褪黑素誘導(dǎo)油菜耐硒性的生理生化、解剖及分子表達(dá)譜研究
發(fā)布時(shí)間：2020-11-03 04:12
硒(Se)是一種普遍存在的元素,土壤中的平均含量為0.1-2 mg kg-1。硒具有豐富的營(yíng)養(yǎng)價(jià)值,是人和動(dòng)物所必需的非金屬元素。較低劑量的硒有利于植物的生長(zhǎng)發(fā)育,并具有減輕重金屬脅迫的能力。但是,過(guò)量的硒對(duì)植物有毒性,尤其對(duì)高等植物的影響還有待商榷。油菜(Brassica napus L.)是主要的食用油料作物,在一定范圍內(nèi),還具有很強(qiáng)的抵抗重金屬、類金屬毒性的能力。因此,甘藍(lán)型油菜對(duì)抵抗硒毒性具有較高的潛在價(jià)值。目前,許多研究專注于使用植物生長(zhǎng)調(diào)節(jié)劑來(lái)減輕重金屬、類金屬的植物毒性。鑒此,本研究旨在通過(guò)使用褪黑素(MT)作為生長(zhǎng)激素來(lái)評(píng)估硒的雙重特性以及甘藍(lán)型油菜對(duì)硒毒性的耐受性。為此,進(jìn)行了相關(guān)實(shí)驗(yàn),所得結(jié)果主要如下:(1)探究亞硒酸鈉形式的硒(0、25、50和100 μM)對(duì)四個(gè)甘藍(lán)型油菜品種('浙大619'、'浙大622'、'浙雙758'和'浙油50')10日齡幼苗(水培生長(zhǎng))在生理生化、細(xì)胞超微結(jié)構(gòu)和基因組變化方面的潛在影響。結(jié)果表明,過(guò)量的Se(ⅣV)(特別是100 μM)顯著降低了植物生長(zhǎng)參數(shù)、色素含量以及水溶性蛋白質(zhì)水平,并且產(chǎn)生了過(guò)量的H202和MDA。在葉片和根部都檢測(cè)到抗氧化酶活性和超氧化物歧化酶(SOD)、過(guò)氧化物酶(POD)、抗壞血酸過(guò)氧化物酶(APX)、谷胱甘肽還原酶(GR)以及還原性谷胱甘肽(GSH)的轉(zhuǎn)錄水平均顯著增加,而過(guò)氧化氫酶(CAT)和根部的氧化型谷胱甘肽(GSSG)水平并沒(méi)有顯著增加。此外,過(guò)量的Se(ⅣV)會(huì)導(dǎo)致葉肉組織和根尖細(xì)胞的超微結(jié)構(gòu)受損�？傮w而言,在50μ的Se(ⅣV)水平下沒(méi)有觀測(cè)到顯著的植物毒性作用。在25μ的Se(ⅣV)水平下則表現(xiàn)出邊際效應(yīng),主要表現(xiàn)為植株生長(zhǎng)增強(qiáng)、色素含量和蛋白水平增加,以及限制H202和MDA過(guò)量產(chǎn)生。同時(shí),也檢測(cè)到ROS解毒酶活性(CAT活性除外)邊際增加或減少以及GSH和GSSG水平升高。與葉相比,Se(ⅣV)在根中的富集要更高。這種富集在'浙大622'中最高,在'浙雙758'中最低,其次是'浙大619'和,浙油50'�？傮w結(jié)果表明,'浙大622'對(duì)Se(ⅣV)毒性最敏感,而'浙雙758'耐受性最強(qiáng)。(2)研究了Se(ⅣV)(0、25、50和100 μM)對(duì)不同甘藍(lán)型油菜品種(即'浙大619'、'浙大622'、'浙油50'和'浙雙758')28日齡幼苗(溫室田間條件)生理生化、解剖結(jié)構(gòu)和分子變化的雙重效應(yīng)。結(jié)果表明,添加硒可通過(guò)促進(jìn)礦物質(zhì)吸收從而促進(jìn)植株生長(zhǎng)并提高植株生物量,同時(shí)還提高了水溶性蛋白質(zhì)和糖的水平以及與色素和氣體交換參數(shù)相關(guān)的光合效率。較高的硒水平降低了光合效率,阻礙了營(yíng)養(yǎng)物質(zhì)吸收,而脯氨酸積累導(dǎo)致的滲透脅迫和根中較高的硒積累導(dǎo)致了植株生長(zhǎng)減緩和生物量減少。通過(guò)激活參與AsA-GSH循環(huán)和ROS-MG解毒的酶,添加硒使ROS(過(guò)氧化氫、超氧化物自由基)、丙二醛和甲基乙二醛(MG)的積累最小化。但是,通過(guò)降低抗壞血酸水平、過(guò)氧化氫酶、谷胱甘肽還原酶和脫氫抗壞血酸還原酶的活性和表達(dá)水平,硒水平的升高破壞了抗氧化劑的氧化代謝。次級(jí)代謝物基因(PAL、PPO)的上調(diào)揭示了硒調(diào)控參與氧化應(yīng)激的轉(zhuǎn)錄網(wǎng)絡(luò)的作用。葉和根超微結(jié)構(gòu)的受損揭示了硒的植物毒性�？傊�,研究結(jié)果揭示了硒(不高于25μM)的保護(hù)機(jī)制,這體現(xiàn)在硒能增強(qiáng)植物形態(tài)、光合作用、滲透保護(hù)和氧化還原平衡,以及通過(guò)減少ROS和MG組分從而增強(qiáng)ROS-MG解毒相關(guān)酶的酶活性。硒過(guò)量則會(huì)通過(guò)削弱上述參數(shù)而增強(qiáng)植物毒性,特別是在濃度為100 μM時(shí)。在上述甘藍(lán)型油菜品種中,'浙大622'對(duì)硒脅迫最敏感,而'浙雙758'則表現(xiàn)出最大耐受性。(3)本研究針對(duì)生理特征、硫醇生物合成和抗氧化系統(tǒng),評(píng)估了外源性褪黑素(MT)(0、50和100μM)對(duì)甘藍(lán)型油菜28日齡幼苗(田間條件)Se(ⅣV)(0、50、100和200 μM)脅迫的防御作用。結(jié)果表明,在Se(ⅣV)脅迫條件下,植物生長(zhǎng)和生物量積累均呈劑量依賴性抑制,同時(shí),也觀測(cè)到色素含量、凈光合速率(Pn)和PSⅡ光化學(xué)效率(Fv/Fm)受到影響,在水培液中添加MT后可有效緩解脅迫。此外,外源施用MT降低了氧化損傷和脂質(zhì)過(guò)氧化,并保護(hù)了細(xì)胞膜免受硒毒性的負(fù)面影響,這主要是通過(guò)降低硒誘導(dǎo)的活性氧積累實(shí)現(xiàn)的。褪黑素還通過(guò)恢復(fù)葉片水分和糖水平來(lái)緩解滲透壓。與單獨(dú)硒脅迫相比,使用褪黑素和硒(MT + Se)共同處理,以下物質(zhì)都有更高水平,包括:ROS解毒酶(SOD、APX、GR、CAT)、脯氨酸、游離氨基酸、硫醇組分(GSH、GSSG、NPT、PC和Cys)及其代謝酶(γ-ECS、GST和PCS)。這表明MT在清除硒誘導(dǎo)的氧化損傷方面具有更大的潛力。褪黑素誘導(dǎo)根中螯合劑的積累表明它更有可能在根中對(duì)硒進(jìn)行解毒,從而減少硒轉(zhuǎn)移到葉中。整體研究結(jié)果表明,MT誘導(dǎo)滲透保護(hù)、抗氧化防御和硫醇配體生物合成的提高,最終通過(guò)增強(qiáng)油菜對(duì)Se(ⅣV)脅迫的耐受性,有助于硒解毒或隔離。(4)在本研究中,我們采用RNA測(cè)序分析深入了解了外源應(yīng)用褪黑素(MT)時(shí),甘藍(lán)型油菜對(duì)硒脅迫的抵抗反應(yīng)和耐受機(jī)制。使用數(shù)字基因表達(dá)(DGE)和轉(zhuǎn)錄組譜分析(TPA)技術(shù)揭示了兩個(gè)甘藍(lán)型油菜品種中硒脅迫和MT誘導(dǎo)的緩解作用所涉及的機(jī)制。不同表達(dá)基因(DEG)的數(shù)據(jù)顯示,與對(duì)照組相比,Se顯著減少了DEG的數(shù)量(在'浙大622'中有更多DEG下調(diào)),并且,施用MT比單獨(dú)的硒處理增加了DEG的數(shù)量。在基因本體論(GO)分析中,在生物過(guò)程和細(xì)胞組分部分,應(yīng)激反應(yīng)光刺激、對(duì)karrikin的響應(yīng)、跨膜受體蛋白酪氨酸激酶信號(hào)傳導(dǎo)途徑、蠟生物合成過(guò)程以及脂肪酸生物合成是主要突出過(guò)程。添加硒可抑制KEGG途徑,這在'浙大622'的脂肪酸代謝、卟啉和葉綠素代謝以及浙雙758的光合作用、光合作用-天線生物合成、乙醛酸和二羧酸代謝中被觀測(cè)到。褪黑素能在硒脅迫下改善或恢復(fù)上述特性,并且對(duì)'浙雙758'耐受性的增強(qiáng)比'浙大622'高。
【學(xué)位單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位年份】：2019
【中圖分類】：S565.4
【文章目錄】：
ACKNOWLEDGEMENT
Abbreviations
Abstract
摘要
CHAPTER 1 Introduction and Literature Review
1.1 Significance of oilseed rape
1.2 Selenium occurrence in the environment
1.3 Selenium uptake, accumulation and metabolism in plants
1.3.1 Selenium uptake and transport
1.3.2 Selenium accumulation in plants
1.3.3 Selenium metabolism in plants
1.4 Dual behavior of selenium in plants
1.4.1 Beneficial effects of selenium and its role in heavy metals tolerance
1.4.2 Selenium phytotoxicity
1.5 Mechanisms of selenium detoxification
1.6 Discovery and biosynthesis of melatonin in plants
1.7 Mechanisms of melatonin-mediated heavy metals/metalloids tolerance and detoxification
1.8 Research objectives
1.9 Overview of the whole study
CHAPTER 2 Comparative response of Brassica napus cultivars against selenium stress by morpho-physiological,and antioxidants system
2.1 Introduction
2.2 Material and methods
2.2.1 Plant materials and experimental conditions
2.2.2 Morphological parameters
2.2.3 Determination of pigment contents and total soluble protein
2.2.4 Analysis of Se content in leaf and root blades
2.2.5 Quantification of lipid peroxidation and hydrogen peroxide content
2.2.6 Biochemical assay of enzyme activities
2.2.7 Determination of non-enzymatic based antioxidants
2.2.8 Ultra-structural observation by transmission electron microscopy
2.2.9 Assays of total RNA extraction, cDNA synthesis, and quantitative real-time PCR（RT-qPCR）
2.2.10 Statistical analysis
2.3 Results
2.3.1 Effects of Se （Ⅳ） on plant growth parameters
2.3.2 Effects of Se （Ⅳ） on the Se contents and its translocation in plant tissues
2.3.3 Effects of Se （Ⅳ） on pigment contents and total soluble protein
2.3.4 Variations in oxidative markers and glutathione under Se （Ⅳ） stress
2.3.5 Effect of Se （Ⅳ） on the ROS-detoxifying enzyme activities
2.3.6 Effect of Se （Ⅳ） on the antioxidant genes expression
2.3.7 Ultra-structural alterations under Se （Ⅳ） stress
2.4 Discussion
2.5 Conclusion
CHAPTER 3 Dual behavior of selenium:Insights into physio-biochemical, anatomical and molecular analysesof four Brassica napus cultivars
3.1 Introduction
3.2 Materials and methods
3.2.1 Plant material and growth conditions
3.2.2 Morphological parameters
3.2.3 Estimation of Se contents
3.2.4 Pigment contents and gas exchange parameters
3.2.5 Assessment of micro and macro elements uptake
3.2.6 Total water-soluble protein and total sugar content
3.2.7 Quantification of oxidative markers with histochemical staining
3.2.8 Assays of antioxidants and total ascorbic acid
3.2.9 Measurement of methylglyoxa content
3.2.10 Ultra-structural observations
3.2.11 Assays of total RNA extraction, cDNA synthesis, and quantitative real-time PCR（RT-qPCR）
3.2.12 Statistical analysis
3.3 Results
3.3.1 Effects of Se （Ⅳ） on the phenotypical appearance
3.3.2 Dual impacts of Se （Ⅳ） on plant growth and gas exchange parameters
3.3.3 Dual effects of Se （Ⅳ） on the mineral homeostasis and osmotic adjustments
3.3.4 Dual effects of Se （Ⅳ） on oxidative markers and glyoxalase system
3.3.5 Regulation of ROS detoxifying enzymes under Se （Ⅳ） stress
3.3.6 Total ascorbic acid,GSH and GSSG levels under Se （Ⅳ） stress
3.3.7 Effects of Se （Ⅳ） on the transcript levels of ASA-GSH cycle enzymes and secondarymetabolites
3.3.8 Ultra-structure changes in response to Se （Ⅳ） stress
3.4 Discussion
3.5 Conclusions
CHAPTER 4 Protective mechanisms of melatonin against selenium toxicity in Brassica napus throughphysiological, thiols and antioxidant machinery
4.1 Introduction
4.2 Materials and methods
4.2.1 Plant materials and experimental design
4.2.2 Morphological parameters and relative water content （RWC）
4.2.3 Pigment contents, gas exchange and chlorophyll fluorescence measurement
4.2.4 Extraction and quantification of endogenous Se and Me by HPLC
4.2.5 Soluble Sugar, free amino acids and proline contents
4.2.6 Quantification of MDA levels, ROS contents, and relative electrolyte leakage
4.2.7 ROS-detoxifying enzymes
4.2.8 Estimation of thiol compounds and observation of leaf stomata
4.2.9 Extraction of total RNA and quantitative real-time PCR （qRT-PCR） assays
4.2.10 Statistical analysis
4.3 Results
4.3.1 Se-induces the endogenous MT and exogenous MT reduces the Se uptake
4.3.2 MT recovers the plant growth and photosynthesis under Se stress
4.3.3 MT improves the metabolic adjustments and mitigates the oxidative damages underSe stress
4.3.4 MT enhances the antioxidant enzymes and phosphate/silicon transporters
4.3.5 MT stimulates the biosynthesis of chelating compounds under Se stress
4.3.6 Exogenous MT helps in stomata opening
4.4 Discussion
4.5 Conclusions
CHAPTER 5 Transcriptome profiling of Brassica napus against selenium toxicity and its alleviation byexogenous melatonin
5.1 Introduction
5.2 Methods
5.2.1 Plant materials and growth conditions
5.2.2 Estimation of plant biomass,and endogenous MT and Se contents
5.2.3 Establishment of de novo assembly
5.2.4 Screening and expression pattern of differentially expressed genes （DEGs）
5.2.5 Gene Ontology （GO） functional enrichment analysis （WEGO） of DEGs
5.2.6 KEGG pathway enrichment analysis
5.2.7 Statistical analysis
5.3 Results
5.3.1 Exogenous MT minimizes the Se-uptake and improves the plant biomass
5.3.2 de novo assembly and genome mapping
5.3.3 Screening and expression analysis of differentially expressed genes （DEGs）
5.3.4 Gene ontology （GO） functional classification （WEGO） of DEGs
5.3.5 KEGG metabolic pathway enrichment analysis
CHAPTER 6 Major Findings and Future Perspectives
6.1 Major findings
6.2 Novel findings
6.3 Future perspectives
References
List of Publications
本文編號(hào)：2868075
【學(xué)位單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位年份】：2019
【中圖分類】：S565.4
【文章目錄】：
ACKNOWLEDGEMENT
Abbreviations
Abstract
摘要
CHAPTER 1 Introduction and Literature Review
1.1 Significance of oilseed rape
1.2 Selenium occurrence in the environment
1.3 Selenium uptake, accumulation and metabolism in plants
1.3.1 Selenium uptake and transport
1.3.2 Selenium accumulation in plants
1.3.3 Selenium metabolism in plants
1.4 Dual behavior of selenium in plants
1.4.1 Beneficial effects of selenium and its role in heavy metals tolerance
1.4.2 Selenium phytotoxicity
1.5 Mechanisms of selenium detoxification
1.6 Discovery and biosynthesis of melatonin in plants
1.7 Mechanisms of melatonin-mediated heavy metals/metalloids tolerance and detoxification
1.8 Research objectives
1.9 Overview of the whole study
CHAPTER 2 Comparative response of Brassica napus cultivars against selenium stress by morpho-physiological,and antioxidants system
2.1 Introduction
2.2 Material and methods
2.2.1 Plant materials and experimental conditions
2.2.2 Morphological parameters
2.2.3 Determination of pigment contents and total soluble protein
2.2.4 Analysis of Se content in leaf and root blades
2.2.5 Quantification of lipid peroxidation and hydrogen peroxide content
2.2.6 Biochemical assay of enzyme activities
2.2.7 Determination of non-enzymatic based antioxidants
2.2.8 Ultra-structural observation by transmission electron microscopy
2.2.9 Assays of total RNA extraction, cDNA synthesis, and quantitative real-time PCR（RT-qPCR）
2.2.10 Statistical analysis
2.3 Results
2.3.1 Effects of Se （Ⅳ） on plant growth parameters
2.3.2 Effects of Se （Ⅳ） on the Se contents and its translocation in plant tissues
2.3.3 Effects of Se （Ⅳ） on pigment contents and total soluble protein
2.3.4 Variations in oxidative markers and glutathione under Se （Ⅳ） stress
2.3.5 Effect of Se （Ⅳ） on the ROS-detoxifying enzyme activities
2.3.6 Effect of Se （Ⅳ） on the antioxidant genes expression
2.3.7 Ultra-structural alterations under Se （Ⅳ） stress
2.4 Discussion
2.5 Conclusion
CHAPTER 3 Dual behavior of selenium:Insights into physio-biochemical, anatomical and molecular analysesof four Brassica napus cultivars
3.1 Introduction
3.2 Materials and methods
3.2.1 Plant material and growth conditions
3.2.2 Morphological parameters
3.2.3 Estimation of Se contents
3.2.4 Pigment contents and gas exchange parameters
3.2.5 Assessment of micro and macro elements uptake
3.2.6 Total water-soluble protein and total sugar content
3.2.7 Quantification of oxidative markers with histochemical staining
3.2.8 Assays of antioxidants and total ascorbic acid
3.2.9 Measurement of methylglyoxa content
3.2.10 Ultra-structural observations
3.2.11 Assays of total RNA extraction, cDNA synthesis, and quantitative real-time PCR（RT-qPCR）
3.2.12 Statistical analysis
3.3 Results
3.3.1 Effects of Se （Ⅳ） on the phenotypical appearance
3.3.2 Dual impacts of Se （Ⅳ） on plant growth and gas exchange parameters
3.3.3 Dual effects of Se （Ⅳ） on the mineral homeostasis and osmotic adjustments
3.3.4 Dual effects of Se （Ⅳ） on oxidative markers and glyoxalase system
3.3.5 Regulation of ROS detoxifying enzymes under Se （Ⅳ） stress
3.3.6 Total ascorbic acid,GSH and GSSG levels under Se （Ⅳ） stress
3.3.7 Effects of Se （Ⅳ） on the transcript levels of ASA-GSH cycle enzymes and secondarymetabolites
3.3.8 Ultra-structure changes in response to Se （Ⅳ） stress
3.4 Discussion
3.5 Conclusions
CHAPTER 4 Protective mechanisms of melatonin against selenium toxicity in Brassica napus throughphysiological, thiols and antioxidant machinery
4.1 Introduction
4.2 Materials and methods
4.2.1 Plant materials and experimental design
4.2.2 Morphological parameters and relative water content （RWC）
4.2.3 Pigment contents, gas exchange and chlorophyll fluorescence measurement
4.2.4 Extraction and quantification of endogenous Se and Me by HPLC
4.2.5 Soluble Sugar, free amino acids and proline contents
4.2.6 Quantification of MDA levels, ROS contents, and relative electrolyte leakage
4.2.7 ROS-detoxifying enzymes
4.2.8 Estimation of thiol compounds and observation of leaf stomata
4.2.9 Extraction of total RNA and quantitative real-time PCR （qRT-PCR） assays
4.2.10 Statistical analysis
4.3 Results
4.3.1 Se-induces the endogenous MT and exogenous MT reduces the Se uptake
4.3.2 MT recovers the plant growth and photosynthesis under Se stress
4.3.3 MT improves the metabolic adjustments and mitigates the oxidative damages underSe stress
4.3.4 MT enhances the antioxidant enzymes and phosphate/silicon transporters
4.3.5 MT stimulates the biosynthesis of chelating compounds under Se stress
4.3.6 Exogenous MT helps in stomata opening
4.4 Discussion
4.5 Conclusions
CHAPTER 5 Transcriptome profiling of Brassica napus against selenium toxicity and its alleviation byexogenous melatonin
5.1 Introduction
5.2 Methods
5.2.1 Plant materials and growth conditions
5.2.2 Estimation of plant biomass,and endogenous MT and Se contents
5.2.3 Establishment of de novo assembly
5.2.4 Screening and expression pattern of differentially expressed genes （DEGs）
5.2.5 Gene Ontology （GO） functional enrichment analysis （WEGO） of DEGs
5.2.6 KEGG pathway enrichment analysis
5.2.7 Statistical analysis
5.3 Results
5.3.1 Exogenous MT minimizes the Se-uptake and improves the plant biomass
5.3.2 de novo assembly and genome mapping
5.3.3 Screening and expression analysis of differentially expressed genes （DEGs）
5.3.4 Gene ontology （GO） functional classification （WEGO） of DEGs
5.3.5 KEGG metabolic pathway enrichment analysis
CHAPTER 6 Major Findings and Future Perspectives
6.1 Major findings
6.2 Novel findings
6.3 Future perspectives
References
List of Publications
本文編號(hào)：2868075
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