sn RK2 family members in liverwort and provides a new perspective to study the evolution of ABA signaling in terrestrial plants.