The Molecular Basis Of Atriplex Canescens,A Secretohalophyte With Salt Bladders,In Response To NaCl

Salinity is one of the most severe abiotic factors threatening agricultural productivity and ecological environment throughout the world.Most forages and crops are glycophytes with limited genetic potential of salt tolerance because they have been cultivated under more favorable conditions.In contrast,desert halophytes have evolved multiple adaptation strategies in terms of morphological structure,physiological and biochemical characters as well as molecular genetics to deal with harsh saline environments,which contain abundant stress-resistant gene resources and have important research value.Our previous study found that Atriplex canescen,a secretohalophyte with salt bladders,was extremely salt-tolerant.In saline environments,this species can accumulate a large amount of Na⁺in leaf salt bladders and excrete it from the plant by rupture to maintain K⁺homeostasis of the mesophyll tissues,and synthesize more organic osmolytes,thereby reducing metabolic cell damage,maintaining the water balance in plants and finally improving the photosynthetic efficiency.However,the related molecular basis of these important physiological mechanisms is poorly understood.Therefore,the objectives of this thesis are to systematically analyze the transcriptome response related to Na⁺accumulation in salt bladders,ion homeostasis,water balance and photosynthesis of A.canescens under NaCl treatment through RNA-Seq.The key genes involved in Na⁺accumulation in salt bladders were screened and identified,and the mechanism by which salt bladders regulated plant water balance was preliminary analyzed.The main findings are as follows:1.Under 100 mM NaCl treatment,the abundance of transcripts encoding transporters/channels for essential elements such as K⁺,Ca²⁺,Mg²⁺,N,P and microelements such as Cu²⁺,Zn²⁺was significantly increased,which was conducive to promote the uptake and transport of nutrient elements in A.canescens.In addition,some genes related to Na⁺transport(such as AcSOS1 and AcHKT1)in leaves of A.canescens were induced by NaCl treatment,and several related transcripts of proteins that provided the energy for Na⁺transport were also up-regulated,which might play crucial roles in the excretion of Na⁺via salt bladders.2.The transcripts of a number of genes related to the synthesis of organic osmolytes in leaves were significantly upregulated by 100 mM NaCl treatment,which was beneficial to accumulate more organic solutes to protect cell structures and enhance the ability of osmotic adjustment under salt treatment.Furthermore,100 mM NaCl was conducive to promote water transport and maintain water balance in A.canescens by inducing the expression of aquaporin genes in leaves.3.After treated with 100 mM NaCl for 6 h,the key transcripts participating in the C₄ photosynthetic pathway were preferentially up-regulated in A.canescens,and the transcript levels of other genes encoding complexes/proteins related to chlorophyll biosynthesis,electron transport and C₃ carbon fixation were significantly increased under NaCl treatment for 24 h.It was indicated that moderate NaCl could gradually activate the key parts of photosynthesis,and then improve the photosynthetic capacity of A.canescens.4.The gene encoding the plasma membrane Na⁺/H⁺antiporter,AcSOS1,was cloned from A.canescens.It was localizated on the plasma membrane by transient expression in tobacco,and functioned as a Na⁺efflux transporter in Saccharomyces cerevisiae as well as helped to maintain intracellular K⁺homeostasis.The expression of AcSOS1 in mature leaves was strongly induced by NaCl.Besides,brushing salt bladders resulted in a rapid up-regulated expression of AcSOS1 in mature leaves,and the amplitude of change enhanced with increasing NaCl concentrations.It was shown that AcSOS1 involved in the Na⁺accumulation of salt bladders in A.canescens,and might mediate Na⁺exclusion from epidermal cells.5.Two genes encoding AcHKT1;1 and AcHKT1;2 were cloned from A.canescens,which all belong to the HKT subfamily I.AcHKT1;1 was mainly expressed in roots of A.canescens and significantly induced by salt treatment,which might participate in root Na⁺absorption.While AcHKT1;2 was mainly expressed in mature leaves and significantly induced by NaCl.AcHKT1;2 was located on the plasma membrane and functioned as a selective Na⁺uptake transporter in Saccharomyces cerevisiae and Xenopus laevis oocytes.Moreover,brushing salt bladders significantly reduced the expression of AcHKT1;2 in mature leaves,indicating that AcHKT1;2 might be preferentially localized on the salt bladders and mediated Na⁺into epidermal bladder cells.6.The volume of leaf salt bladders in A.canescens was increased and swelled under NaCl treatment,concomitantly salt bladders began to break and their rupture rate increased with the intensity of NaCl treatment,indicating that a large amount of water entered into the salt bladders as Na⁺accumulation under salt treatment.After brushing the salt bladders,the water potential gradient of A.canescens from root to stem and from stem to leaf were significantly decreased under NaCl treatment,non-stomatal water loss was obviously increased,as well as leaf succulence and root activity were significantly reduced,indicating that salt bladders participated in the establishment and maintenance of the water potential gradient from root to shoot,thereby regulating the water balance in plants.7.Brushing salt bladders directly decreased the expression of AcTIP2;2 in mature leaves of A.canescens,while significantly stimulated a high up-regulated expression of AcTIP4;1 and AcTIP1;3,indicating that AcTIP2;2 involved in the water accumulation of salt bladders,and might mediate the water compartmentalization to the vacuole of epidermal bladder cell and AcTIP4;1 and AcTIP1;3 might involved in regulating the accumulation of water in mesophyll cells.In summary,all above results laid a theoretical foundation for systematically elucidating the salt-tolerant mechanism of the secretohalophyte with salt bladders.