| Leymus chinensis is a perennial important grass,rhizome plant,rich in nutrition,palatability,mainly in Russia,Mongolia,North Korea,China's eastern inner Mongolia,northeast and northwest and other countries.In recent years,the distribution of L.chinensis tends to expand southward.Due to climate change drought,saline and other adverse factors affect habitat of L.chinensis seriously,especially severe drought will hampered its growth and development and even lead to plant death.In this paper,the effects of plant growth regulators and foliar application of nutrients on the growth,physiological and biochemical reactions of L.chinensis under drought stress were studied in field and laboratory experiment.At the same time,transcriptome technique was used to understand the physiological and molecular mechanism.The research results will provide a useful reference for the production of technical measures to regulate the L.chinensis growth,restoration of plant productivity.Below are main research findings:1.Effects of plant growth regulators on the growth and physiological characteristics of L.chinensis under field conditionsIn the laboratory L.chinensis had been treated with more than 10 kinds of different plant growth regulators,and the 5 kinds of the better efficacious reagents were screened for field experiment.The field experiments were carried for two consecutive years in Xilin Gore under field conditions.The different concentrations of naphthalene acetic acid?NAA?,6-benzylamino adenine?6-BA?,brassinolide?BR?,gibberellin?GA3?and5-aminolevulinic acid?ALA?were used to carry out foliar treatment of the natural grassland inside the fence and the same amount of water as the control?CK?.?1?The results showed that proper concentration of plant growth regulators could increase the plant growth and improve its phsiologcal status of L.chinensis compared with CK.Among the treatments,BR and ALA showed best effectively.?2?The results showed that plant growth regulators could increase the plant height,fresh weight and dry weight of L.chinensis compared with CK.The plant height,fresh weight and dry weight was the highest when spraying ALA?50.0 mg/L?.Besides,the plant growth regulators had the effect on the photosynthetic pigment content of L.chinensis.The contents of chlorophyll a,chlorophyll b,carotenoids and total photosynthetic pigments were increased significantly.Highest content of chlorophyll a,chlorophyll b and total photosynthetic pigments were observed under ALA?50.0 mg/L?treatment,which had significant difference compared with CK.The soluble sugar,soluble protein and free amino acid of plant were increased with the application of plant growth regulators,and the trend was consistent with the morphological index.The maximum amount of soluble sugar and free amino acid were observed when L.chinensis was treated with ALA?50.0 mg/L?,nevertheless the soluble protein was maximum under BR?0.20 mg/L?treatments.The activity of several antioxidant enzymes of L.chinensis was affected by plant growth regulators.The trend was similar to that of morphological indexes,and the activities of superoxide dismutase?SOD?,catalase?CAT?and glutathione reductase?GR?reached the maximum under ALA?50.0 mg/L?treatment,while the best effect peroxidase?POD?and ascorbate peroxidase?APX?were when BR?0.20 mg/L?treatment applied.?3?In brief,ALA?50.0 mg/L?and BR?0.20 mg/L?had the best effect.We selected BR and ALA as the basis for the pot experiment of drought stress ultimately,and further study was carried out.2.Effects of plant growth regulators and foliar nutrition on growth of L.chinensis under drought stressIn pot experiment under drought stress,the effects of BR,ALA and BR or ALA with foliar nutrition on the growth of L.chinensis were studied.The results showed that water deficit was precarious environmental constraint restricting plant growth and productivity,and under stress application of growth regulators could ameliorate damaging effects of drought stress and increase the plant height,fresh weight,dry weight and root activity.The effects of plant growth regulators exhibited more significant effect on plants when combined with foliar nutrition.?1?BR,ALA,BR+foliar nutrition and ALA+foliar nutrition in different concentrations were used to spray seedlings respectively,and their effects on the growth of L.chinensis were studied under drought stress.The results sh owed that the plant height,fresh weight,dry weight and root activity of plant seedlings increased first and then decreased in all conditions,and the growth rate reached the maximum when BR?0.10 mg/L?or ALA?50.0 mg/L?only.Maximum plant height and root activity were observed when BR?0.10 mg/L?sprayed,while fresh weight and dry weight were in ALA?50.0 mg/L?sprayed treatments.?2?Growth rate was maximum when urea?1%?+potassium dihydrogen phosphate?1%?+BR?0.10 mg/L?or urea?1%?+potassium dihydrogen phosphate?1%?+ALA?50.0 mg/L?were used under drought stress.In addition,plant height was maximum when treatment of urea?1%?+potassium dihydrogen phosphate?1%?+BR?0.10 mg/L?applied,while the fresh weight,dry weight and root activity were maximum when treatment of urea?1%?+potassium dihydrogen phosphate?1%?+ALA?50.0 mg/L?applied.3.Effects of plant growth regulators and foliar nutrition on photosynthetic characteristics and chlorophyll fluorescence characteristics of L.Chinensis under drought stressThe results showed that plant growth regulators could improve the photosynthetic efficiency of L.chinensis and maintain its photosynthetic apparatus.?1?Under drought stress,BR,ALA,BR+foliar nutrition and ALA+foliar nutrition was used to treat the L.chinensis.The results showed that the chlorophyll a,chlorophyll b and carotenoids of plant increased first and then decreased after treatment with different concentrations.Maximum chlorophyll a,chlorophyll b,chlorophyll a/b and carotenoids contents were showed under BR?0.10 mg/L?spraying.Urea?1%?+potassium dihydrogen phosphate?1%?+BR?0.10 mg/L?or urea?1%?+dihydrogen phosphate?1%?mixed with ALA?50.0 mg/L?,the indicators reached the maximum under drought stress.In addition,the contents of Chlorophyll a,chlorophyll b,carotenoids and chlorophyll a/b were maximum when urea?1%?+potassium dihydrogen phosphate?1%?+ALA?50.0 mg/L?treatment applied.?2?Net photosynthetic rate?Pn?,stomatal conductance?Gs?and carboxylation utilization?CUE?of plant were decreased,and water use efficiency?WUE?increased first and then decreased with the enhancement of drought stress.Pn,Gs,transpiration rate?Tr?,SUE,WUE and limiting value of stomata?Ls?increased after treatment with BR?0.10 mg/L?or ALA?50.0 mg/L?accompanied by decreasing of intercellular CO2concentration?Ci?.The limit value?Ls?increases while Ci decreases.The potential activity of PS??Fv/Fo?and the light system?effective quantum yield??PS??was decreased,while minimal fluorescence yield?Fo?,maximal fluorescence yield?Fm?,optimal/maximal quantum yield of PSII photochemistry?Fv/Fm?,Fv/Fo and?PS?were increased after treatment with BR?0.10 mg/L?or ALA?50.0 mg/L?.4.Effects of plant growth regulators and foliar nutrition on osmotic adjustment substances in L.Chinensis under drought stressThe role of plant growth regulators on osmotic adjustment ability of L.chinensis under drought stress were studied by using different concentrations of BR,ALA,BR+foliar nutrition and ALA+foliar nutrition in combination.The results showed that plant growth regulators decreased membrane lipid peroxidation,enhanced osmotic adjustment substance contents and cell osmotic adjustment ability of plant.?1?Under drought stress BR,ALA,BR+foliar nutrition and ALA+foliar nutrition was used to treat L.chinensis seedlings with different concentrations,and their effects on osmotic adjustment of plant were studied.After treatment,malondialdehyde?MDA?and leaf conductivity of L.chinensis were decreased compared with drought stress?CK2?,and the contents of proline,soluble protein and soluble sugar were increased.When the BR?0.10 mg/L?or ALA?50.0 mg/L?was sprayed,the MDA content and leaves conductivity decreased to the lowest,and the increase of proline,soluble protein and soluble sugar contents reached the maximum.Furthermore,compared with BR and ALA,the noticeable change of MDA content,leaf conductivity and proline was treatment of BR?0.10 mg/L?,while soluble protein and soluble sugar under treatment of ALA?50.0 mg/L?were observed.?2?When spraying urea?1%?+potassium dihydrogen phosphate?1%?+BR?0.10mg/L?or urea?1%?+potassium dihydrogen phosphate?1%?+ALA?50.0 mg/L?,the MDA content and leaves conductivity decreased to the lowest,and the increase of proline,soluble protein and soluble sugar reached the maximum.Among the effects of BR,ALA and foliar nutrient treatments,MDA,leaf conductivity and proline decreased most with urea?1%?+potassium dihydrogen phosphate?1%?+ALA?50.0 mg/L?,while soluble protein and soluble sugar increased by urea?1%?+potassium dihydrogen phosphate?1%?+ALA?50.0 mg/L?.5.Effects of plant growth regulators and foliar nutrition on activities membrane protective enzymes of L.Chinensis under drought stressStudies have shown that one of the mechanisms by which plant growth regulators improve the drought resistance of L.chinensis is to enhance the activity of cell membrane protective enzymes and to enhance the ability to scavenge free radicals.It is also possible increases the contents of NPK and changes in activity of nutrition and metabolism related enzymes.?1?Under drought stress BR,ALA,BR+foliar nutrition and ALA+foliar nutrition in different concentrations were used to treat L.chinensis seedlings,and their effects on the enzyme activity and NPK content of plant were studied.After treatment with drought stress?CK2?,the activities of POD,SOD,CAT,GR and APX of L.chinensis increased.The activities of POD,SOD,CAT,GR and APX were increased first then decreased after different concentrations of BR and ALA compared with CK2.The activities of POD,SOD,CAT,GR and APX were the highest in BR?0.10 mg/L?or ALA?50.0 mg/L?alone.By comparing the effects of BR and ALA,the activities of POD,SOD and CAT were maximum when BR?0.10 mg/L?applied,and the effect of GR and APX under ALA?50.0 mg/L?treated plants were maximum.When spraying urea?1%?+potassium dihydrogen phosphate?1%?+BR?0.10 mg/L?or urea?1%?+potassium dihydrogen phosphate?1%?+ALA?50.0 mg/L?,the activities of SOD,CAT,GR and APX were noticeably changed.Activity of CAT increased significantly only with urea?1%?+potassium dihydrogen phosphate?1%?+ALA?50.0 mg/L?application and the activity of other others enzymes was maximum when urea?1%?+potassium dihydrogen phosphate?1%?+BR?0.10 mg/L?applied.?2?The activities of nitrate reductase?NR?and malate dehydrogenase?MD?decreased and the activity of acid phosphatase?ACP?increased under drought stress?CK2?.After treatment with different concentrations of BR,ALA and foliar nutrition,the activities of NR and MD decreased first and then increased,and the activity of ACP showed opposite trend,regardless of the normal condition?CK1?or drought stress?CK2?.?3?N,P and K of L.chinensis seedlings decreased under drought stress?CK2?.After treatment with different concentrations of BR,ALA and foliar nutrition,the content of N,P and K decreased first and then increased under normal condition?CK1?or drought stress?CK2?.It was also found that the N and K contents were proportional to the activities of NR and MD,respectively,while the P content was inversely proportional to the activity of ACP.6.Effects of plant growth regulators and foliar nutrition on endogenous hormones in L.Chinensis under drought stressThe results showed that the treatment of exogenous plant growth regulators and foliar nutrition had great influence on the endogenous hormones and their ratio,and the plant growth regulators and foliar nutrition could control the growth of L.chinensis and improve the stress resistance of the plants may relate to signal transduction pathways and the induction of plant endogenous hormones.?1?Under drought stress,different concentrations of BR and ALA were used to treat L.chinensis seedlings,and their effects on plant were studied.The abscisic acid?ABA?and indole acetic acid?IAA?content in L.chinensis increased under drought stress?CK2?,and gibberellin?GA3?and zeatin?ZR?decreased.The contents of ABA increase first and then decreased with the increase of IAA,GA3 and ZR after treatment with BR+foliar nutrition,and the contents of ABA and IAA increased first and then decreased along with GA3 and ZR content that increased after treatment with ALA+foliar nutrition under normal?CK1?and drought?CK2?conditions?2?The ratio of IAA/ABA increased,and the ratio of ZR/ABA and GA3/ABA decreased with BR or foliar nutrition under drought stress.The ratio of IAA/ABA decreased first and then increased after the treatment of BR and ALA or leaf nutrition under normal condition?CK1?or drought stress?CK2?.ZR/ABA and GA3/ABA ratio were first decrease after the increase in normal case.Under drought stress,the ratio of IAA/ABA increased by ALA+foliar nutrition application,and other treatments were reduced.7.Transcriptome analysis in L.Chinensis under drought stressThe transcriptions were analyzed by normal?control?,drought stress and drought stress+ALA treated L.chinensis.The results showed that the changes of many physiological indexes and the effect of plant growth regulator ALA on the drought resistance of L.chinensis were closely related to the gene expression of transcriptome.?1?Under drought stress 1373 genes were significantly different,and 733 genes were down-regulated and 640 genes were up-regulated compared with normal samples.The GO enrichment analysis and the KEGG metabolic pathway were significantly enriched by the differential gene expressed in drought stress.The results showed that L.chinensis reflected the metabolic process,catalytic activity,redox reaction and other metabolic pathways significantly reduced expression,which were the important reasons of plant growth and physiological inhibition.These significant down-regulated genes mainly include the following categories.I)Carbohydrate and respiratory metabolism related genes:genes such as starch and sucrose metabolism,carbohydrate metabolism,glucose catabolism,hexose metabolism,monosaccharide decomposition and carbohydrate decomposition in a number of related metabolic processes were significantly down-regulated under drought stress.The expression of NADH-coenzyme Q oxidoreductase?complex I?,F0F1-type ATP synthase?complex??,which is an important component of the respiratory electron transport chain and oxidative phosphorylation,was significantly down-regulated in the aerobic respiratory metabolic pathway,besides,coenzyme Q-cytochrome c oxidoreductase?complex III?expression was also significantly reduced.Down-regulation of these gene expressions will greatly reduce the ability of cells to produce ATP and reducing power,affecting carbohydrate metabolism,resulting in the plant can't meet the various physiological processes of the material and energy needs.II)Photosynthetic apparatus and photosynthetic capacity related genes:photosynthesis-related gene expressions in many metabolic pathways were significantly reduced in drought stress.Such as photosynthetic system,chloroplast,chloroplast composition,chloroplast coating,photosynthetic membrane,plastid,plastid composition,plastid membrane,thylakoid,thylakoid membrane and other related gene expression,which affect the photosynthetic characteristics to the photosynthetic cell structure of the integrity and stability,and with the formation of photosynthetic capacity of the light system I,ribulose bisphosphate carboxylase complex,photosynthetic system II CP47 reaction center protein,cytochrome b6f complex and other related genes significant down-regulation of the expression which will directly affect the photosynthetic efficiency of the plant,including the photosynthetic reaction and CO2assimilation ability in the photosynthetic process,resulting in decreased productivity and blocked growth.III)Membrane protection system and scavenging free radical enzyme-related genes:important antioxidant enzyme such as APX,POD gene expression was significantly reduced under drought stress,may lead to free radical production and clearance of the balance was broken,resulting in many reactive oxygen species in the cell accumulates and reducing the ability to adapt the adversity.IV)Secondary metabolic material formation-related genes:many of secondary metabolic substance-related gene expression under drought stress were significantly down-regulated.Such as the pathway combined with tetrapyrrole metabolism were down-regulated,which will affect a variety of important physiological substances synthesis like the chlorophyll,heme,protokinin,phytochrome,thus affecting the physiological role.?-linolenic acid metabolism and linolenic acid metabolic pathway gene expression was significantly reduced may change the membrane lipid unsaturated fatty acid content,thus affecting the membrane mobility.Phenylpropane biosynthetic pathway significantly reduced,the plant tissue flavonoids and other protective substances accumulated adverse,weaken its tolerance for adversity.?2?With the drought stress as the control,1315 genes were significantly different from the drought stress+ALA treatment,among which 639 genes were down-regulated and 676 genes were up-regulated.The enrichment analysis of GO enrichment and KEGG in ALA treatment material under drought stress showed that the metabolic pathway of L.chinensis under metabolic process,redox reaction and so on significantly decreased under drought condition,their expression was significantly up-regulated after spraying ALA which was consistent with the effect of exogenous ALA treatment could greatly alleviate the damage of drought stress to its physiological process.These significantly upregulated genes mainly include the following categories.I)Carbohydrate and respiratory metabolic related genes:the samples treated with ALA under drought stress were significantly upregulated in the aerobic respiratory metabolic pathway compared to the control,while respiratory electron transport?Complex I?,F0F1-type ATP synthase?complex??,coenzyme Q-cytochrome c oxidoreductase?complex III?,and the expression of NADH-coenzyme Q oxidoreductase assume up regulation.The up-regulation of these genes can ensure the normal production of ATP and reducing power,maintain the relative respiration and carbohydrate metabolism,and enhance the level of material and energy metabolism in the physiological processes of plants.II)Photosynthetic apparatus and photosynthetic capacity-related genes:under drought stress,the down-regulation of photosynthetic genes in L.chinensis was improved by ALA treatment,and the photosynthetic metabolism and photosynthetic antenna protein metabolic pathway were up-regulated.Such as increasing the expression of psbA gene in L.chinensis,can accelerate the synthesis of D1 protein and contribute to the repair of PSII function in key parts of tolerance stress.This is very beneficial for improving photosynthetic efficiency,increasing carbon source and promoting plant growth.III)Membrane protection system and scavenging free radical enzyme-related genes:the expression of antioxidant activity metabolic pathway increased significantly after ALA-treated under drought conditions,including glutathione peroxidase?GSH-Px?,catalase?CAT?,ascorbate peroxidase?APX?,peroxidase?POD?and superoxide dismutase?SOD?,to clear the excess ROS produced by drought stress in plants,thus effectively prevent membrane lipid peroxidation chain reaction,protecting the normal metabolism and enhancing the plant's drought resistance.IV)Secondary metabolic material formation-related genes:genes related to the formation in the secondary metabolic substances had down-regulated while it was reversed due to ALA treatment under drought stress.The expression of linolenic acid was significantly down-regulated after drought stress,which was opposite after ALA treatment and the fatty acid synthesis pathway and glycosphingolipid biosynthesis were also up-regulated.Significant upregulation of these genes can promote the synthesis of the corresponding substances increase the mobility of the membrane alleviate the damage of the drought to the cell membrane,and promote the stability and resistance of the membrane structure and function.?3?The results showed that brassinolide?BR?synthesis was significantly up-regulated by ALA treatment under drought stress.BR was one of the six major hormones in promoting plant growth,improving plant resistance and signal transduction.We inferred the mechanism of ALA is inducing the up-regulation of genes in BR synthesis pathway,increasing the content of endogenous BR in the cells,regulating other physiological processes and to improve the drought resistance of L.chinensis and promote plant growth. |
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