He-Ne激光輻照對高羊茅幼苗耐鹽性響應(yīng)的調(diào)控及作用機理
發(fā)布時間：2018-08-03 15:21
【摘要】：鹽脅迫是影響全球農(nóng)作物和其它植物生長發(fā)育及其品質(zhì)產(chǎn)量的常見的環(huán)境脅迫因子之一。環(huán)境的鹽漬化污染往往導(dǎo)致農(nóng)業(yè)耕地土壤品質(zhì)退化,土壤物化特性改變,土壤疏松程度和多孔性性狀降低。鹽漬化的土壤環(huán)境不僅難以為植物提供充足的水分和氧氣,而且,也使大量毒性離子(如鈉離子、氯離子等)在植物細(xì)胞內(nèi)高度積累,這樣,必然會對植物生長造成毒害作用,最終導(dǎo)致農(nóng)作物產(chǎn)量下降。本研究選取生長發(fā)育狀態(tài)良好的13日齡的高羊茅(Festuca arundinacea Schreb.)幼苗為生物材料,分別經(jīng)鹽脅迫(添加半致死濃度的氯化鈉,N)和He-Ne激光(λ=632.8nm,光斑直徑d=100.0mm,輻照劑量5.0m W·mmm~(-2))輻照(H),或外源硝普鈉SNP(NO供體)處理(S),或普通冷白光(LED燈提供,PAR=100μmol mm~(-2) s-1)照射(W)等的復(fù)合處理(即分別為N+H,N+S,N+W處理組)后,研究了He-Ne激光輻照對幼苗早期發(fā)育階段抗鹽性能和響應(yīng)行為的影響,探討了激光對植物耐鹽性響應(yīng)和代謝的誘導(dǎo)效應(yīng),并利用SNP處理和普通冷白光照射作為對照組進一步進行了功能驗證。通過瓊脂糖凝膠電泳、生物微量檢測儀和核基因組DNA Apopladder分析技術(shù)檢測激光輻照對幼苗核基因結(jié)構(gòu)與功能損傷的保護效應(yīng);通過傅立葉變換紅外光譜儀、紫外吸收光譜儀、激光共聚焦掃描顯微鏡分析系統(tǒng)等技術(shù)對植物細(xì)胞壁多糖的物化特性和生物學(xué)活性進行分析檢測,以期揭示He-Ne激光輻照調(diào)控高羊茅幼苗耐鹽性響應(yīng)的物理化學(xué)機理或分子機制。主要研究結(jié)果如下:(1)一定程度的鹽脅迫處理會導(dǎo)致高羊茅幼苗早期生長發(fā)育受抑,植株表觀表型發(fā)育異常,葉片變窄變細(xì),顏色發(fā)黃或呈棕黃,主根系變粗變短,側(cè)根較發(fā)達,生物量驟減,生長速率緩慢等。然而,采用He-Ne激光輻照處理后,可以明顯緩解植株生長發(fā)育遲緩的狀態(tài),幼苗的表型和葉片顏色逐漸接近正常對照組植株,因此,我們認(rèn)為適宜劑量和輻照時間的He-Ne激光處理在一定程度上能誘導(dǎo)植物體對鹽脅迫生長環(huán)境表現(xiàn)出耐鹽性響應(yīng)行為。(2)通過設(shè)置外源硝普鈉(SNP)處理組(作為NO供體,陽性對照組)和普通冷白光照射處理組(陰性對照組)作為對照處理,比較了各處理組中幼苗的生長狀態(tài)、表型變化和農(nóng)藝學(xué)性狀等參數(shù)指標(biāo),結(jié)果進一步表明He-Ne激光輻照對鹽脅迫植物幼苗生長發(fā)育具有積極的生物學(xué)效應(yīng)。(3)He-Ne激光輻照可以促進植物細(xì)胞外Ca~(2+)發(fā)生內(nèi)流,使得細(xì)胞質(zhì)中Ca~(2+)分布濃度明顯升高,這樣當(dāng)Ca~(2+)與鈣調(diào)蛋白(CaM)結(jié)合后,繼而激活了胞內(nèi)一氧化氮合酶(NOS)的催化活性,促進了內(nèi)源一氧化氮(NO)分子的合成和釋放;NO信號分子通過特定的信號轉(zhuǎn)導(dǎo)途徑,進一步啟動和激活了植物細(xì)胞內(nèi)的抗氧化防御體系,即增強了抗氧化物的合成和抗氧化物酶的催化活性,這樣,就可以分解胞內(nèi)超量積累的活性氧(ROS)產(chǎn)物,避免或降低了鹽脅迫對植物細(xì)胞造成的氧化脅迫和氧化損傷。(4)He-Ne激光輻照也可以使部分抗氧化物酶基因(包括Cu/Zn SOD,POD,CAT,APX,GR)的轉(zhuǎn)錄和表達上調(diào),這些被激活的抗氧化物酶也有利于清除多余的ROS產(chǎn)物,從而緩解鹽脅迫導(dǎo)致的氧化脅迫。我們還發(fā)現(xiàn),激光輻照也使植物細(xì)胞的光受體—光敏色素B(即PHYB)基因表達發(fā)生上調(diào),那么,phy B與He-Ne激光輻照誘導(dǎo)的植物耐鹽性響應(yīng)是否有聯(lián)系,或phy B是否參與此過程的調(diào)控有待進一步深入研究探討。(5)鹽脅迫處理抑制高羊茅植物生長發(fā)育的另一原因是造成了胞內(nèi)大量毒性離子(如Na+和Cl-)的分布和積累,引起了離子毒性效應(yīng)。而He-Ne激光輻照可以激活質(zhì)膜上的PM H~+-ATPase和囊泡膜上的V-型質(zhì)子泵的催化活性,介導(dǎo)質(zhì)子在膜兩側(cè)形成了一定的質(zhì)子電化學(xué)梯度,進而驅(qū)動了Na~+/Cl~-/K+等陰、陽離子的運輸過程,降低了胞內(nèi)毒性離子(Na~+/Cl~-)的毒害作用,有效緩解了鹽脅迫對植物生長發(fā)育的抑制效應(yīng)。(6)鹽脅迫處理后,高羊茅葉片出現(xiàn)顯著的細(xì)胞死亡現(xiàn)象,根系組織中則同時表現(xiàn)出了明顯的細(xì)胞死亡和細(xì)胞凋亡兩種情況。臺盼蘭染色法結(jié)果顯示,與正常對照組和單獨激光輻照處理組相比較,鹽脅迫幼苗葉片和根系細(xì)胞活力明顯降低,而He-Ne激光輻照處理進一步提高了鹽脅迫幼苗的細(xì)胞活力。通過流式細(xì)胞技術(shù)檢測線粒體膜電位的變化,結(jié)合Annexin-V-FITC和碘化丙啶PI雙標(biāo)染色分析發(fā)現(xiàn),鹽脅迫導(dǎo)致根系組織發(fā)生了細(xì)胞凋亡和細(xì)胞死亡,而葉片組織僅僅檢測到明顯的細(xì)胞死亡現(xiàn)象,細(xì)胞凋亡頻率極低。推測其原因可能是根系直接與含有鹽分的營養(yǎng)液接觸導(dǎo)致根細(xì)胞毒性離子積累更嚴(yán)重,細(xì)胞活力受抑更加明顯;但根系通常較葉片表現(xiàn)出更強的耐逆性,所以,根系組織細(xì)胞出現(xiàn)了細(xì)胞凋亡現(xiàn)象,而葉組織細(xì)胞中只出現(xiàn)細(xì)胞死亡,幾乎沒有細(xì)胞凋亡的發(fā)生。He-Ne激光輻照均可以有效緩解根系和葉片中的細(xì)胞凋亡和細(xì)胞死亡現(xiàn)象的發(fā)生,說明激光可以誘導(dǎo)植株的耐鹽性響應(yīng)行為。(7)通過瓊脂糖凝膠電泳和DNA Apopladder分析,發(fā)現(xiàn)鹽脅迫導(dǎo)致幼苗核基因組DNA結(jié)構(gòu)和活性嚴(yán)重?fù)p傷,完整核基因DNA含量下降,凝膠電泳圖譜中檢測到明顯的“DNA smear”和“DNA laddering”現(xiàn)象。He-Ne激光輻照可以有效緩解鹽脅迫引起的核基因序列的結(jié)構(gòu)損傷,表明激光輻照保護了核基因DNA結(jié)構(gòu)的完整性,為抗性基因的高效轉(zhuǎn)錄和表達提供了先決條件。(8)通過對不同處理組幼苗的細(xì)胞壁多糖含量、寡糖組成比例和物理化學(xué)特性鑒定,結(jié)果表明,He-Ne激光輻照可以保護植物細(xì)胞壁和細(xì)胞壁多糖,避免了不利脅迫因子對其產(chǎn)生的結(jié)構(gòu)破壞和物化活性損傷,即可能激光輻照協(xié)助植物細(xì)胞壁在逆境中快速進行了結(jié)構(gòu)重建,維持了細(xì)胞壁結(jié)構(gòu)的完整性和較高的生物學(xué)活性。這樣,就保護了細(xì)胞核基因組DNA免收傷害。綜上所述,He-Ne激光輻照顯著激活了高羊茅幼苗的各類抗氧化物酶基因的轉(zhuǎn)錄活性和表達水平,促進了內(nèi)源NO信號分子的生物合成,改善了植物幼苗的抗氧化防御體系,降低了ROS的積累,有效地緩解了鹽脅迫對高羊茅幼苗造成的氧化損傷;同時維持了高活性的質(zhì)膜和液泡膜質(zhì)子泵活性,調(diào)控了細(xì)胞中陰、陽離子的運輸,解除了毒性離子對植物細(xì)胞造成的毒害作用,從而誘導(dǎo)了植物的耐鹽性。
[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.
【學(xué)位授予單位】：山西師范大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q945.78
，
本文編號：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.
【學(xué)位授予單位】：山西師范大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q945.78
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本文編號：2162158
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