Physiological And Gene Expression Regulation Mechanism Of Leymus Chinensis In Response To Alkaline Environment

Alkaline soil exists widely and is one of the important environmental factors restricting agricultural development.Especially in the western part of Songnen Plain in Northeast China,most saline-alkali soils are alkaline.Soil salinization is an important environmental factor limiting crop and forage production in Northeast China.Leymus chinensis is the dominant species of alkalized grassland in Northeast China.Because of its high alkali resistance and high forage value,Leymus chinensis has been widely used in the restoration and utilization of alkalized land.Therefore,studying the alkali tolerance physiological and molecular mechanism of this species can not only enrich the theory of plant alkali resistance,but also develop its genetic resources to provide support for the improvement of alkali resistance of other forages and crops.In this paper,in order to ensure that the samples of the control and treatment groups are genetically highly uniform,a Leymus chinensis was randomly collected under field conditions,and different ramets were generated by asexual reproduction for all physiological and molecular experiments.Different ramets from the same plant were divided into control group and alkali stress group,and were treated with 200 m M alkaline salt solution(Na HCO3∶Na2CO3=9∶1)for alkali stress treatment,and the control group was irrigated with nutrient solution for 30 days.The anatomical structure,photosynthetic parameters,photosynthetic pigment content,chloroplast ultrastructure,osmotic adjustment substances(10 soluble sugars,19free amino acids,4 polyols,K~+concentration,Na~+concentration and soluble protein)were compared between the control group and the stress group,content and transcriptional profiles,and explored the physiological and gene expression regulatory mechanisms of Leymus chinensis in response to an alkaline environment.The main results and conclusions are as follows:(1)The inhibition of alkaline stress on the growth and photosynthesis of Leymus chinensis was mainly caused by the restriction of gas exchange.Alkaline stress significantly reduced the net photosynthetic rate and transpiration rate of Leymus chinensis leaves,but did not reduce the content of photosynthetic pigments in leaves,nor did it destroy the ultrastructure of chloroplasts.By comparing the leaf cross-section structure of the control group and the stress group,it was found that the volume and number of the aerenchyma in the leaves treated with alkali stress were significantly smaller.These data indicated that alkali stress did not cause obvious ion damage to Leymus chinensis leaves,and the decrease of photosynthetic rate and growth inhibition may be mainly caused by the decrease of aerenchyma volume and stomatal conductance.(2)Leymus chinensis adapts to the alkaline environment by regulating the content of Na~+and K~+and the accumulation of osmotic regulators such as sucrose.Alkaline stress significantly increased the accumulation of Na~+in leaves and roots,while decreasing the content of K~+in both organs.Alkaline stress did not cause obvious ion toxicity to Leymus chinensis.The increase of Na~+content could reduce the water potential of Leymus chinensis roots and leaves,and increase the water absorption capacity to resist the osmotic stress caused by alkali stress.Of the 10 soluble sugars detected in the leaves,the contents of 7 were significantly increased.Trehalose in the leaves of the stress group was 11.79 times higher than that in the leaves of the control group.Sucrose and Fructose were the highest in the control group,respectively.6.32times and 6.29 times that of glucose(Glucose)and ribose(Ribose)were 4.67 times and 4.39times that of the control group,respectively.In the roots,the contents of 6 kinds of soluble sugars were increased,and the sucrose content increased by a maximum of 5.4 times that of the control group,followed by the glucose content significantly increased to 3.94 times that of the control group,and fructose and trehalose were 2.81 times and 2.24 times that of the control group,respectively.times.Four polyols of pinitol,inositol,xylitol,sorbitol/mannitol were detected in leaves,and only pinitol content was increased by 2.46 times.Three polyols,pinitol,inositol,and sorbitol/mannitol,were detected in the roots,and only the sorbitol/mannitol content was increased.In leaves,the contents of 12 amino acids increased,and the contents of histidine and glycine increased the most,which were 3.58 times and 3.42 times that of the control,respectively.However,only 3 kinds of free amino acids increased in root,proline,aspartic acid and glutamic acid increased to 3.99 times,1.42 times and 1.49 times of the control.In order to explore the role of each organic solute in osmoregulation,the percentage of each solute molar content to the total solute molar content was calculated.In terms of the contribution rate,sucrose in the leaves of Leymus chinensis under stress was the substance with the highest contribution rate,accounting for 33.34%of the total solute,followed by pinitol and fructose with contribution rates of 10.5%and 9.92%,and asparagine and serine in free amino acids.,Alanine contributed the highest rate of 5.64%,5.52%,5.67%,respectively.In the roots treated with stress,sucrose was also the organic solute that contributed the most to osmotic regulation,accounting for 37.2%of the total solute,followed by asparagine with 19.08%,pinitol with6.73%,and aspartic acid with 6.07%.In general,sucrose was the most important osmotic regulator of Leymus chinensis under alkali stress,and asparagine was the organic solute with the greatest osmotic regulation among free amino acids.(3)Leymus chinensis resists ion toxicity by increasing the expression of key resistance genes HKT8,NHX,Dehydrin,LEA,and NR.In order to explore the gene expression regulation mechanism of Leymus chinensis in response to alkali stress,control treatment and alkaline stress treatment were applied to Leymus chinensis ramets,and Leymus chinensis samples under different conditions,developmental stages and organs were collected,including roots,stems,leaves and buds.,spikelets,flower organs and other 38 samples.After extracting RNA from each tissue,they were mixed in equal proportions,and then the mixed samples were sequenced with full-length transcriptome using Pac Bio Sequel sequencing technology(third-generation sequencing technology).Reference sequences for transcriptome sequencing.This paper pays special attention to the expression changes of alkali tolerance genes.The results show that alkali stress up-regulates the expression levels of NHX and HKT8 genes in leaves,up-regulates the expression of alkali-tolerant genes such as NHX,Dehydrin and LEA in roots,and also up-regulates the expression of NRT genes in roots.Physiological analysis of this paper showed that alkali stress did not cause obvious ion damage to Leymus chinensis leaves,and Leymus chinensis had strong Na~+regulation ability under alkali stress.Under alkaline stress,Leymus chinensis may resist Na~+poisoning by up-regulating the expression level of HKT8 gene in leaves.HKT8 gene has been proved to have the function of expelling Na~+into conducting tissues.High expression of HKT8 gene in leaves will promote the excretion of Na~+from leaf cells to transporting tissue.in the guiding organization.The up-regulation of NHX gene in leaves under alkali stress will promote the separation of accumulated Na~+into vacuoles in leaves,avoiding ion toxicity.The up-regulation of NHX in roots will promote Na~+compartmentalization and reduce the toxic effect of Na~+on Leymus chinensis root cells.The up-regulation of Dehydrin and LEA gene expression levels in roots will improve the ability of root cells to cope with osmotic regulation,and the up-regulation of NRT in roots will promote NO3~-.The absorption,increase the ability of osmotic regulation,which is beneficial to nitrogen metabolism.An important contribution of this paper is to use Pac Bio Sequel sequencing technology to establish a reference sequence for the analysis of Leymus chinensis transcriptome,which lays a foundation for the study of Leymus chinensis gene expression and related molecular biology.Alkali stress did not cause obvious ion toxicity to Leymus chinensis,and the inhibition of Leymus chinensis growth and photosynthesis was mainly caused by restricting gas exchange.Biochemical analysis showed that Leymus chinensis mainly accumulated sucrose and asparagine,the most important organic osmotic regulators.Based on transcriptome sequencing data,it was found that Leymus chinensis improved the ability to resist ion toxicity and osmotic stress by enhancing key resistance genes HKT8,NHX,Dehydrin,LEA and NRT.