抗碱盐生植物虎尾草和甜土植物棉花应对各种盐碱胁迫时DNA甲基化调节的研究
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摘要
土壤盐碱化是一个世界性的环境问题，越来越受到人们的重视。在自然界中，土壤的盐化和碱化常常相伴而生，同时存在。许多研究证明盐胁迫和碱胁迫是两种性质不同的胁迫，这两种胁迫对植物的生长及生理都有不同的影响，并且植物对这两种胁迫的反应机制也不完全相同。前期虽然有关于虎尾草和棉花的抗盐碱机制报道，但大多集中在生理响应机制方面。目前还没有关于盐、碱胁迫对植物DNA甲基化影响的研究。虎尾草（Chloris virgata Swartz）是一种较为抗碱的盐生植物，棉花（Gossypium spp）是一种重要的非盐生经济作物。本文利用两种处理方式，从叶和根两种器官水平，从生理响应及DNA甲基化响应两方面来探讨虎尾草和棉花的抗盐、碱机制，其主要实验结果及结论如下：
1.盐、碱胁迫在不同的处理时间长度下，对虎尾草体内离子平衡和渗透调节影响各有不同。
采用两种中性盐（NaCl和Na_2SO_4）的复合盐和两种碱性盐（NaHCO_3和Na_2CO_3）的复合盐分别处理虎尾草幼苗，通过比较两种胁迫在不同的处理时间长度下，虎尾草体内光合色素、离子含量和有机酸含量的差异，发现随着盐、碱胁迫时间的增加，虎尾草叶片Na~+含量逐渐上升，并且碱胁迫下的上升幅度更大。K+含量并没有随着盐、碱胁迫时间的增长而出现明显变化。碱胁迫下随着时间的增长无机阴离子含量逐步降低。虎尾草主要靠琥珀酸的积累来调节长期的盐胁迫，而主要靠积累柠檬酸来调节长期的碱胁迫。总体而言，碱性盐胁迫的影响显著大于中性盐胁迫，并且随着胁迫时间的延长，这种影响差异性越来越显著。
2.与盐胁迫相比，碱胁迫对虎尾草、棉花甲基化的影响更为显著。
采用两种中性盐（NaCl和Na_2SO_4）的复合盐和两种碱性盐（NaHCO_3和Na_2CO_3）的复合盐分别处理虎尾草幼苗，利用MSAP（甲基化敏感扩增多态性）技术，检测到在中性复合盐胁迫后的叶中，DNA甲基化变异频率为0-0.24%，根中为0.09-1.85%，而在碱性复合盐胁迫后的叶中，DNA甲基化变异频率为0-0.41%，根中为0.09-5.48%。在棉花中也发现，盐胁迫对甲基化水平没有影响，而碱胁迫显著降低了棉花的总体甲基化水平。盐胁迫下叶的总甲基化变异频率为1.38%，根的甲基化变异频率为2.20%；碱胁迫下叶的总甲基化变异频率为2.59%，根的总甲基化变异频率为11.44%。
本实验从DNA甲基化的角度证实了盐胁迫和碱胁迫是两种性质截然不同的胁迫，因为高pH值的影响，碱胁迫对植物DNA甲基化的影响更为严重。由此说明，在长期的盐碱环境中生长，植物可能通过DNA甲基化的方式来调节相关基因的表达，从而在不影响DNA序列和基因组稳定性的前提下，来适应和抵抗外界盐、碱环境，这也是一种快速的应对胁迫并对植物体基因组影响较小的进化模式。
3.四种单盐及混合盐对虎尾草DNA甲基化的影响明显不同。
采用四种单盐（NaCl、Na_2SO_4、NaHCO_3、Na_2CO_3）及这四种单盐的混合盐（摩尔数比1:1:1:1）分别处理虎尾草幼苗，利用MSAP技术，检测到碱性盐（NaHCO_3、Na_2CO_3）的影响显著大于中性盐的影响（NaCl、Na_2SO_4）；四种单盐混合后对DNA甲基化的影响，在叶中显著大于中性盐和碱性盐的影响，而在根中却表现出了一定的缓解作用。根据不同阴离子的作用机制不同，可将所有的甲基化变异条带分为8种类型：Na~+特异的条带；Cl-特异的条带；SO42-特异的条带；HCO_3-特异的条带；CO_32-特异的条带；Mix特异的条带；高pH特异的条带；SS（中性盐特异的条带）。在叶中以Mix特异的条带为主，而在根中以高pH特异的条带为主。本文从DNA甲基化的角度证实四种单盐虽然均为Na盐，但因为其特异阴离子的存在，对DNA甲基化的影响也有其特异性，四种单盐混合后作用机制复杂，存在一定的缓解效应，而土壤的高pH环境对根的影响较为显著。
4.盐、碱胁迫下，植物根的DNA甲基化变异更为显著。
分析盐、碱胁迫下虎尾草和棉花的根和叶中发生的DNA甲基化变异情况可以得知，无论在中性复合盐胁迫、碱性复合盐胁迫、单盐单独胁迫还是在单盐混合胁迫下， DNA甲基化变异在根中发生的更为频繁。并且在甲基化的变异中，基本都以甲基化降低为主。在盐、碱环境中，根是直接接触离子毒害和渗透压力的器官，它对外界胁迫的响应更为直接和迅速，根中DNA的低甲基化和在胁迫下较高的变异率恰恰说明了根在调节碱胁迫的过程中可能起着非常重要的作用。
Soil salinization and alkalization is a worldwide environmental problem, and isdrawing more and more people’s attentions. Soil salinization and alkalization is oftenco-occurred in nature. Many studies have proved that salt stress and alkali stress aretwo different stresses with different properties, they have different effects on plantgrowth and physiology, and the mechanisms that plants reacted to them are alsodifferent. Although there were many reports about the resistance mechanism of C.virgata and cotton, most of them concentrated in the physiological response. Therewas still no study about the effects on plant DNA methylation of salt stress and alkalistress. Chloris virgata is a kind of alkali-resistant halophyte, while cotton is a kind ofimportant economic glycophyte. Two kinds of salt and alkali stresses were used in thispaper, and we discussed the resistance mechanisms of C. virgata and cotton fromphysiological response and DNA methylation response, and the experiments werebased on two organ levels that were leaf and root. The main results and conclusionswere as follows:
1. Salt stress and alkali stress have different effects on ionic balance and osmoticregulation of C. virgata under different treated time.
Two kinds of neutral salts (NaCl and Na_2SO_4) and alkaline salts (NaHCO_3andNa_2CO_3) were used to treat C. virgata seedlings. By comparing the different effects onphotosynthetic pigment, ion content and organic acids content, we found that Na~+increased with the increased treated time of both stresses, and the increasment underalkali stress was more obvious. K+content didn’t change under both stresses. Thecontents of anions were decreased with the increament of treated time of alkali stress.The mainly accumulated organic acid was succinic acid to adjust the long-term saltstress, and was citric acid to adjust long-term alkali stress. Overall, alkali stress hadmore significant influence on C. virgata than salt stress, and the effects were more andmore significant along with the extension of the treated time.
2. Compared with salt stress, alkali stress had more significant influence on DNAmethylation of C. virgata and cotton.
Two kinds of neutral salts (NaCl and Na_2SO_4) and alkaline salts (NaHCO_3andNa_2CO_3) were used to treat C. virgata seedlings, and through the analysis of MSAP(methylation sensitive amplified polymorphism) technology, we found that DNAmethylation variation frequency was0-0.24%in leaf and0.09-1.85%in roots undersalt stress, while DNA methylation variation frequency was0-0.41%in leaf and0.09-5.48%in roots under alkali stress. In cotton we also found that salt stress had noeffect on the overall level of methylation, while alkali stress significantly decreasedthe overall methylation level. The total variation frequency was1.38%in leaf undersalt stress, and the frequencies were2.20%in root respectively. Meanwhile, the total variation frequency was2.59%in leaf under alkali stress, and the frequencies were11.44%respectively.
Therefore, this experiment confirmed that salt stress and alkali stress are reallytwo different stresses from the point of DNA methylation. The effect of the alkalistress on plant DNA methylation was more serious with higher pH value. Under thelong-term salt and alkali stress, plants could regulate stress-related gene expressionthrough DNA methylation, which has no impact on the DNA sequence and genomestability. It was also a quick response to stress and a kind of evolution model with theleast effect on plant genome.
3. Four kinds of single salt and their mixed salt had significantly different effectson C. virgata DNA methylation.
Using four kinds of single salt (NaCl, Na_2SO_4, NaHCO_3, Na_2CO_3) and the mixedfour salts (molar ration was1:1) to treat C. virgata seedlings, and through theanalysis of MSAP technology, we found that alkaline salts (NaHCO_3and Na_2CO_3),had more significant effect than neutral salts (NaCl, Na_2SO_4). After the treatment ofthe mixed four salts, the influence in the leaves was much more serious than neutralsalts and alkaline salts, while in root it showed a soothing effect. According to thedifferent properties of different anions, all the methylation variation were classifiedinto8types: Na~+specific bands; Cl-specific bands; SO42-specific bands;HCO_3-specific bands; CO_32-specific bands; Mix specific bands; Higher pH specificbands; SS (neutral salts specific) bands. The main variation bands were Mix specificbands in leaf, while that were high pH specific bands in roots. This article showed thatalthough four kinds of single salt were sodium salts, but they had different influenceon DNA methylation because of its specific anion, and the mixed four salts hadcomplex effects on plants, there were some alleviation effects, and the impact ofhigher pH in the soil surrounding root was significant.
4. Under salt stress and alkali stress, DNA methylation variation was moremarked in roots.
The variation frequencies occurred in C. virgata and cotton showed that nomatter in a neutral salt stress or in alkaline salt stress or in single salt stress or inmixed salt stress, the phenomenon of DNA methylation variations was more markedin root than in leaf. And among all the methylation variations, the hypomethylationwas the main type. In salt and alkali environment, roots was the primary organ whichcontacted directly with ion poison and osmotic pressure, it’s responses to the outsidestresses were more directly and quickly than other organs. The lower DNAmethylation level and the higher variation frequencies in root justified the importantrole of root in the process of adjusting alkali stress.
1.盐、碱胁迫在不同的处理时间长度下，对虎尾草体内离子平衡和渗透调节影响各有不同。
采用两种中性盐（NaCl和Na_2SO_4）的复合盐和两种碱性盐（NaHCO_3和Na_2CO_3）的复合盐分别处理虎尾草幼苗，通过比较两种胁迫在不同的处理时间长度下，虎尾草体内光合色素、离子含量和有机酸含量的差异，发现随着盐、碱胁迫时间的增加，虎尾草叶片Na~+含量逐渐上升，并且碱胁迫下的上升幅度更大。K+含量并没有随着盐、碱胁迫时间的增长而出现明显变化。碱胁迫下随着时间的增长无机阴离子含量逐步降低。虎尾草主要靠琥珀酸的积累来调节长期的盐胁迫，而主要靠积累柠檬酸来调节长期的碱胁迫。总体而言，碱性盐胁迫的影响显著大于中性盐胁迫，并且随着胁迫时间的延长，这种影响差异性越来越显著。
2.与盐胁迫相比，碱胁迫对虎尾草、棉花甲基化的影响更为显著。
采用两种中性盐（NaCl和Na_2SO_4）的复合盐和两种碱性盐（NaHCO_3和Na_2CO_3）的复合盐分别处理虎尾草幼苗，利用MSAP（甲基化敏感扩增多态性）技术，检测到在中性复合盐胁迫后的叶中，DNA甲基化变异频率为0-0.24%，根中为0.09-1.85%，而在碱性复合盐胁迫后的叶中，DNA甲基化变异频率为0-0.41%，根中为0.09-5.48%。在棉花中也发现，盐胁迫对甲基化水平没有影响，而碱胁迫显著降低了棉花的总体甲基化水平。盐胁迫下叶的总甲基化变异频率为1.38%，根的甲基化变异频率为2.20%；碱胁迫下叶的总甲基化变异频率为2.59%，根的总甲基化变异频率为11.44%。
本实验从DNA甲基化的角度证实了盐胁迫和碱胁迫是两种性质截然不同的胁迫，因为高pH值的影响，碱胁迫对植物DNA甲基化的影响更为严重。由此说明，在长期的盐碱环境中生长，植物可能通过DNA甲基化的方式来调节相关基因的表达，从而在不影响DNA序列和基因组稳定性的前提下，来适应和抵抗外界盐、碱环境，这也是一种快速的应对胁迫并对植物体基因组影响较小的进化模式。
3.四种单盐及混合盐对虎尾草DNA甲基化的影响明显不同。
采用四种单盐（NaCl、Na_2SO_4、NaHCO_3、Na_2CO_3）及这四种单盐的混合盐（摩尔数比1:1:1:1）分别处理虎尾草幼苗，利用MSAP技术，检测到碱性盐（NaHCO_3、Na_2CO_3）的影响显著大于中性盐的影响（NaCl、Na_2SO_4）；四种单盐混合后对DNA甲基化的影响，在叶中显著大于中性盐和碱性盐的影响，而在根中却表现出了一定的缓解作用。根据不同阴离子的作用机制不同，可将所有的甲基化变异条带分为8种类型：Na~+特异的条带；Cl-特异的条带；SO42-特异的条带；HCO_3-特异的条带；CO_32-特异的条带；Mix特异的条带；高pH特异的条带；SS（中性盐特异的条带）。在叶中以Mix特异的条带为主，而在根中以高pH特异的条带为主。本文从DNA甲基化的角度证实四种单盐虽然均为Na盐，但因为其特异阴离子的存在，对DNA甲基化的影响也有其特异性，四种单盐混合后作用机制复杂，存在一定的缓解效应，而土壤的高pH环境对根的影响较为显著。
4.盐、碱胁迫下，植物根的DNA甲基化变异更为显著。
分析盐、碱胁迫下虎尾草和棉花的根和叶中发生的DNA甲基化变异情况可以得知，无论在中性复合盐胁迫、碱性复合盐胁迫、单盐单独胁迫还是在单盐混合胁迫下， DNA甲基化变异在根中发生的更为频繁。并且在甲基化的变异中，基本都以甲基化降低为主。在盐、碱环境中，根是直接接触离子毒害和渗透压力的器官，它对外界胁迫的响应更为直接和迅速，根中DNA的低甲基化和在胁迫下较高的变异率恰恰说明了根在调节碱胁迫的过程中可能起着非常重要的作用。
Soil salinization and alkalization is a worldwide environmental problem, and isdrawing more and more people’s attentions. Soil salinization and alkalization is oftenco-occurred in nature. Many studies have proved that salt stress and alkali stress aretwo different stresses with different properties, they have different effects on plantgrowth and physiology, and the mechanisms that plants reacted to them are alsodifferent. Although there were many reports about the resistance mechanism of C.virgata and cotton, most of them concentrated in the physiological response. Therewas still no study about the effects on plant DNA methylation of salt stress and alkalistress. Chloris virgata is a kind of alkali-resistant halophyte, while cotton is a kind ofimportant economic glycophyte. Two kinds of salt and alkali stresses were used in thispaper, and we discussed the resistance mechanisms of C. virgata and cotton fromphysiological response and DNA methylation response, and the experiments werebased on two organ levels that were leaf and root. The main results and conclusionswere as follows:
1. Salt stress and alkali stress have different effects on ionic balance and osmoticregulation of C. virgata under different treated time.
Two kinds of neutral salts (NaCl and Na_2SO_4) and alkaline salts (NaHCO_3andNa_2CO_3) were used to treat C. virgata seedlings. By comparing the different effects onphotosynthetic pigment, ion content and organic acids content, we found that Na~+increased with the increased treated time of both stresses, and the increasment underalkali stress was more obvious. K+content didn’t change under both stresses. Thecontents of anions were decreased with the increament of treated time of alkali stress.The mainly accumulated organic acid was succinic acid to adjust the long-term saltstress, and was citric acid to adjust long-term alkali stress. Overall, alkali stress hadmore significant influence on C. virgata than salt stress, and the effects were more andmore significant along with the extension of the treated time.
2. Compared with salt stress, alkali stress had more significant influence on DNAmethylation of C. virgata and cotton.
Two kinds of neutral salts (NaCl and Na_2SO_4) and alkaline salts (NaHCO_3andNa_2CO_3) were used to treat C. virgata seedlings, and through the analysis of MSAP(methylation sensitive amplified polymorphism) technology, we found that DNAmethylation variation frequency was0-0.24%in leaf and0.09-1.85%in roots undersalt stress, while DNA methylation variation frequency was0-0.41%in leaf and0.09-5.48%in roots under alkali stress. In cotton we also found that salt stress had noeffect on the overall level of methylation, while alkali stress significantly decreasedthe overall methylation level. The total variation frequency was1.38%in leaf undersalt stress, and the frequencies were2.20%in root respectively. Meanwhile, the total variation frequency was2.59%in leaf under alkali stress, and the frequencies were11.44%respectively.
Therefore, this experiment confirmed that salt stress and alkali stress are reallytwo different stresses from the point of DNA methylation. The effect of the alkalistress on plant DNA methylation was more serious with higher pH value. Under thelong-term salt and alkali stress, plants could regulate stress-related gene expressionthrough DNA methylation, which has no impact on the DNA sequence and genomestability. It was also a quick response to stress and a kind of evolution model with theleast effect on plant genome.
3. Four kinds of single salt and their mixed salt had significantly different effectson C. virgata DNA methylation.
Using four kinds of single salt (NaCl, Na_2SO_4, NaHCO_3, Na_2CO_3) and the mixedfour salts (molar ration was1:1) to treat C. virgata seedlings, and through theanalysis of MSAP technology, we found that alkaline salts (NaHCO_3and Na_2CO_3),had more significant effect than neutral salts (NaCl, Na_2SO_4). After the treatment ofthe mixed four salts, the influence in the leaves was much more serious than neutralsalts and alkaline salts, while in root it showed a soothing effect. According to thedifferent properties of different anions, all the methylation variation were classifiedinto8types: Na~+specific bands; Cl-specific bands; SO42-specific bands;HCO_3-specific bands; CO_32-specific bands; Mix specific bands; Higher pH specificbands; SS (neutral salts specific) bands. The main variation bands were Mix specificbands in leaf, while that were high pH specific bands in roots. This article showed thatalthough four kinds of single salt were sodium salts, but they had different influenceon DNA methylation because of its specific anion, and the mixed four salts hadcomplex effects on plants, there were some alleviation effects, and the impact ofhigher pH in the soil surrounding root was significant.
4. Under salt stress and alkali stress, DNA methylation variation was moremarked in roots.
The variation frequencies occurred in C. virgata and cotton showed that nomatter in a neutral salt stress or in alkaline salt stress or in single salt stress or inmixed salt stress, the phenomenon of DNA methylation variations was more markedin root than in leaf. And among all the methylation variations, the hypomethylationwas the main type. In salt and alkali environment, roots was the primary organ whichcontacted directly with ion poison and osmotic pressure, it’s responses to the outsidestresses were more directly and quickly than other organs. The lower DNAmethylation level and the higher variation frequencies in root justified the importantrole of root in the process of adjusting alkali stress.
引文
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