| Soil salinization is considered one of the most severe environmental hazards that lead to ecological environment deterioration and restrict agricultural development.Soil salinity causes low water potential,specific ion toxicity effects and oxidative stress on plants,which seriously affects the normal growth and development of plants.Cotyledon is an important component of plant seed and plays a key role in seed germination and early seedling growth.It has many functions such as nutrition storage,photosynthetic capacity and stress defense.Hence,cotyledon plays an important role in seed germination,seedling establishment and the completion of plant life cycle.Castor bean(Ricinus communis L.)is one of the most important oilseed crop species and belongs to the Euphorbiaceae family.It has many excellent characteristics,such as resistance to drought,saline-alkali and poor nutrients environment,and has attracted great attention in biological improvement of saline-alkali land in recent years.Castor bean is a dicotyledons whose cotyledons have photosynthetic capacity.The hypocotyl of the castor bean elongates causes the cotyledons to protrude from the soil surface during skotomorphogenesis.It plays an important role in early seedling development.However,the mechanism and function of the cotyledon,especially in response to salt stress,are not clear in the early growth stage.In this study,the cotyledon of the oil crop castor bean were studied and the following problems were revealed,including the effects of salt stress on development dynamics and photosynthetic capacity of cotyledon,functional differences between cotyledon and leaf in response to salt stress,the important function of cotyledon in seedling response to salt stress by cotyledon removal.The main results are as follows:(1)During photomorphosis phase,the fresh weight in the cotyledon decreased significantly under salinity stress.The photosynthetic pigments of cotyledon in salinity treatment were higher than control treatment in the early photomorphosis phase.In addition,salinity stress inhibits stomata development,destroys chloroplast membrane structure,and affects thylakoid membrane protein synthesis in cotyledon.The results showed that salinity stress significantly inhibited the growth process of castor bean cotyledon and reduced its mobilization ability to store nutrients.The photosystemⅡof cotyledon also showed strong adaptability to salt stress in castor bean seedling.Cotyledon can maintain normal physiological function of PSⅡunder salt stress by improving turnover efficiency of D1 protein.Moreover,in order to maintain the growth and development of castor bean seedlings under salt stress,cotyledon can provide photoassimilates for castor bean seedling through coordination of PSⅠand PSⅡto maintain photosynthetic activity,affects the assembly and quantity of Cyt b6f complex,adjust LEF to CEF ratio and affects the synthesis of ATP and NADPH.(2)Physiological analysis of castor bean cotyledon and leaf in response to salt stress showed that the biomass and photosynthetic parameters of both cotyledon and leaf decreased with increasing salt stress,and the those parameters of leaf were much greater than that of cotyledon.The Na~+contents in cotyledon and leaf were both increased with increasing salinity concentration.In addition,accumulation of Na~+in cotyledon was much greater than that in leaf.The results showed that the tolerance of cotyledon to neutral salt stress was higher than that of leaf in early seedling stage of castor bean,and the growth inhibition of cotyledon and leaf under alkaline salt stress were stronger than that of neutral salt stress.The cotyledon of castor bean seedlings accumulated a large amount of Na~+under salt stress,which alleviated the degree of ion toxicity in the photosynthetic system and maintained a high content of photosynthetic pigments in the leaf.Through comparative analysis of physiological parameters between cotyledon and leaf in response to salt stress,the different physiological response of cotyledon and leaf during early seedling stage of castor bean plant were illuminated.(3)Proteomic analysis revealed 30 and 42 differentially accumulated protein spots in castor cotyledon and leaf under salt stress,respectively.According to mass spectrometry result,there were 11 proteins overlapped in the cotyledon and leaf under salt stress.The physiological and proteomic results highlighted that cotyledon could improved the activity of TCA and provided more energy to help leaf cope with salt stress.The leaf saved carbon structures to synthesize osmotic substances,and the enhancement of chlorophyll synthesis and electron transfer in leaf could also maintain photosynthesis under salt stress.(4)Through physiological and proteomic research,it was confirmed that there were different mechanisms between castor bean cotyledon and leaf in response to salt stress,and the cotyledon removal was further used to clarify the role of cotyledon in this process.Physiological analysis of castor seedling responses to salt stress before and after removal of cotyledons showed that cotyledon had positive effects on salt stress.Cotyledon removal significantly inhibited the growth and photosynthesis of leaf under salt stress.The ultrastructure of chloroplasts was greatly altered and distorted in the leaf of plants whose cotyledons were removed,and cotyledon removal dramatically increased the Na~+content with increasing salinity.In addition,cotyledon removal also affected the contents of osmotic adjustment substances in the leaf of plants under salt stress.These results showed that cotyledon could alleviate the reduction in photosynthetic capability caused by stomatal closure in the leaf of castor bean plants.Moreover,cotyledon could also counteract Na~+toxicity and maintain the K~+/Na~+to alleviate osmotic stress caused by salt stress.In addition,the existence of cotyledon could reduce osmotic stress of leaf under salt stress and reduce carbohydrate consumption caused by osmotic adjustment substance synthesis of leaf,thus promoting the growth of leaf in castor bean seedling under salt stress.(5)On the basis of physiological analysis in the responses of leaf to salt stress at different cotyledon removal levels,transcriptomics and metabolomics analysis were carried out for further analysis.Transcriptomics analysis showed that 730 genes were differentially expressed in leaf of the NR seedling under salt stress,while 838 genes were differentially expressed in that of the TR seedlings.In metabolomics analysis,115 differently accumulated metabolites were found in leaf of the NR seedling under salt stress,while 161 differently accumulated metabolites were found in that of the TR seedlings.In order to further clarify the effects of cotyledon removal on the salt stress response of castor bean seedlings,339 differential genes and 103 differential metabolites were identified by comparing the transcriptome and metabolome data of castor bean seedling before and after cotyledon removal under salt stress.Combined with KEGG pathway analysis and KGML network analysis,metabolome and transcriptome data were integrated to comprehensively analyze the gene and metabolite levels in leaf of castor seedlings after cotyledon removal under salt stress.It was found that after cotyledon removal,salt stress leads energy deficit in leaf of castor bean.The leaf need to activate glycolysis and tricarboxylic acid cycle to cope with the energy deficit caused by cotyledon removal under salt stress.After cotyledon removal,the substrate consumption was reduced by inhibiting the biosynthesis of terpenoid skeleton,and more metabolites were retained for glycolysis and tricarboxylic acid cycle,which further maintained the energy metabolism process.In addition,cotyledon removal also significantly inhibited the glycerophospholipid and linoleic acid metabolisms in leaf under salt stress,which seriously affected the process of stress defense and oxidative stress in leaf under salt stress,and adversely affected the salt stress response process of castor bean seedling. |
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