Physiological Mechanisms Of Salt Tolerance In The Early Seedling Stage Of Rapeseed And A Special Profile Of The Effects Using Melatonin

Seedling survival rate under salinity stress,vegetative growth,yield,and yield quality of rapeseed（Brassica napus.L）are highly related to seed germination and early growth performance.In this study,morpho-physiological traits,biochemical responses,and anatomical changes of different cultivars of rapeseed were investigated under different levels of sodium chloride for evaluation and selection of the most important traits correlated with the salt tolerance in rapeseed at the germination and seedling stages.The results indicated a big difference between the two stages concerning the salt tolerance of rapeseed cultivars.Therefore,due to the importance of the germination stage,we performed a transcriptome profiling analysis using the most salt-tolerant cultivar and the most salt-sensitive one at the germination stage to identify potential candidate genes involved in the salt stress responses.Moreover,the potentiality of melatonin（MT）application（0,25,50,or 100μM）as a seed priming agent in mediating K⁺/Na⁺homeostasis and attenuating the salinity stress-mediated oxidative damage,photosynthetic inhibition,and anatomical changes,besides improving the yield and quality was studied.Thus,the study of morphological,physiological,biochemical,anatomical,and molecular responses to salt stress could be helpful for further breeding and development of tolerated genotypes,which have a good performance against salinity conditions.We summarized the main results as follows:（1）Assessment of morphological,physiological,biochemical,and anatomical traits for evaluating salt tolerance in rapeseed cultivarsIn this study,we investigated the sodium chloride tolerance at the seedling stage among 10 rapeseed cultivars based on membership function values（MFV）and Euclidean cluster analyses by exposing seedlings to 0,100,or 200 m M Na Cl.The Na Cl toxicity significantly（p<0.05）reduced growth,biomass,endogenous K⁺levels,relative water content,and increased electrolyte leakage,soluble sugar levels,proline levels,and antioxidant enzyme activities.SPAD values were highly variable among rapeseed cultivars.The 10 cultivars were identified into three divergent（tolerant,moderately tolerant,and sensitive）groups.We found that the cultivars with higher MFV values（Hua6919 and Yunyoushuang2;0.90 and 0.84,respectively）were classified as the most salt-tolerant cultivars.In contrast,cultivars with the lower MFV values（Yangyou9 and Zhongshuang11;0.17 and 0.14,respectively）were classified as the salt-sensitive cultivars at the seedling stage.The rapeseed cultivars were further subjected to photosynthetic gas exchange and anatomical trait analyses.Among the photosynthetic gas exchange and anatomical traits,the stomatal aperture area was the most highly significant correlated（p<0.01）with salinity tolerance in rapeseed cultivars,and thus was important for future studies aiming to improve salinity tolerance in rapeseed.In the second experiment,we evaluated the performance of rapeseed cultivars against salt stress using MFV of certain germination traits,including the germination rate,vigor index,besides the lengths,fresh weights,and dry weights of plant shoot and root at the germination stage.Based on the MFV of certain germination traits,we have calculated the salt tolerance coefficients among rapeseed cultivars.A great difference in the salt tolerance was found in rapeseed cultivars between the germination and seedling stages.Interestingly,the results of the germination stage showed that Yangyou9（YY9）achieved the highest value of MFV,while Zhongshuang11（ZS11）achieved the lowest value,thus they were selected as the most salt-tolerant cultivar and the most salt-sensitive cultivar at the germination stage,respectively.Therefore,YY9 and ZS11 at the germination stage were selected for identifying potential mechanisms involved in the salt tolerance by using the transcriptome profiling analysis.（2）Comparative transcriptome profiling of seed germination phases in two rapeseed cultivars contrasting in salinity toleranceIn this study,results of the moisture content indicated that the four-time points,i.e.,0,12,24,and 48h showed a significant phenotypic difference between the most salt-tolerant cultivar and the most salt-sensitive one.Moreover,the quantification assay of biochemical indices including hydrogen peroxide（H₂O₂）accumulation,besides catalase（CAT）,superoxide dismutase（SOD）,and ascorbate peroxidase（APX）activities indicated a highly significant（p<0.05）difference among both cultivars under salt stress conditions at the selected time points.Therefore,a dynamic transcriptome analysis was carried out at the selected four-time points of seed germination in the selected two rapeseed cultivars under normal and Na Cl stress conditions.The identified differentially expressed genes（DEGs）between the two cultivars were 5325,6941,5835,and 9114 down-regulated and4463,4710,5431 and 10997 up-regulated in Yangyou9＿Salt stress（YY9＿SS）/Zhongshuang11＿Salt stress（ZS11＿SS）at 0,12,24,and 48h,respectively.Furthermore,in each germination phase,we specified the expression profiles and main enriched pathways.For the seed germination process,a considerable DEGs number,including those implicated in the“pentose phosphate pathway”,“protein processing in endoplasmic reticulum”,“pyruvate metabolism”,and“glycolysis/gluconeogenesis”pathways were specifically and transiently induced at the first 12 hours.In the comparison of salinity tolerance between both cultivars,the“MAPK signaling pathway-plant”,“plant hormone signal transduction”,and carbohydrate metabolism related-pathways were significantly enriched.We found many DEGs related to starch/sucrose,glycolysis/gluconeogenesis,fermentation,and tricarboxylic cycle（TCA）among the tolerant and sensitive cultivars.More specifically,in the plant hormone transduction pathway,genes related to auxin（AUX）signal transduction,i.e.,Auxin1（AUX1）,Transport Inhibitor Responses 1（TIR1）,Gretchen Hagen 3（GH3）,and Small Auxin-Upregulated RNA（SAUR）exhibited significant upregulation in the tolerant samples to regulate cell enlargement and activate the germination process.Genes related to cytokinin（Arabidopsis Histidine Phosphotransfer（AHP）and Type-A Arabidopsis Responses Regulator（A-ARR））,gibberellin（GA;Phytochrome-Interacting Factor（PIF））,brassinosteroids（BRI1-Associated receptor Kinase1（BAK1）,BR-Signaling Kinase 1（BSK）,Xyloglucan:xyloglucosyl transferase（TCH4）,Brassinazole resistant1/2（BZR1/2）,and Cyclin-D3（CYCD3））,and salicylic acid（Nonexpressor of Pathogenesis-related genes（NPR1）,Pathogenesis-related genes（PR1）and TGACG-BINDING FACTOR（TGA））signaling transduction were also up-regulated in the salt-tolerant samples.While genes related to abscisic acid（Protein Phosphatase 2C（PP2C）and ABA-responsive element（ABRE）-binding factor（ABF））,and Ethylene Response Factor1/2（ERF1/2）)and ethylene（ETHYLENE INSENSITIVE3（EIN3））signaling relatively highly up-regulated in the dry seeds and under salt stress of the sensitive cultivar.Therefore,these genes could be participating in the salt sensitivity and seed dormancy in rapeseed.（3）Melatonin enhances salt tolerance and yield quality of rapeseed plants by improving the antioxidant system,ion homeostasis,photosynthetic activities,and anatomical changesWe found that 50μM melatonin（MT）application imparted a very prominent impact on growth,metabolism of antioxidants,photosynthesis,osmolytes,secondary metabolites,yield,and fatty acids composition.Days required for the appearance of the first flower and 50%flowering were decreased by MT application.Exogenous MT treatment effectively decreased the oxidative damage by significantly declining the generation of superoxide and hydrogen peroxide under saline and non-saline conditions,as reflected in lowered lipid peroxidation,heightened membrane stability,and up-regulation of antioxidant enzymes（CAT,SOD,and APX）.Furthermore,MT application enhanced the chlorophyll content,photosynthetic rate,relative water content（RWC）,K⁺/Na⁺homeostasis,soluble sugars,and proline content.Moreover,MT application obviously improved the oil quality of rapeseed cultivars by reducing glucosinolates,saturated fatty acids（palmitic and arachidic acids）,and enhancing unsaturated fatty acids（linolenic and oleic acids except erucic acid were reduced）.Yield related-traits such as silique traits,seed yield per plant,1000 seeds weight,seed oil content,and yield biomass traits were enhanced by MT application.The anatomical analysis of leaf and stem showed that stomatal and xylem vessels traits are associated with sodium chloride tolerance,yield,and seed fatty acid composition.These results suggest the supportive role of MT on the quality and quantity of rapeseed oil yield.