【作者】 谷文英；

【导师】 高洪文；

【作者基本信息】 扬州大学 ， 草业科学， 2013， 博士

【摘要】 盐胁迫对植物种子萌发和生长有抑制作用,一氧化氮(nitric oxide, NO)作为信号分子或效应分子参与植物的多种生理过程而发挥重要作用。本文以将军菊苣(Cichorium intybus L. cv. Commander)为试验材料,考察不同浓度的NaCl (0～280mmol/L)对菊苣种子萌发、幼苗生长和生理响应的影响,评估外源性NO供体硝普钠(SNP)对不同浓度NaCl胁迫下菊苣(Cichorium intybus L.)营养生长期幼苗渗透调节物质的影响,以及SNP对NaCl胁迫下萌发期幼苗的抗氧化酶活性、抗应激蛋白基因表达及其含量的影响。主要研究结果如下：1.100mmol/L盐胁迫明显降低了菊苣种子的发芽势、发芽率、发芽指数和活力指数,且随着盐胁迫浓度的升高,这种抑制作用越明显(P<0.05).180mmol/L可使种子萌发率降低50%；而280mmol/L则完全抑制了种子的萌发。盐胁迫对胚芽、株高、初生根和次生根的抑制作用也具有明显的浓度(剂量)-效应,在种子萌发期对初生根的抑制作用强于胚芽,在营养生长期对株高的抑制作用强于次生根。盐胁迫对萌发期和营养生长期幼苗新生叶的长、宽和面积都有抑制作用且随着盐浓度升高均呈显著性降低(P<0.05),但对幼苗根的鲜重、干重表现为低浓度(50,70mmol/L)促进而高浓度(>100mmol/L)抑制。2.与对照组相比,随着盐浓度的升高,菊苣幼苗中丙二醛(MDA)含量、质膜透性(MP)和脯氨酸含量均逐渐升高,而根系活力则逐渐下降,其中MDA和脯氨酸含量在70mmol/L时即显著高于对照组,MP则在140mmol/L处理时显著高于对照组(P<0.05)。盐胁迫影响了叶绿素的合成与代谢,菊苣幼苗中叶绿素的含量随着盐胁迫浓度的升高呈现先升高后降低的趋势。处理组菊苣根和叶中的Na+含量均明显升高而K+含量降低,其中根部显著降低(P<0.05)；Ca2+含量变化不明显；根和叶的K+/Na+以及叶的Ca2+/Na+比值均显著降低(P<0.05)。3.不同浓度的SNP (0.10～0.30mmol/L)预处理均可以缓解盐胁迫对种子萌发和幼苗生长的抑制作用,其中0.2mmol/L SNP处理组达到最佳效果,使种子的发芽率、发芽势、发芽指数和活力指数均显著高于其相应的单盐处理组(P<0.05)。此外,0.2mmol/L SNP预处理也使盐胁迫(140mmol/L NaCl胁迫15d)对幼苗生长的抑制降到最低。4.对盐胁迫下渗透调节物质的测定表明,与空白对照相比,随着盐胁迫时间的延长(6-15d),菊苣相对含水量(RWC)明显降低,但脯氨酸含量显著上升(P<0.05)。HPLC分析表明,根中果糖、葡萄糖、蔗糖和1-蔗果三糖含量均随着盐胁迫时间的延长而降低,而蔗果四糖的含量则升高。SNP预处理不仅缓解了盐胁迫对菊苣叶RWC的抑制,而且使脯氨酸含量和蔗果四糖含量急剧增加(P<0.05),同时降低了果糖、葡萄糖和蔗糖的含量。5.抗氧化酶系统参与了植物盐胁迫下的生理响应和适应性。盐胁迫使菊苣幼苗中超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性减弱,而使过氧化物酶(POD)活性增强,MDA含量升高。SNP预处理可缓解盐胁迫对菊苣幼苗根长、鲜重的抑制作用,并促进MDA含量显著下降(P<0.05); SOD和POD同工酶谱带增多,且酶活性显著增强(P<0.05), CAT活性也有增强趋势。过氧化氢(H2O2,0.5mmol/L)预处理也有上述效应。6.实时荧光定量PCR和酶联免疫分析表明,盐胁迫下菊苣幼苗的热激蛋白(HSP90)和脱水素(CiDHN1)基因的mRNA相对表达量增加且后者在胁迫2h显著高于空白对照组(P<0.01)；随着盐胁迫时间的延长(2～48h), HSP90含量无明显变化,但CiDHN1的含量则逐渐升高,并在24h和48h后分别在0.001和0.01水平极显著高于空白对照组。SNP预处理使盐胁迫下菊苣幼苗内HSP90和CiDHN1的mRNA表达进一步增多,其中HSP90和CiDHN1的mRNA含量分别在盐胁迫8h和24h明显高于其对应的单盐处理组(P<0.05)。盐胁迫期间(2～48h), SNP预处理菊苣幼苗中HSP90含量无明显变化,而CiDHN1蛋白含量则呈现逐渐上升的趋势。过氧化氢(0.5mmol/L)预处理后,菊苣幼苗中HSP90和CiDHN1的mRNA相对表达量和蛋白含量也表现出与SNP处理后相似的变化趋势,但效应不及SNP明显。以上结果表明,70mmol/L的NaCl是将军菊苣种子萌发期和营养生长期幼苗的耐受浓度；0.2mmol/L SNP不但能有效缓解盐胁迫对菊苣种子萌发的抑制,而且还能明显提高盐胁迫下幼苗地上部分的生物量,增强菊苣的盐适应；这种缓解效应可能是通过以下几种途径来实现的：1.SNP预处理增强了菊苣幼苗的保水能力,促进渗透调节物质脯氨酸的合成与积累,同时促进小分子糖类向果聚糖特别是蔗果四糖转化,使果聚糖含量增加；2.SNP增强了菊苣幼苗中POD同工酶的活性,并使其同工酶谱带增加,同时促使SOD和CAT活性增加,而减少MDA含量；3.SNP促进了菊苣幼苗中抗应激蛋白HSP90和CiDHN1mRNA的表达,并提高了幼苗中CiDHN1的含量。由此可见,SNP预处理可以通过菊苣幼苗内渗透调节物质、抗氧化酶系统与抗应激蛋白等途径发挥协同作用以提高菊苣幼苗的盐适应能力。更多还原

【Abstract】 Seed germination and plant growth can be largely inhibited under salt stress. Nitric oxide (NO) as a signaling molecule or effector molecule participates in a variety of physiological processes in plants and plays an important role. This paper was aimed at investigating the impacts of NaCl (0～280mmol/L) on seed germination, growth and physiological response of chicory (Cichorium intybus L. cv. Commander) seedlings, evaluating the effects of sodium nitroprusside (SNP, common used as NO donor,0.2mmol/L), on osmotic regulation substances of chicory seedlings in vegetative stage(VS) under different concentrations of NaCl (140or280mmol/L), and the roles of SNP (0.1mmol/L) on antioxidant enzyme activity, gene expression and protein contents of HSP90and CiDHNl. The main research results are as follows:1The germination energy, germination percentage, germination index and vigor index were all inhibited significantly under salt stress (100～280mmol/L) in profound dose-dependent and time-dependent manners. Germination percentage in180mmol/L NaCl group was decreased by50%compared with the control, and the seed germination could be completely inhibited by salt stress (280mmol/L NaCl).The plumules, plant heights, primary and secondary roots of chicory seedlings were also inhibited in an obvious concentration (dose)-dependent effect. The inhibition effect of salt stress on primary roots was stronger than on that of plumule, and this inhibiting effect on plant heights was more obvious than that of secondary roots at vegetative stage. Fresh weights and dry weights of roots of seedlings were improved under low salt concentration (50～70mmol/L), but inhibited under high salt concentration (>100mmol/L). The length, width and area of new leaves were all decreased significantly with the increase of salt concentrations (P<0.05). Fresh weight and dry weight of seedling leaves had the similar performance to seedling roots.2As for the anti-oxidant parameters, the contents of malondialdehyde (MDA), plasma membrane permeability (MP) and proline in chicory seedlings under salt stress were increased compared with the control group. Meanwhile, the vigor of seedling roots decreased gradually with the increase of salt concentrations. Both the MDA content and proline content were remarkably higher than those of the control group at70mmol/L, whereas the MP was significantly higher than the control group at140mmol/L (P<0.05). Salt stress disturbed the process of chlorophyll synthesis and metabolism. Chlorophyll content of chicory seedlings showed a trend of increase at first and then decrease with the rise of salt concentrations of salt stress. Na+contents in chicory roots and leaves of treatment group were significantly increased while K+contents decreased, which decreased significantly in roots (P<0.05). There was no obvious change of Ca2+contents in the tested groups. Both the K+/Na+ratio in roots, leaves and Ca2+/Na+ratio in leaves were all significantly decreased (P<0.05).3Treatment with different SNP concentrations (0.10～0.30mmol/L) supplemented to salt solution could alleviate the inhibition of salt stress on seed germination and seedling growth, and the optimal concentration of SNP was0.2mmol/L. The seed germination percentage, germination energy, germination index and vigor index were all significantly higher than the corresponding salt exposing alone group (P<0.05). In addition, SNP pretreatment (0.2mmol/L) alleviated the inhibition role on seedlings growth induced by salt stress (140mmol/L for15d) to a maximum level in all SNP-treated groups.4The data from detecting the osmotic regulation substances showed that chicory relative water content (RWC) was decreased significantly while the proline content was significantly increased (P<0.05) under salt stress (6～15d) compared with the control. The results of high-performance liquid chromatography (HPLC) analysis further indicated that the contents of fructose, glucose, sucrose and1-kestose increased while nystose content decreased in a time-dependent manner (6～15d). However, the declination of RWC of chicory seedling leaves induced by salt stress was significantly alleviated by SNP pretreatment with a dramatic rise in the contents of proline and nystose (P<0.05) compared with the group treated by salt alone, while with a decline of fructose, glucose and sucrose.5Antioxidant enzymes system involved in the physiological responses and acclimation when plants under salt stress. The activities of superoxide dismutase (SOD) and catalase (CAT) were weakened while peroxidase (POD) activity and MDA content were obviously increased under salt stress. The inhibition of salt stress on root length, fresh weight and dry weight of chicory seedlings could be alleviated by SNP pretreatment, and MDA content of chicory seedlings pretreated with SNP decreased significantly(P<0.05). SOD and POD isozyme bands increased, and their activities were significantly increased (P<0.05), CAT activity has also an increased trend. Pretreatment with hydrogen peroxide (0.5mmol/L) has the same effect as above. 6Real-time fluorescent quantitative PCR and enzyme linked immune analysis indicate that the relative expression of heat shock protein (HSP90) and dehydration (CiDHNl) mRNA increased under salt stress, and the relative expression of CiDHNl mRNA was significantly higher than the control (P<0.01) at2h after exposure to salt stress. HSP90content has no obvious change, but the content of CiDHN1gradually increased in a time-dependent manner (2-48h), and showed extremely significant difference at24h (P<0.001) and very significant (P<0.01) difference at48h compared with the control. The relative expression of mRNA of HSP90and CiDHN1in chicory seedlings pretreated with SNP prior to salt stress were further increased, and they were significantly increased at8h and24h respectively compared with the corresponding single salt treatment group (P<0.05). HSP90content had no obvious change in chicory seedlings pretreated with SNP while CiDHN1content gradually increased during salt stress(2-48h). The relative expression of HSP90and CiDHNl mRNA and protein contents showed the similar tendency in chicory seedlings pretreated with hydrogen peroxide (H2O2,0.5mmol/L), but the effect is less than the SNP.The results indicated that the tolerance level of chicory seedlings at germination and vegetative stage to salinity was70mmol/L NaCl. Treatment or pretreatment with0.2mmol/L SNP not only alleviated the inhibition on seeds germination, but also promoted biomass on the ground part of seedlings under salt stress and improved salt adaptation of chicory. The inhibition-alleviating effect of SNP pretreatment may be worked through the following ways:1. Pretreated with SNP, water retention capacity of chicory seedlings was strengthened, the synthesis and accumulation of proline was promoted, while small molecular sugars transforming into fructan, especially nystose was also promoted, and resulted in increased fructan content.2. Pretreated with SNP, the activity of POD isozyme in chicory seedlings was enhanced, and POD isozyme bands were also increased; at the same time, SOD and CAT activities were also increased while the MDA content was reduced.3. Pretreated with SNP, the relative expression of HSP90and CiDHNl mRNA were promoted and CiDHNl contents were also significantly increased. Therefore, the salt adaptability of chicory seedlings was improved through the combination results of osmotic regulation substances, antioxidant enzyme system and anti-stress proteins system.更多还原