Functional Analysis Of Ion Transporter Genes In A Rare Relic Recretohalophyte Reaumuria Trigyna

Soil salinization is the main environmental limiting factor for plant growth.Studying the salt tolerance mechanism of plants and using genetic engineering methods to improve the salt tolerance of plants is an economic and effective way to harness and utilize large-scale salinized land.Under salt stress conditions,the ion transporters on the surface of the membrane synergistically completed the excessive salt ions efflux and intracellular compartmentation,and reconstructed or maintained the Na⁺-K⁺ions homeostasis under salt stress.However,the current researches on plant ion transport systems are mostly focused on model plants and crops,and the study of wild recretohalophyte have rarely been reported.Reaumuria trigyna?Tamaricaceae?,as an ancient,narrow distributed and endangered recretohalophyte,has strong adaptability to salinized desert environment.In this work,based on the transcriptome data,we screened and cloned 4 ion transporter genes?RtNHX1,RtVP1,RtHKT1 and RtKCO1?.The molecular characteristics,expression pattern and functions of these proteins were analyzed in order to investigate how ion transporters synergistically reconstruct ion homeostatic balance of plants and improve the salt tolerance of plants.The results of this study are as follow:1.The vacuolar Na⁺/H⁺antiporter from R.trigyna,RtNHX1,contains a1,662-bp open reading frame?ORF?encoding a 553-amino-acid protein,which was located on the vacuolar membrane and rapidly up-regulated by NaCl and exogenous abscisic acid treatment.RtNHX1 can enhanced the salt tolerance of transgenic AXT3 yeast,while it can sequester excess Na⁺and K⁺from the cytoplasm into the vacuole,and maintained a low Na⁺/K⁺ratio of the yeast cell.In addition,RtNHX1 can restore the salt-sensitive growth phenotype of Arabidopsis nhx1 mutants.Under salt stress,transgenic Arabidopsis overexpressing RtNHX1 showed more vigorous growth compared to WT plants,lower leaf Na⁺/K⁺ratio,higher antioxidant enzyme activity and more proline accumulation,confirming that R.trigyna RtNHX1 is a functional antiporter that could confer salt tolerance on transgenic Arabidopsis plants by maintaining Na⁺-K⁺homeostasis and enhancing osmotic and antioxidant regulatory capacity.2.The vacuolar H⁺-pyrophosphatase gene was isolated from R.trigyna by RACE-PCR and RT-PCR base on RtVP1 cDNA fragment from transcriptome database,which contains a 2292-bp ORF encoding a protein of 763 amino acids with the location of plasma membrane and tonoplast,and rapidly up-regulated by NaCl and exogenous abscisic acid treatment.Over-expression of RtVP1 in avp1 mutant can rescued the phenotype of sucrose-sensitive.Over-expression of RtVP1 in WT Arabidopsis can enhanced the accumulation of more glucose,sucrose,and soluble sugars,and improved various plant growth parameters,confirming that RtVP1 can enhanced the growth and development of transgenic Arabidopsis by accelerating carbohydrates metabolism.In addition,the lower Na⁺/K⁺ratio were observed in transgenic yeast cells and transgenic Arabidopsis expressing Rt VP1 under salt stress,indicating that RtVP1 may play an important role in maintaining Na⁺-K⁺homeostasis.The antioxidant enzyme activity and proline accumulation were significantly increased in WT Arabidopsis expressing RtVP1,confirming that this gene confers salt tolerance on transgenic Arabidopsis by enhancing osmotic and antioxidant regulatory capacity.3.Overexpression of RtHKT1 in yeast conferred tolerance to high Na⁺or low K⁺,while transgenic Arabidopsis overexpressing RtHKT1 exhibited tolerance to high Na⁺and sensitivity to high K⁺,or high Na⁺-low K⁺conditions,confirming that Na⁺tolerance in transgenic Arabidopsis depends on a sufficient external K⁺concentration.Transgenic yeast accumulated more Na⁺-K⁺,Na⁺,and K⁺under external high Na⁺,high K⁺,and low K⁺conditions,respectively;transgenic Arabidopsis accumulated less Na⁺and more K⁺,more Na⁺,and more Na⁺-K⁺in the presence of high Na⁺,high K⁺,and low K⁺,respectively.These results indicate that the ion transport properties of RtHKT1 depend on the external Na⁺-K⁺environment.Salt stress induced upregulation of some ion-transporter genes,as well as downregulation of some genes,revealing that multi-ion-transporter synergism maintains Na⁺/K⁺homeostasis under salt stress in transgenic Arabidopsis.Furthermore,overexpression of RtHKT1 enhanced seed germination,biomass accumulation,and chlorophyll content in transgenic Arabidopsis during salt stress.POD and CAT activities,proline content,and relative water content all increased significantly in salt-stressed transgenic Arabidopsis plants,whereas the H₂O₂ and MDA contents decreased dramatically.These results suggest that RtHKT1 confers salt tolerance on transgenic Arabidopsis for maintenance of Na⁺/K⁺homeostasis,antioxidant regulation,and osmotic adjustment.4.Transgenic overexpression of RtKCO1 in yeast conferred tolerance to high Na⁺or high K⁺,while transgenic Arabidopsis overexpressing RtKCO1exhibited tolerance to high Na⁺or low K⁺conditions,confirming that RtKCO1may play different roles in transgenic yeast and Arabidopsis.Although the ion distribution patterns are different,they all maintain a relatively low Na⁺/K⁺ratio.These results indicate that the ion transport properties of RtKCO1 depend on the external Na⁺-K⁺environment,and this gene can maintain a relatively stable Na⁺/K⁺ratio to confer the ability of transgenic yeast cells and Arabidopsis plants to quickly adapt to the external environment.Furthermore,overexpression of RtKCO1 enhanced biomass accumulation,POD and CAT activities,and proline accumulation,indicating that RtKCO1 enhanced salt tolerance on transgenic Arabidopsis by improving the capacity of antioxidant regulation and osmotic adjustment.5.In R.trigyna,RtNHX1 is a functional antiporter that sequesters Na⁺and/or K⁺in the vacuole and pumps out H⁺from vacuole;whereas,Rt VP1utilizes the energy of inorganic pyrophosphate?PPi?hydrolysis as the driving force for H⁺movement across biological membranes to create proton electrochemical potential,which provides a motive source for energizing RtNHX1 to maintain Na⁺-K⁺homeostatic balance in responds to salt stress;RtHKT1 mediates the translocation of K⁺across the plasma membrane,and prevents the Na⁺transfer to shoots cells;whereas,plasma membrane channel protein,RtKCO1,promotes Na⁺and K⁺efflux from the cytoplasm.These ion-transporters synergism reconstructs and maintains the Na⁺-K⁺homeostasis in responding to salt stress,and enhanced the capacity of osmoregulation and antioxidant regulation,which in turn improves salt tolerance to plant.