| Nicotiana tabacum is an important cash crop widely cultivated all over the world.Tobacco seedlings are the basis and guarantee for tobacco field production.The quality of tobacco seedlings directly determines the growth and development of tobacco plants and the quality of tobacco leaves after harvesting.Because most of the seedlings are cultivated in the early spring,and this time most of the southern tobacco areas in China are colder.The rainy climate and the meteorological conditions of low temperature and low light have a relatively unfavorable impact on the cultivation of tobacco seedlings,which seriously restricts the early growth and normal development,and stabilizes the economic benefits of tobacco farmers and the sustainable development of tobacco industry.In this experiment,the feasibility of 5-aminolevulinic acid?ALA?in tobacco seedling application was investigated by means of laboratory simulation and field validation.A series of morphological,physiological and photosynthetic parameters were analyzed after ALA treatment in tobacco seedlings.The application concentration and transcriptome analysis technology were used to understand the mechanism of ALA on improving the stress resistance of plant,provide a practical application of ALA to tobacco production,and solve the problem of tobacco seedlings caused by low temperature and low light weather with new and reliable ideas and methods.Below are main research results:1.Effects of ALA on tobacco seedling resistance and photosynthetic physiology under low light stressIn light incubators of laboratory,simulate low light condition,at light 12 hours/25°C;dark 12 hours/15°C,illumination intensity 100?mol·m-2·s-1,ALA?5 mg/L,10 mg/L,20 mg/L,40 mg/L,80 mg/L?was used to treat 5-leaf stage of tobacco seedlings,and the agronomic traits,chlorophyll content,osmotic adjustment and chlorophyll fluorescence of each treatment was studied.The changes of parameters and other indicators revealed that the low light stress had harmful effects on biomass accumulation,root morphological characteristics,membrane stability,osmotic adjustment,photosynthetic pigment content and chlorophyll fluorescence of tobacco seedlings and the growth of tobacco seedlings was substantially inhibited.However,the application of exogenous ALA effectively alleviated the adverse effects of low light stress on the growth and development of plants.In addition,ALA also has the concentration effect on growth just like other plant growth regulate substances.When the ALA concentration is too high?40 mg/L-80 mg/L?,it also inhibited the growth and development of tobacco seedlings,while the lower concentration of ALA?5 mg/L?the result is not good.Comprehensive considerations of agronomic traits,stress resistance and photosynthetic capacity of the conditions,the concentration of ALA applicationis preferably 20mg/L for tobacco seedlings.2.Effects of ALA on tobacco seedling resistance and photosynthetic physiology under low temperature stressIn light incubators of laboratory,simulate low temperature condition,at light 12hours/15°C;dark 12 hours/10°C,illumination intensity 200?mol·m-2·s-1,ALA 20mg/L,the ALA was used to treat 5-leaf stage tobacco seedlings,and the changes of agronomic traits,chlorophyll content,osmotic adjustment,defense enzymatic activities and chlorophyll fluorescence parameters of each treatment were studied.The results showed that low temperature had a negative impact on the development of tobacco seedlings.However,the root-shoot ratio of plants was greatly increased under low temperature conditions.Low temperature also promoted the accumulation of membrane lipid peroxidation productmalondialdehyde?MDA?,and proline,soluble sugars and soluble proteins,increased activities of antioxidant enzymes.It was found that application of ALA at low temperature would reduce the content of MDA,significantly increase the contents of osmotic adjustment substances such as proline,soluble sugars and soluble proteins,significantly increase activities of antioxidant enzymes and the content of indole acetic acid?IAA?.In terms of photosynthetic characteristics,ALA increased the photosynthetic pigments content of plants,and significantly improved Fm,Fm',Y?NPQ?,NPQ,the chlorophyll fluorescence parameters such as qN,qP,qL,?PS?and ETR,have no significant effect on Y?NO?.3.Effects of ALA on tobacco seedling resistance and photosynthetic physiology under low temperature and low light stressIn the light incubator of laboratory,simulate the double stress of low temperature and low light,under the conditions of illumination of 12 hours/15°C;dark 12 hours/10°C,illumination intensity of 100?mol·m-2·s-1,20 mg/L ALA was used to treat 5-leaf stage tobacco seedlings.The agronomic traits,photosynthetic pigment content,osmotic adjustment substances,defense enzyme activity and chlorophyll fluorescence parameters of each treatment were studied.The results showed that the conditions of low temperature and low light double stress had very negative impact on the growth and development of tobacco seedlings.Under the same conditions,ALA significantly increased the content of osmotic adjustment substances such as proline,soluble sugar and soluble protein in plants,significantly reduced the content of MDA,and significantly increased the activity of SOD,POD,CAT,APX and GR antioxidative enzymes.The treatment of ALA had great influence on the endogenous hormones and their ratio,the content of endogenous hormone abscisic acid?ABA?and IAA in tobacco seedlings resulted in limited defensive physiological changes in tobacco seedlings under low temperature and low light stress,which better ensured the survival and morphogenesis of tobacco seedlings under adverse conditions.4.Sequencing and analysis of transcriptome on tobacco seedlings under low temperature and low light stressALA solution was sprayed to plants under low temperature and low light conditions,through differential expression analysis,GO function analysis,differential gene KEGG enrichment analysis,the mechanism of ALA at the transcriptome level to improve the resistance of tobacco seedlings was studied.The results showed that:?1?Transcriptomics study of tobacco under low temperature stressLow temperature stress caused significant down-regulation of material metabolism,energy metabolism and photosynthesis of tobacco seedlings.Secondary biosynthesis and flavonoid biosynthesis pathways were also significantly down-regulated,while antioxidant-related metabolic pathways was significantly up-regulated and enriched.?2?Transcriptional study of tobacco under low temperature and low light stressLow temperature and low light seriously affected the normal metabolic activities of tobacco seedlings,and their important metabolic pathways were significantly down-regulated.Low temperature and low light stress not only affected the important metabolic processes of the plant,but also affect the morphogenesis and development of the cells,as well as the synthesis pathway of flavonoid organisms.Under low temperature and low light stress,photosynthesis and chloroplast-related metabolic pathways of tobacco seedlings were significantly enriched,including porphyrin and chlorophyll anabolic pathways and photosynthetic related proteins.?3?Molecular mechanism of ALA improving tobacco seedling resistance under low temperature stressAfter spraying ALAunder low temperature stress,the significantly up-regulated gene was significantly enriched in the KEGG metabolic pathway to the photosynthetic antenna protein pathway,the important gene for chlorophyll synthesis of proto-chlorophyllate reductase gene,chloroplast oxygen-enhancing protein,light-harvesting pigment protein?LHC?.The expression of the chlorophyll a/b binding protein gene was also significantly upregulated.In addition,ALA induces glutathione metabolic pathway genes and antioxidant enzyme POD expression,omega-3 fatty acid desaturase,palmitoyl monogalactosyldiester 7 desaturase expression,LRR-type receptor protein in tobacco seedlings.The expression of kinase gene and the enzymes of two important synthetic pathways of ABA were significantly up-regulated,which produced a series of physiological and biochemical changes in tobacco seedlings and improved tobacco resistance.?4?Molecular mechanism of ALA improving tobacco seedling resistance under low temperature and low light stressAfter spraying ALA under low temperature and low light stress,the expression of many important metabolic related genes in tobacco seedlings were significantly up-regulated,including peroxidase gene,glutathione transferase in glutathione metabolism pathway,and glutathione specificity,glutamyl transferase,cytochrome P450,sucrose synthase,amino acid permease,DNA melting enzyme,carboxy terminal ubiquitin hydrolase,nucleic acid polymerase,pyruvate decarboxylase,lipid transfer protein,histidine decarboxylase.The gene of the peptide growth substance-phytosulfonin was significantly up-regulated.In addition,the expression of the gene phosphatase 2C and SnRK2 in the ABA signaling pathway was significantly up-regulated,and it was concluded that ALA was involved in the signal transduction pathway of ABA by up-regulating the expression of SnRK2 gene.To regulate the expression of a large number of genes related to stress resistance downstream of the signaling pathway,this experiment also found that ABA-dependent signaling pathways regulate plant expression of abiotic stress transcription factors MYB48,MYB4,and NAC genes significantly up-regulated,while plant MYB is an important regulator of stress tolerance suggests that ALA may be involved in the regulation of ABA signaling pathway and may initiate the synthesis and metabolism of a series of downstream stressors by regulating transcription factors.5.Effects of ALA treating tobacco seedlings on tobacco growth and physiological characteristics under field conditionsIn order totest and verify the effect of ALA under the conditions of field,thetobacco seedlings were translated to field,which come from the nursery greenhouse used in the actual production of the tobacco area,under the conditions of low temperature and low light weather in the nursery season,the closed seedlings that were not subjected to low temperature and low light stress were used as controls.The morphogenesis of tobacco seedlings,photosynthetic physiology,early rapid growth performance after planting,and economic traits after post-harvesting were analyzed.The results showed that after ALA treatment,tobacco seedlings was significantly improved,and the agronomic traits were substantially upgrade than those of unsprayed ALAcontrols.ALA also significantly improved photosynthesis and SPAD values of tobacco seedlings.The dry matter accumulation and agronomic traits of ALA treatment in the 20 days after transplanting to the field are significantly better than those of the unsprayed ALA.The yield and quality of the harvested tobacco in the late field are basically the same as those of the control,and it is also significantly better than the treatment of unsprayed ALA.It indicates that ALA can better improve the ability of tobacco seedlings to resist low temperature and low light in the tobacco breeding process.The research resultssuggestthat ALA is a regulator with applicable and potentialvalue,and its application concentration of 20 mg/L has also proved to be suitable. |
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