Comparison Of Annexins In Medicago Truncatula And The Functional Analysis Of MtANN2 Under Salt Stress

Annexins present in eukaryotes consist of multiple phylogenetically conserved members and participate in diverse stress-related biological processes by binding membrane phospholipids in a Ca²⁺-dependent way.There is scarce information,however,on annexins in legume,particularly forage legume.According to the phylogenetic analysis based on the conserved functional domains and gene structure of annexins from seven representative plant species,this study firstly compared the expression pattern and salt response of three Medicago truncatula annexins from different evolutionary branches.We then focused on the biological function of MtANN2,which showed continuous and relatively stronger induction by Na Cl,in salt response using molecular biology.The main results are as the followings:Annexins from the seven plants were divided into three main branches.Besides the members shared with lower plants,higher plants have two additional branches with more members in dicot than in monocot.These members shared a conserved domain named annexin repeat and the Ca²⁺binding motif of K/GXGX.Expression analysis showed that three annexin genes representing individual branch were expressed in the tested tissues with a relatively higher level in root and lower level in leaf.The transcriptional level of the three genes was increased by Na Cl treatment and displayed different patterns under the treatment of La Cl₃,a calcium channel inhibitor.Among the three,MTR＿0276s0050,which was progressively induced by Na Cl treatment,shared the highest homology（70.65%）with At ANN2 in Arabidopsis,and hereafter,was named MtANN2.When transiently expressed in tobacco,the fluorescence of the MtANN2-GFP recombinant protein was predominantly distributed on membrane of the epidermal cells,indicating that MtANN2 is a membrane protein.Moreover,we identified a T-DNA insertion line of atann2 in Arabidopsis.Compared with the wild type,the homologous atann2 mutant was shorter and weaker,and statistical analysis revealed lower number of the lateral roots and less fresh weight of root.Under Na Cl treatment,both the wild type and atann2 delayed germination and growth of the lateral roots.The latter was more severely affected by salt stress than the former.Ectopic expression of MtANN2 partly rescued the morphologic defects of atann2 and its hypersensitivity to salt treatment,suggesting that At ANN2 is involved in plant growth and salt response.Under normal conditions,there was no obvious phenotypic difference between the transgenic Arabidopsis lines overexpressing MtANN2 and the wild type.Upon salt treatment（200 m M）,leaves of the wild type grown in soil turned wilted earlier than the MtANN2overexpression Arabidopsis.Examination of the content of chlorophyll or Na⁺showed that relative to the wild type,the MtANN2 overexpression lines had higher content of chlorophyll and lower content of Na⁺in leaves.These results suggest that overexpression of MtANN2 in Arabidopsis enhanced salt tolerance.MtANN2 was predicted to possess four potential phosphorylation sites:T₃,Y₁₂₅,T₂₇₀and Y₃₀₃.To mimick different phosphorylation status of MtANN2,T₃ alone or the four above mentioned amino acids were mutated simultaneously to A or D for non-phosphorylated（T₃A or 4A）or phosphorylated（T₃D or4D）MtANN2,respectively,by site-directed mutagenesis.The constructs for constitutive expression were transformed separately into atann2 or wild type,and dozens of positive lines have obtained for individual construct.For the MtANN2-4D overexpression plants,which showed no obvious morphological abnormality,the content of chlorophyll or Na⁺in leaves was similar to that in the MtANN2 overexpression lines.Importantly,we have obtained the transgenic Medicago truncatula overexpressing MtANN2 or the mutated MtANN2.Further analysis of these genetic materials would help to reveal the biological function of MtANN2 in Medicago truncatula.In conclusion,the study investigated the expression of representative genes in three evolutionary branches of annexins in Medicago truncatula and the biological function of MtANN2.The results indicate that MtANN2 plays a positive role in salt response.This research provides a candidate gene for breeding alfalfa varieties with improved salt tolerance.