硼缓解柑橘枳壳砧木铝毒的机理研究

论文摘要

酸性土壤占世界可耕地面积的40%。铝（A1）是地壳中继氧和硅之后的第三丰富元素,约占地壳的7%。另外,红壤面积占全国总土地面积的20%以上,且主要分布在中国的热带和亚热带地区。在酸性土壤中,铝毒害是限制作物生长发育和生产力的主要环境因素。其中,铝毒对植物的毒害症状最初和最明显的症状是抑制植物根系伸长。硼（B）是高等植物生长发育最重要的微量营养素之一,缺硼通常诱导植物根系的异常生长,侧根发育受阻。硼定义了细胞壁结构并确保植物受到逆境胁迫时细胞壁的稳定性。高等植物的细胞壁含有大量的多糖,包括鼠李半乳糖醛酸（RG-Ⅱ）,硼通过双酯键与RG-Ⅱ交联结合以稳定细胞壁结构,充分堆积的细胞网络形成稳定的细胞壁,孔径减小,细胞壁收紧,限制大分子/元素进入细胞。由于植物的细胞壁是铝积累的主要部位,因此细胞壁在植物抗铝毒机制中具有重要作用。据报道,具有羧酸盐基团的初生细胞壁是铝吸附的主要位点。目前已研究出几种农艺措施可以减轻铝的毒害,比如说酸性土壤中施用石灰。然而,施用石灰会降低必需元素的可用性而对植物生长产生不利影响,并且在经济上并不总是可行的。另外,一些学者已经提出硼可以减轻铝对植物生长的毒性作用,并改善植物在酸性土壤中的性能。然而,目前为止并没有关于硼对枳壳砧木铝毒改善作用的研究,以及硼如何减轻铝毒的具体机制尚不清楚。如果可以在柑橘中验证这一假设,那么硼肥施用将是改善酸性土壤中柑橘的铝毒性的候选措施之一。柑橘属于常绿亚热带果树,主要种植在湿润和半湿润的热带、亚热带和温带地区。在中国,柑橘是一种重要的水果作物,其主要种植在酸性土壤中,特别是江西省赣州市。该地区柑橘种植面积居世界第一,但该地区高降雨量可冲掉根区碱性元素,从而产生酸性条件,使得82.1%的土壤为酸性红壤（pH≤5）。本研究以枳壳砧木实生苗为试验材料,研究了硼在减轻铝毒性及其潜在机制中的作用。主要结果总结如下:（1）铝毒严重阻碍了植物的根系生长和生理特性,缺硼且铝毒条件下引起植株氧化应激,膜损伤和根系损伤。然而,铝胁迫下加硼明显改善根伸长和光合作用参数。另外,加硼可以通过调节抗氧化酶（超氧化物歧化酶（SOD）,过氧化物酶（POD）,过氧化氢酶（CAT）和抗坏血酸过氧化物酶（APX））,脯氨酸,次级代谢产物（苯丙氨酸氨裂解酶和多酚氧化酶）的活性,稳定蛋白质的完整性,减少根系损伤。无论有无铝处理,植株硼浓度并无明显差异,说明铝并不影响植株对硼的吸收。因此,结果表明,硼通过减少植物活性氧（ROS）的积累和铝浓度来促进根系生长,从而减轻了铝对植株的毒害。（2）铝胁迫下显著增加幼苗根系中H202、果胶和去甲基酯化果胶含量。然而,铝胁迫下,相比于缺硼处理,加硼处理的根系中H202和02·-的含量分别降低了48.11%和44.87%。硼供应减弱铝毒害引起的氧化应激、膜脂过氧化、膜渗漏和细胞死亡。铝毒害在根和根细胞壁中积累了更多的铝,并且在根细胞壁中积累的铝占根中总铝的50%以上。铝胁迫下,相比于缺硼处理,加硼明显降低根和根细胞壁中的铝浓度,且分别降低59%和58%。（3）在缺硼处理,铝胁迫下细胞壁中铝积累量明显增加,并且lumogallion荧光分子探针定位分析明确得出大部分铝与细胞壁的外围以及细胞核结合,表明铝对细胞壁和细胞质的吸附非常明显。铝胁迫下,相对于缺硼处理（BO）,B1和B2处理下的荧光强度明显降低。透射电镜（TEM）观察表明B0Al-和B0A1+处理均导致细胞的不规则性。B0A1+处理观察到显著增厚和肿胀的根细胞壁。加硼减少了淀粉颗粒的积累,并且观察到细胞壁厚度明显减小。铝胁迫下,JIM5和JIM7单克隆抗体作用下显示,HG（高聚半乳糖醛酸）表位的分布紊乱,且积累较高的质外体铝。傅立叶变换红外光谱（FTIR）结果表明,铝胁迫明显影响各吸收峰的强度,尤其是蛋白质,纤维素,半纤维素和果胶的峰值,相比于缺硼处理,两种浓度的硼添加均降低与果胶和半纤维素相关的峰值。（4）与正常处理相比,铝胁迫下根系细胞壁中果胶和半纤维素含量明显增加,且分别增加了 60.04%和149.10%。相对于缺硼处理（BO）,B1和B2处理下的非甲基化果胶分别减少了 37.05%和46.78%,半纤维素分别减少了 31.34%和50%。B1和B2处理下果胶甲基酯化度分别增加了 197.18%和221.61%。另外,铝胁迫下,加硼处理减少了胼胝质积累和细胞壁组分的变化。结果表明,细胞壁组分含量的变化以及果胶中提供结合铝离子的负电荷基团,使得细胞壁中积累较多的铝,从而抑制根伸长。综上所述,硼可以通过减弱质外体铝离子和细胞壁组分的变化来减少铝对植株诱导的毒害作用,最终减少根系损伤而改善植株的生长。该结果明确揭示了硼对铝毒害的缓解机制,有助于酸性土壤中柑橘的农业生产,特别是为酸性红壤中硼肥施用措施提供科学的理论依据。

论文目录

• 摘要
• Abstract
• CHAPTER 1 Introduction
•   1.1 Acid soils and aluminum toxicity
•     1.1.1 Acid soils and distribution of acid soils
•     1.1.2 Occurrence of aluminum
•   1.2 Symptoms of A1 toxicity
•     1.2.1 Root growth inhibition
•     1.2.2 Alterations in the cell wall structure
•     1.2.3 Induction of callose
•     1.2.4 Plasma membrane damages
•     1.2.5 Nutritional imbalance
•     1.2.6 Oxidative stress
•   1.3 Measures of alleviating Al toxicity
•     1.3.1 Importance of B
•     1.3.2 Role of B in the cell wall
•     1.3.3 RG-Ⅱ borate complexes in the primary cell wall
•     1.3.4 Bio-synthesis of pectic wall polymers （pectin methyltransferase）
•     1.3.5 B induced alleviation of Al toxicity
•     1.3.6 B induced regulation of apoplastic binding of Al and root injury
•     1.3.7 B induced activation of antioxidant defense system
•   1.4 Trifoliate orange
•   1.5 Research objectives
• CHAPTER 2 Boron alleviates aluminum toxicity in trifoliate seedling by regulatingantioxidant defense system and reducing root cell injury
•   2.1 Introduction
•   2.2 Objective
•   2.3 Materials and methods
•     2.3.1 Plant material and growth conditions
•     2.3.2 Experimental treatments
•     2.3.3 Leaf gas exchange parameters and root length measurement
•     2.3.4 FDA-PI double staining, hematoxylin and morin staining
• 2O₂ content'>    2.3.5 Antioxidant enzyme assay, and determination of MDA, H₂O₂content
•     2.3.6 Assay of phenylalanine ammonia lyase and polyphenol oxidase
•     2.3.7 Measurement of total soluble proteins and free proline contents
•     2.3.8 Determination of B and A1 concentrations
•     2.3.9 Statistical analysis
•   2.4 Results
•     2.4.1 Effect of B on the plant growth parameters under Al toxicity
•     2.4.2 Influence of B on leaf gas exchange parameters under Al toxicity
•     2.4.3 FDA-PI double staining, hematoxylin and morin staining
• 2O₂ contents andantioxidant enzymes activities under Al toxicity'>    2.4.4 Effect of B on the total soluble protein, proline, MDA, H₂O₂ contents andantioxidant enzymes activities under Al toxicity
•     2.4.5 Effect of B on the PAL and PPO contents under Al toxicity
•     2.4.6 B and Al concentration in plant parts
•   2.5 Discussion
•   2.6 Conclusion
• CHAPTER 3 Boron reduces aluminum-induced growth inhibition and oxidativedamages in roots of trifoliate seedling
•   3.1 Introduction
•   3.2 Objectives
•   3.3 Materials and methods
•     3.3.1 Plant material
•     3.3.2 Growth conditions
•     3.3.3 Experimental treatments
•     3.3.4 Measurement of root growth and dry biomass
•     3.3.5 Extraction of cell wall
•     3.3.6 Quantification of total B and A1 concentrations
•     3.3.7 Assessment of plasma membrane （PM） integrity and electrolyte leakage in roots
•     3.3.8 Measurement of reactive oxygen species, and malondialdehyde contents
•     3.3.9 Statistical analysis
•   3.4 Results
•     3.4.1 Effect of B on root growth, dry weight, and visible symptoms of A1 toxicity
•     3.4.2 B and A1 distribution in roots and root cell wall
•     3.4.3 Influence of B on ROS levels in roots under A1 stress
•     3.4.4 Influence of B and Al on the MDA contents and root electrolyte leakage
•   3.5 Discussion
•   3.6 Conclusion
• CHAPTER 4 Boron increases root growth by reducing aluminum-induced disorganizeddistribution of HG epitopes and alterations in the subcellular cell wall structure oftrifoliate orange roots
•   4.1 Introduction
•   4.2 Objectives
•   4.3 Material and methods
•     4.3.1 Plant materials and growth conditions
•     4.3.2 Experimental treatments
•     4.3.3 Extraction of cell wall
•     4.3.4 Determination of B and Al concentrations
•     4.3.5 Lumogallion staining and confocal microscopy
•     4.3.6 Immunolabelling of low and high-methyl esterified pectin epitope
•     4.3.7 Transmission electron microscopy of roots
•     4.3.8 FTIR sample preparation and analysis
•     4.3.9 Statistical analysis
•   4.4 Results
•     4.4.1 Root growth response to Al toxicity
•     4.4.2 Localization of A1 with lumogallion staining
•     4.4.3 Transmission electron microscope of roots
•     4.4.4 Effect of A1 toxicity on JIM5 and JIM7 homogalacturonan epitopes
•     4.4.5 FT-IR analysis of root cell wall
•   4.5 Discussion
•   4.6 Conclusion
• CHAPTER 5 Boron supply maintains efficient antioxidant system, cell wall componentsand reduces aluminum concentration in roots of trifoliate orange
•   5.1 Introduction
•   5.2 Objectives
•   5.3 Materials and methods
•     5.3.1 Plant material and growth condition
•     5.3.2 Experimental treatments
•     5.3.3 Plant sampling and specimen preparation
•     5.3.4 Extraction of cell wall material
•     5.3.5 Quantification of Al and B in root apex and cell wall
•     5.3.6 Fractionation of cell wall components
•     5.3.7 Measurement of monoamine oxidase,xanthine,T-AOC,and VC concentrations
•     5.3.8  Histochemical analysis of callose deposition
•     5.3.9 Statistical analysis
•   5.4 Results
•     5.4.1 Effect of B on the root growth under A1 toxicity
•     5.4.2 Localization of A1 in the root tips through confocal laser microscope
•     5.4.3 Fractionation of Al
•     5.4.4 Cell wall components
•     5.4.5 Activities of monoamine oxidase, xanthine oxidase, vitamin C （VC） and totalantioxidant capability （T-AOC） in leaves
•     5.4.6 Effect of A1 on callose deposition
•     5.4.7 Principal component analysis
•   5.5 Discussion
•   5.6 Conclusion
• CHAPTER 6 General conclusion
•   6.1 Plant growth characteristics
•   6.2 Al concentration in roots
•   6.3 Alteration in cell wall components
•   6.4 Distribution of HG epitopes
•   6.5 Efficient antioxidant system
•   6.6 Oxidative stress and root injuries
•   6.7 Current issues and future directions
• Reference
• Major Achievements during PhD
• Achievements/Awards
• Acknowledgement

文章来源

类型: 博士论文

作者: 利阿兹（MUHAMMAD RIAZ）

导师: 姜存仓

关键词: 酸性土壤,枳壳砧木,活性氧,细胞壁

来源: 华中农业大学

年度: 2019

分类: 基础科学,农业科技

专业: 自然地理学和测绘学,农业基础科学,农艺学,园艺

单位: 华中农业大学

分类号: S666;S156.6

DOI: 10.27158/d.cnki.ghznu.2019.000823

总页数: 122

文件大小: 9361K

下载量: 94