He-Ne激光辐照对高羊茅幼苗耐盐性响应的调控及作用机理
发布时间：2018-08-03 15:21
【摘要】：盐胁迫是影响全球农作物和其它植物生长发育及其品质产量的常见的环境胁迫因子之一。环境的盐渍化污染往往导致农业耕地土壤品质退化,土壤物化特性改变,土壤疏松程度和多孔性性状降低。盐渍化的土壤环境不仅难以为植物提供充足的水分和氧气,而且,也使大量毒性离子(如钠离子、氯离子等)在植物细胞内高度积累,这样,必然会对植物生长造成毒害作用,最终导致农作物产量下降。本研究选取生长发育状态良好的13日龄的高羊茅(Festuca arundinacea Schreb.)幼苗为生物材料,分别经盐胁迫(添加半致死浓度的氯化钠,N)和He-Ne激光(λ=632.8nm,光斑直径d=100.0mm,辐照剂量5.0m W·mmm~(-2))辐照(H),或外源硝普钠SNP(NO供体)处理(S),或普通冷白光(LED灯提供,PAR=100μmol mm~(-2) s-1)照射(W)等的复合处理(即分别为N+H,N+S,N+W处理组)后,研究了He-Ne激光辐照对幼苗早期发育阶段抗盐性能和响应行为的影响,探讨了激光对植物耐盐性响应和代谢的诱导效应,并利用SNP处理和普通冷白光照射作为对照组进一步进行了功能验证。通过琼脂糖凝胶电泳、生物微量检测仪和核基因组DNA Apopladder分析技术检测激光辐照对幼苗核基因结构与功能损伤的保护效应;通过傅立叶变换红外光谱仪、紫外吸收光谱仪、激光共聚焦扫描显微镜分析系统等技术对植物细胞壁多糖的物化特性和生物学活性进行分析检测,以期揭示He-Ne激光辐照调控高羊茅幼苗耐盐性响应的物理化学机理或分子机制。主要研究结果如下:(1)一定程度的盐胁迫处理会导致高羊茅幼苗早期生长发育受抑,植株表观表型发育异常,叶片变窄变细,颜色发黄或呈棕黄,主根系变粗变短,侧根较发达,生物量骤减,生长速率缓慢等。然而,采用He-Ne激光辐照处理后,可以明显缓解植株生长发育迟缓的状态,幼苗的表型和叶片颜色逐渐接近正常对照组植株,因此,我们认为适宜剂量和辐照时间的He-Ne激光处理在一定程度上能诱导植物体对盐胁迫生长环境表现出耐盐性响应行为。(2)通过设置外源硝普钠(SNP)处理组(作为NO供体,阳性对照组)和普通冷白光照射处理组(阴性对照组)作为对照处理,比较了各处理组中幼苗的生长状态、表型变化和农艺学性状等参数指标,结果进一步表明He-Ne激光辐照对盐胁迫植物幼苗生长发育具有积极的生物学效应。(3)He-Ne激光辐照可以促进植物细胞外Ca~(2+)发生内流,使得细胞质中Ca~(2+)分布浓度明显升高,这样当Ca~(2+)与钙调蛋白(CaM)结合后,继而激活了胞内一氧化氮合酶(NOS)的催化活性,促进了内源一氧化氮(NO)分子的合成和释放;NO信号分子通过特定的信号转导途径,进一步启动和激活了植物细胞内的抗氧化防御体系,即增强了抗氧化物的合成和抗氧化物酶的催化活性,这样,就可以分解胞内超量积累的活性氧(ROS)产物,避免或降低了盐胁迫对植物细胞造成的氧化胁迫和氧化损伤。(4)He-Ne激光辐照也可以使部分抗氧化物酶基因(包括Cu/Zn SOD,POD,CAT,APX,GR)的转录和表达上调,这些被激活的抗氧化物酶也有利于清除多余的ROS产物,从而缓解盐胁迫导致的氧化胁迫。我们还发现,激光辐照也使植物细胞的光受体—光敏色素B(即PHYB)基因表达发生上调,那么,phy B与He-Ne激光辐照诱导的植物耐盐性响应是否有联系,或phy B是否参与此过程的调控有待进一步深入研究探讨。(5)盐胁迫处理抑制高羊茅植物生长发育的另一原因是造成了胞内大量毒性离子(如Na+和Cl-)的分布和积累,引起了离子毒性效应。而He-Ne激光辐照可以激活质膜上的PM H~+-ATPase和囊泡膜上的V-型质子泵的催化活性,介导质子在膜两侧形成了一定的质子电化学梯度,进而驱动了Na~+/Cl~-/K+等阴、阳离子的运输过程,降低了胞内毒性离子(Na~+/Cl~-)的毒害作用,有效缓解了盐胁迫对植物生长发育的抑制效应。(6)盐胁迫处理后,高羊茅叶片出现显著的细胞死亡现象,根系组织中则同时表现出了明显的细胞死亡和细胞凋亡两种情况。台盼兰染色法结果显示,与正常对照组和单独激光辐照处理组相比较,盐胁迫幼苗叶片和根系细胞活力明显降低,而He-Ne激光辐照处理进一步提高了盐胁迫幼苗的细胞活力。通过流式细胞技术检测线粒体膜电位的变化,结合Annexin-V-FITC和碘化丙啶PI双标染色分析发现,盐胁迫导致根系组织发生了细胞凋亡和细胞死亡,而叶片组织仅仅检测到明显的细胞死亡现象,细胞凋亡频率极低。推测其原因可能是根系直接与含有盐分的营养液接触导致根细胞毒性离子积累更严重,细胞活力受抑更加明显;但根系通常较叶片表现出更强的耐逆性,所以,根系组织细胞出现了细胞凋亡现象,而叶组织细胞中只出现细胞死亡,几乎没有细胞凋亡的发生。He-Ne激光辐照均可以有效缓解根系和叶片中的细胞凋亡和细胞死亡现象的发生,说明激光可以诱导植株的耐盐性响应行为。(7)通过琼脂糖凝胶电泳和DNA Apopladder分析,发现盐胁迫导致幼苗核基因组DNA结构和活性严重损伤,完整核基因DNA含量下降,凝胶电泳图谱中检测到明显的“DNA smear”和“DNA laddering”现象。He-Ne激光辐照可以有效缓解盐胁迫引起的核基因序列的结构损伤,表明激光辐照保护了核基因DNA结构的完整性,为抗性基因的高效转录和表达提供了先决条件。(8)通过对不同处理组幼苗的细胞壁多糖含量、寡糖组成比例和物理化学特性鉴定,结果表明,He-Ne激光辐照可以保护植物细胞壁和细胞壁多糖,避免了不利胁迫因子对其产生的结构破坏和物化活性损伤,即可能激光辐照协助植物细胞壁在逆境中快速进行了结构重建,维持了细胞壁结构的完整性和较高的生物学活性。这样,就保护了细胞核基因组DNA免收伤害。综上所述,He-Ne激光辐照显著激活了高羊茅幼苗的各类抗氧化物酶基因的转录活性和表达水平,促进了内源NO信号分子的生物合成,改善了植物幼苗的抗氧化防御体系,降低了ROS的积累,有效地缓解了盐胁迫对高羊茅幼苗造成的氧化损伤;同时维持了高活性的质膜和液泡膜质子泵活性,调控了细胞中阴、阳离子的运输,解除了毒性离子对植物细胞造成的毒害作用,从而诱导了植物的耐盐性。
[Abstract]:Salt stress is one of the common environmental stress factors that affect the growth and quality of crops and other plants in the world. The salinization of the environment often leads to the degradation of soil quality, the change of soil physical and chemical properties, the decrease of soil porosity and the porosity of the soil. The soil environment of salinization is not only difficult to provide for plants. Sufficient water and oxygen are provided, and a large number of toxic ions, such as sodium ions, chloride ions, etc., are highly accumulated in plant cells. This will inevitably cause toxic effects on plant growth and eventually lead to the decline of crop yield. This study selects the 13 day old tall fescue (Festuca arundinacea Schreb.) seedlings of good growth and development. For biological materials, the combined treatment of salt stress (adding half lethal concentration of sodium chloride, N) and He-Ne laser (lambda =632.8nm, spot diameter d=100.0mm, radiation dose 5.0m W mmm~ (-2)) irradiation (H), or exogenous sodium nitroprusside SNP (NO donor) treatment (S), or ordinary Leng Baiguang After N+S, N+W treatment group), the effect of He-Ne laser irradiation on salt resistance and response behavior at early stage of seedling development was studied. The effect of laser on salt tolerance and metabolism induced by laser was discussed. The function of SNP treatment and common cold white light irradiation as control group was further carried out. Microdetector and nuclear genome DNA Apopladder analysis were used to detect the protective effect of laser irradiation on the structural and functional damage of nuclear gene, and the physicochemical properties and biological activity of plant cell wall polysaccharides were obtained by Fu Liye transform infrared spectrometer, UV absorption spectrometer and laser confocal scanning microscope analysis system. In order to reveal the physicochemical mechanism or molecular mechanism of He-Ne laser irradiation on the response to salt tolerance of tall fescue seedlings, the main research results are as follows: (1) a certain degree of salt stress can lead to the early growth and development of tall fescue seedlings, the apparent phenotypic development of the plant, the narrowing and thinning of the leaves, the yellow or brown yellow color of the leaves. The main root system became shorter, the lateral root was more developed, the biomass decreased rapidly and the growth rate was slow. However, the plant growth retardation was obviously alleviated by He-Ne laser irradiation. The phenotype and leaf color of the seedlings were gradually close to the normal control group. Therefore, we think that the suitable dose and the irradiation time of He-Ne laser treatment are considered. To some extent, the plant body could induce salt tolerance response to salt stress growth environment. (2) by setting exogenous sodium nitroprusside (SNP) treatment group (as NO donor, positive control group) and normal cold white light irradiation treatment group (negative control group) as control treatment, the growth state, phenotypic changes and phenotypic changes of the seedlings in each treatment group were compared. The results showed that He-Ne laser irradiation had positive biological effects on the growth and development of plant seedlings with salt stress. (3) He-Ne laser irradiation could promote the occurrence of Ca~ (2+) internal flow outside of the plant cells, so that the concentration of Ca~ (2+) in the cytoplasm increased obviously, so that when Ca~ (2+) was combined with calmodulin (CaM), Then activation of the catalytic activity of intracellular nitric oxide synthase (NOS) promotes the synthesis and release of endogenous nitric oxide (NO) molecules, and the NO signal molecules further activate and activate the antioxidant defense system in plant cells through specific signal transduction pathways, that is, the synthesis of antioxidants and the catalytic activity of antioxidant enzymes are enhanced. In this way, the active oxygen (ROS) products can be decomposed to avoid or reduce oxidative stress and oxidative damage caused by salt stress on plant cells. (4) He-Ne laser irradiation can also increase the transcriptional and expression of some antioxidant enzyme genes (including Cu/Zn SOD, POD, CAT, APX, GR), and these activated antioxidant enzymes are also available. It is beneficial to remove the superfluous ROS products, thus alleviating the oxidative stress caused by salt stress. We also found that laser irradiation also up-regulated the expression of the light sensitive pigment B (PHYB) gene of plant cells. Then, whether the salt tolerance induced by PHY B and He-Ne laser irradiation should be related, or whether PHY B is involved in the regulation of this process. Further research is needed. (5) another reason for the inhibition of the growth and development of tall fescue plants by salt stress treatment is the distribution and accumulation of a large number of toxic ions (such as Na+ and Cl-), resulting in ionic toxicity. The He-Ne laser irradiation can activate the catalytic activity of the PM H ~+-ATPase on the plasma membrane and the V- type proton pump on the vesicle membrane. The protons formed a certain proton electrochemical gradient on both sides of the membrane, and then drove the transport process of Na~+/Cl~-/K+ equal Yin and cations, reduced the toxic effects of intracellular toxic ions (Na~+/Cl~-), effectively alleviated the inhibition effect of salt stress on plant growth and development. (6) the leaves of tall fescue were significantly thinner after salt stress treatment. Cell death and two cases of cell death and cell apoptosis were shown in the root tissue. Trypan blue staining showed that compared with the normal control group and the single laser irradiation treatment group, the cell vitality of the leaves and roots of the seedlings was significantly reduced by salt stress, while the He-Ne laser irradiation improved the salt stress further. The changes in mitochondrial membrane potential were detected by flow cytometry. Combined with Annexin-V-FITC and propidium iodide PI double staining analysis, it was found that salt stress caused cell apoptosis and cell death in root tissue, and the leaf tissue only detected obvious cell death, and the frequency of cell apoptosis was very low. The reason may be that the root cells directly contact with the salt containing nutrient solution lead to more serious accumulation of toxic ions in the root cells and more obvious inhibition of cell viability, but the root system usually shows stronger resistance to the leaf than the leaves, so the cell apoptosis appears in the root tissue cells, and the cell death in the leaf tissue cells is almost no fine. Apoptosis and.He-Ne laser irradiation can effectively alleviate the apoptosis and cell death in the roots and leaves, indicating that the laser can induce the salt tolerance response behavior of the plant. (7) the analysis of agarose gel electrophoresis and DNA Apopladder showed that salt stress resulted in serious damage to the structure and activity of DNA in the nuclear genome of the seedlings. The content of the complete nuclear gene DNA decreased, and the obvious "DNA smear" and "DNA laddering".He-Ne laser irradiation in the gel electrophoresis atlas could effectively alleviate the structural damage of the nuclear gene sequence caused by salt stress, indicating that the laser irradiation protects the integrity of the nuclear gene DNA structure and provides the efficient transcription and expression of the resistant genes. The preconditions were given. (8) the results showed that He-Ne laser irradiation could protect the cell wall and cell wall polysaccharide of plant cells by the content of polysaccharide, the proportion of oligosaccharides and physicochemical properties of the seedlings of different treatment groups, and the results showed that the structural damage and physical activity damage caused by adverse stress factors were avoided, that is, the possible laser irradiation co - irradiation. The structural reconstruction of the cell wall of the plant helps to maintain the integrity of the cell wall structure and the high biological activity. In this way, the damage of the nuclear genome DNA is protected. In summary, the He-Ne laser irradiation significantly activates the transcriptional activity and expression level of various anti oxide enzyme genes in the seedlings of tall fescue. The biosynthesis of endogenous NO signal molecules improved the antioxidant defense system of plant seedlings, reduced the accumulation of ROS, effectively alleviated the oxidative damage caused by salt stress on tall fescue seedlings, and maintained the activity of high active plasmalemma and vacuolar proton pump, regulating the transport of anion and cation in cells and relieving toxicity. Its toxicity to plant cells induces salt tolerance of plants.
【学位授予单位】：山西师范大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q945.78
，
本文编号：2162158
[Abstract]:Salt stress is one of the common environmental stress factors that affect the growth and quality of crops and other plants in the world. The salinization of the environment often leads to the degradation of soil quality, the change of soil physical and chemical properties, the decrease of soil porosity and the porosity of the soil. The soil environment of salinization is not only difficult to provide for plants. Sufficient water and oxygen are provided, and a large number of toxic ions, such as sodium ions, chloride ions, etc., are highly accumulated in plant cells. This will inevitably cause toxic effects on plant growth and eventually lead to the decline of crop yield. This study selects the 13 day old tall fescue (Festuca arundinacea Schreb.) seedlings of good growth and development. For biological materials, the combined treatment of salt stress (adding half lethal concentration of sodium chloride, N) and He-Ne laser (lambda =632.8nm, spot diameter d=100.0mm, radiation dose 5.0m W mmm~ (-2)) irradiation (H), or exogenous sodium nitroprusside SNP (NO donor) treatment (S), or ordinary Leng Baiguang After N+S, N+W treatment group), the effect of He-Ne laser irradiation on salt resistance and response behavior at early stage of seedling development was studied. The effect of laser on salt tolerance and metabolism induced by laser was discussed. The function of SNP treatment and common cold white light irradiation as control group was further carried out. Microdetector and nuclear genome DNA Apopladder analysis were used to detect the protective effect of laser irradiation on the structural and functional damage of nuclear gene, and the physicochemical properties and biological activity of plant cell wall polysaccharides were obtained by Fu Liye transform infrared spectrometer, UV absorption spectrometer and laser confocal scanning microscope analysis system. In order to reveal the physicochemical mechanism or molecular mechanism of He-Ne laser irradiation on the response to salt tolerance of tall fescue seedlings, the main research results are as follows: (1) a certain degree of salt stress can lead to the early growth and development of tall fescue seedlings, the apparent phenotypic development of the plant, the narrowing and thinning of the leaves, the yellow or brown yellow color of the leaves. The main root system became shorter, the lateral root was more developed, the biomass decreased rapidly and the growth rate was slow. However, the plant growth retardation was obviously alleviated by He-Ne laser irradiation. The phenotype and leaf color of the seedlings were gradually close to the normal control group. Therefore, we think that the suitable dose and the irradiation time of He-Ne laser treatment are considered. To some extent, the plant body could induce salt tolerance response to salt stress growth environment. (2) by setting exogenous sodium nitroprusside (SNP) treatment group (as NO donor, positive control group) and normal cold white light irradiation treatment group (negative control group) as control treatment, the growth state, phenotypic changes and phenotypic changes of the seedlings in each treatment group were compared. The results showed that He-Ne laser irradiation had positive biological effects on the growth and development of plant seedlings with salt stress. (3) He-Ne laser irradiation could promote the occurrence of Ca~ (2+) internal flow outside of the plant cells, so that the concentration of Ca~ (2+) in the cytoplasm increased obviously, so that when Ca~ (2+) was combined with calmodulin (CaM), Then activation of the catalytic activity of intracellular nitric oxide synthase (NOS) promotes the synthesis and release of endogenous nitric oxide (NO) molecules, and the NO signal molecules further activate and activate the antioxidant defense system in plant cells through specific signal transduction pathways, that is, the synthesis of antioxidants and the catalytic activity of antioxidant enzymes are enhanced. In this way, the active oxygen (ROS) products can be decomposed to avoid or reduce oxidative stress and oxidative damage caused by salt stress on plant cells. (4) He-Ne laser irradiation can also increase the transcriptional and expression of some antioxidant enzyme genes (including Cu/Zn SOD, POD, CAT, APX, GR), and these activated antioxidant enzymes are also available. It is beneficial to remove the superfluous ROS products, thus alleviating the oxidative stress caused by salt stress. We also found that laser irradiation also up-regulated the expression of the light sensitive pigment B (PHYB) gene of plant cells. Then, whether the salt tolerance induced by PHY B and He-Ne laser irradiation should be related, or whether PHY B is involved in the regulation of this process. Further research is needed. (5) another reason for the inhibition of the growth and development of tall fescue plants by salt stress treatment is the distribution and accumulation of a large number of toxic ions (such as Na+ and Cl-), resulting in ionic toxicity. The He-Ne laser irradiation can activate the catalytic activity of the PM H ~+-ATPase on the plasma membrane and the V- type proton pump on the vesicle membrane. The protons formed a certain proton electrochemical gradient on both sides of the membrane, and then drove the transport process of Na~+/Cl~-/K+ equal Yin and cations, reduced the toxic effects of intracellular toxic ions (Na~+/Cl~-), effectively alleviated the inhibition effect of salt stress on plant growth and development. (6) the leaves of tall fescue were significantly thinner after salt stress treatment. Cell death and two cases of cell death and cell apoptosis were shown in the root tissue. Trypan blue staining showed that compared with the normal control group and the single laser irradiation treatment group, the cell vitality of the leaves and roots of the seedlings was significantly reduced by salt stress, while the He-Ne laser irradiation improved the salt stress further. The changes in mitochondrial membrane potential were detected by flow cytometry. Combined with Annexin-V-FITC and propidium iodide PI double staining analysis, it was found that salt stress caused cell apoptosis and cell death in root tissue, and the leaf tissue only detected obvious cell death, and the frequency of cell apoptosis was very low. The reason may be that the root cells directly contact with the salt containing nutrient solution lead to more serious accumulation of toxic ions in the root cells and more obvious inhibition of cell viability, but the root system usually shows stronger resistance to the leaf than the leaves, so the cell apoptosis appears in the root tissue cells, and the cell death in the leaf tissue cells is almost no fine. Apoptosis and.He-Ne laser irradiation can effectively alleviate the apoptosis and cell death in the roots and leaves, indicating that the laser can induce the salt tolerance response behavior of the plant. (7) the analysis of agarose gel electrophoresis and DNA Apopladder showed that salt stress resulted in serious damage to the structure and activity of DNA in the nuclear genome of the seedlings. The content of the complete nuclear gene DNA decreased, and the obvious "DNA smear" and "DNA laddering".He-Ne laser irradiation in the gel electrophoresis atlas could effectively alleviate the structural damage of the nuclear gene sequence caused by salt stress, indicating that the laser irradiation protects the integrity of the nuclear gene DNA structure and provides the efficient transcription and expression of the resistant genes. The preconditions were given. (8) the results showed that He-Ne laser irradiation could protect the cell wall and cell wall polysaccharide of plant cells by the content of polysaccharide, the proportion of oligosaccharides and physicochemical properties of the seedlings of different treatment groups, and the results showed that the structural damage and physical activity damage caused by adverse stress factors were avoided, that is, the possible laser irradiation co - irradiation. The structural reconstruction of the cell wall of the plant helps to maintain the integrity of the cell wall structure and the high biological activity. In this way, the damage of the nuclear genome DNA is protected. In summary, the He-Ne laser irradiation significantly activates the transcriptional activity and expression level of various anti oxide enzyme genes in the seedlings of tall fescue. The biosynthesis of endogenous NO signal molecules improved the antioxidant defense system of plant seedlings, reduced the accumulation of ROS, effectively alleviated the oxidative damage caused by salt stress on tall fescue seedlings, and maintained the activity of high active plasmalemma and vacuolar proton pump, regulating the transport of anion and cation in cells and relieving toxicity. Its toxicity to plant cells induces salt tolerance of plants.
【学位授予单位】：山西师范大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q945.78
，
本文编号：2162158
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