【作者】 赵晓菊；

【导师】 满秀玲； 唐中华；

【作者基本信息】 东北林业大学 ， 水土保持与荒漠化防治， 2018， 博士

【摘要】 目前,全球土壤次生盐渍化范围不断扩大,程度逐渐加重,严重影响农业生产和生态环境,盐渍土的改良和植被恢复是国内外研究者关注的热点问题。一氧化氮(nitric oxide,NO)作为一种重要的信号分子,参与调控植物种子萌发、生长、发育和衰老等过程,同时在植物逆境响应中也发挥着重要作用。近年来研究表明,NO能通过改变植物代谢影响植物盐耐受性,但其调控基础还不清楚。长春花(Catharanthus roseus)因体内含有抗癌活性的生物碱类而备受关注。此外,大量研究表明长春花还富含酚类化合物,其中大部分酚类化合物因具有免疫调节、抗肿瘤、抗氧化等多种药理功效,现已成为新药、先导化合物和新化学实体的重要来源。已有研究证实盐胁迫和外源氮素都可促进长春花体内生物碱含量的提高,但对于同样是次生代谢产物的酚类化合物鲜有报道。本研究在外源NO供体硝普钠(sodium nitropptusside,SNP)处理NaCl胁迫下长春花生长、生理指标等分析的基础上,利用液相色谱-质谱联用(LC-MS)仪靶向分析NO对盐胁迫下长春花不同部位中酚类化合物组分及含量水平的变化,从代谢组水平揭示NO调控盐胁迫下酚类化合物的响应,为酚类代谢调控和NO提高长春花耐盐性机理奠定基础,并对盐碱地开发利用具有实践意义。主要研究结果如下:(1)短时间(10 d)25 mM NaCl处理下长春花幼苗(木质部未分化)与对照差异不显著,甚至部分程度促进植株生长,浓度大于50 mMNaCl时,与对照组差异显著,整体上看,NaCl抑制植株生长,且随着浓度的增大和时间的延长抑制效果越明显。长时间(85 d)NaCl处理长春花幼苗(木质部已分化),NaCl胁迫15 d内与对照相比各生长指标差异不显著,20 d后开始有差异显著性。株高主要靠节数目的增加,节间距变化不明显,形成木质部的长春花幼苗,对盐胁迫有一定的抵御能力。(2)NaCl浓度在100 mM以下时,植株上、中、下叶片内脯氨酸(proline,Pro)和丙二醛(malonaldehyde,MDA)含量平均值均低于对照组,叶面积、叶长、叶宽等生长指标与对照相比生长缓慢;125 mMNaCl处理下Pro和MDA含量高于对照组,达到最高值,植株叶片面积、叶长、叶宽等生长指标表示明显抑制了植株生长。100-125 mM NaCl浓度是严重影响植株生长的临界范围。(3)SNP对盐胁迫下长春花种子萌发的影响实验中,50mM NaCl胁迫下的发芽势、发芽率、发芽指数、活力指数、根长、芽长均低于正常对照,盐胁迫抑制了种子萌发;0.1 mM SNP有效缓解盐胁迫对长春花种子萌发的抑制作用,甚至能恢复种子正常发芽率;大于0.5 mM SNP会加重盐胁迫伤害,各项发芽参数、芽长、根长等均低于盐胁迫下。NO在盐胁迫下长春花种子萌发过程中起着重要的信号调控作用,低浓度缓解盐胁迫伤害,高浓度加重胁迫伤害。(4)外源SNP有效提高盐胁迫下长春花叶片中硝酸还原酶(nitrate reductase,NR)活性,4 d后NR活性达到最高值,活性变化情况为4 d>5 d>3 d>2 d>1 d;0.5 mM SNP处理条件下NR活性最高,其中0-0.5 mM SNP提升NR作用显著,1.0-2 mM SNP作用下降。0.1 mM SNP促进了 Pro含量积累,降低了盐胁迫导致的MDA含量上升,可溶性糖含量达到最高,有效缓解盐胁迫引起的伤害,增强植株的耐盐性。(5)SNP对盐胁迫下木质部成熟的长春花植株在生长指标上缓解效果不显著,其中125 μM SNP明显抑制株高生长,且株高的改变主要靠降低节的个数来完成的,同时基茎明显增粗,植物靠降低高度增加茎粗度来提高对外界胁迫的抵御。NO作为一种信号分子参与调节气孔运动,SNP处理盐胁迫下长春花叶片下表皮气孔数量和开放数量增多,施加次数增多,变化幅度加大,0.1 mMSNP处理气孔数量增加幅度最大。(6)采用LC-MS技术分析SNP对盐胁迫下长春花根、茎、叶、花四个部位酚类物质种类和含量变化,鉴定出18种酚类物质和L-苯丙氨酸,按照化学结构式把18种酚类物质分成C6C1酚类5种:香草酸、对羟基苯甲酸、丁香酸、原儿茶酸、没食子酸;C6C3酚类5种:对羟基肉桂酸、绿原酸、阿魏酸、肉桂酸、对香豆酸;C6C3C6(黄酮类化合物)8种:染料木素、木犀草苷、柚皮素、槲皮苷、杨梅苷、槲皮素、山柰酚、芦丁。其中槲皮素、绿原酸、原儿茶酸是长春花体内主要酚类物质,在不同部位中均有存在。(7)不同浓度SNP处理后长春花不同器官酚类化合物响应积累呈现出明显不同的变化。根和茎中积累较多的小分子酚酸类化合物,而叶片以及花中则表现为C3C6C3型酚类化合物的明显富集,相比之下,根和茎中酚类化合物对盐胁迫及SNP处理的响应更敏感一些。(8)酚类化合物在不同部位的分布明显不同,其中C6C3C6结构的黄酮类化合物在根、茎、叶、花中组成和相对含量差异较大,茎中8种黄酮类物质均有检出,而叶片中染料木素、木犀草苷、柚皮素和槲皮苷均未检出。槲皮素在不同部位相对含量均为最高,盐胁迫下茎、叶中含量上升,根和花中下降。(9)75 μM SNP(3#)下植株没有开花,该处理组C6C1酚类化合物在叶中有香草酸和丁香酸出现,而其他开花植株叶片中未出现,酚类与植物成花的内在关系密切。更多还原

【Abstract】 Currently,the scope of soil secondary salinization is expanding in the world with the severity gradually increasing,which seriously affects the agricultural production and ecological environment.The improvement of saline soil and vegetation restoration are the hot issues concerned by researchers around the world.As an important signaling molecule,nitric oxide(NO)participates in the process of plant seed’s germination,growth,development,senescence,It also plays an important role in plant stress response.Recent studies have shown that NO can affect plant salt tolerance by changing plant metabolism,but the regulatory mechanism is not clear.Catharanthus roseus(C.roseus)has attracted much attention because of the alkaloids with anticancer activities in its body.Subsequently,numerous studies have shown that this plant also contains phenolic compounds most of which have a variety of pharmacological effects in immune regulation,anti-tumor,anti-oxidation etc.Now,It has become an important source of new drugs,lead compounds and new chemical entities.Studies have shown that both salt stress and exogenous nitrogen can promote the increase of alkaloid content in C.roseus,but there are few reports on phenolic compounds which are also secondary metabolites.In this study,on the basis of the change of the growth and physiological indexes of the C.roseus under salt stress treated by sodium nitropptusside(SNP)with gradient concentration,LC-MS technology is adopted to measure the components and content of phenolic metabolites in different parts of the C.roseus and to reveal the response of phenolic metabolites under the salt stress in NO regulation from the level of metabolome,and a foundation is laid for further exploring the mechanism that the regulation of phenolic metabolism and NO improves the salt tolerance of the C.roseus,and has practical significance to the development and utilization of saline-alkali land.The major findings are as follows:(1)The difference of the C.roseus seedling(with undifferentiated xylem)was not significant between NaCl(25 mM)treated group and the control group within short time(10 d),NaCl(25 mM)treatment even partly promoted plant growth;as the NaCl concentration exceeded 50mM,the difference was significant compared with the control group;overall,NaCl inhibited plant growth,and with the increase of concentration and the prolongation of time,the inhibitory effect was more conspicuous.Growth and morphological changes of the C.roseus seedling(xylem undifferentiation)under NaCl treatment within long time(85 d),the difference of each growth index was not significant compared with the control group within 15 d under NaCl stress,and the difference significance began after 20 d.The plant height mainly depends on the increase in the number of nodes,and the change of node pitch is not obvious,seedlings of C.roseus with xylem being formed has a certain ability of resistance to salt stress.(2)As the concentration of NaCl is below 100 mM,the average value of proline and malondialdehyde(MDA)content in the upper,middle and lower leaves of the plant were all lower than those of the control group.The leaf area,leaf length,leaf width and other growth indicators were lower than those of the control group;at the concentration of 125 mM NaCl,proline and MDA content were higher than those of the control group,and reached the maximum value;the leaf area,leaf length,leaf width and other growth indicators of the plant stopped growing,which obviously inhabited the growth of the plant.The concentration of 100-125 mM NaCl is the critical range of serious effects on plant growth.(3)In the experiment of the effect of SNP on the C.roseus seed germination under salt stress,the germination potential,germination rate,germination index,vigor index,root length,bud length under 50 mM NaCl stress were all lower than those of the normal control group.Salt stress inhibited the seed germination;0.1 mM SNP effectively relieved the inhibiting effect of salt stress on the c.roseus seed germination,and even restored the normal germination rate of seeds;SNP exceeding 0.5 mM would aggravate salt stress injury;various germination parameters,bud length,and root length were lower than those under salt stress.NO plays an important role in signal regulation under salt stress during the c.roseus seed germination.Low concentration alleviates salt stress injury,and high concentration exacerbates salt stress injury.(4)Exogenous SNP effectively increased the activity of nitrate reductase(NR)in the leaves of the catharanthus roseus under salt stress.After 4d,the activity of NR reached the maximum value,4 d>5 d>3 d>2 d>1 d;the activity of NR was the highest with a concentration of 0.5mM SNP,during which 0-0.5 mM SNP had a significant effect on improving NR,while the effect of 1.0-2 mM SNP declined.As the C.roseus plant being treated by 0.1 mM SNP under salt stress,proline content accumulation was promoted,MDA content rising caused by salt stress was reduced,content of soluble sugar reached the highest,injury caused by salt stress was relieved,and salt tolerance of plants was strengthened.(5)SNP had an insignificant slow-release effect on growth index of the C.roseus plant with xylem maturation under salt stress,in which 125 μM SNP obviously inhibited the plant height growth,and the change of plant height mainly depended on reducing the number of nodes.At the same time,the basal stem obviously thickened,as plants increased the resistance to external stress by reducing height and increasing stem diameter.NO participated in regulating stomatal movement as a kind of signaling molecule.The number of stomata and opening number on lower epidermis of leaves of the C.roseus treated by SNP under salt stress increased.As the number of times applied increased,the range of variation increased.The increase in number of stomata treated by 0.1 mM SNP was the largest.(6)LC-MS technology was adopted to analyze the phenolic substance changes in different positions like roots,stems,leaves,flowers of the C.roseus under salt stress under the effect of SNP,and 18 kinds of phenolic substances and L-phenylalanine were identified.According to the chemical structure formula,the 18 kinds of phenolic substances were classified into 5 kinds of C6C1 phenols:vanillic acid,p-hydroxybenzoic acid,eugenic acid,protocatechuic acid,gallic acid;5 kinds of C6C3 phenols:p-hydroxycinnamic acid,chlorogenic acid,ferulic acid,cinnamic acid,p-coumaric acid;and 8 kinds of C6C3C6(flavonoids):genistein,galuteolin,naringenin,quercitrin,myricetrin,quercetin,kaempferol,rutin,among which the quercetin,chlorogenic acid,protocatechuic acids are the main phenolic substances in the body of the C.roseus,which exist in different positions.(7)The responsive accumulation of the phenolic compounds in different tissues of C.roseus were significantly different under different concentrations of SNP treatment.It is some small molecular phenolic acids that were accumulated in roots and stems;however C3C6C3 phenolic compounds were enriched mainly in surface leaves and flowers.In contrast to flower and leaf,adaptive responses of phenolic compounds in roots and stems were more sensitive to salt stress and SNP treatments.(8)The distribution of phenolic compounds in different positions is significantly different.The composition and relative content of flavonoids with the structure of C6C3C6 in roots,stems,leaves,and flowers are quite different.8 kinds of flavonoids are all detected in stems,but the genistein,galuteolin,naringenin and quercitrin are not detected in leaves.The relative content of the quercitrin is the highest in different positions.Its content increases in stems and leaves under salt stress,but sharply declines in roots and flowers.(9)Under 75 μM SNP(3#),the plant didn’t bloom,C6C1 Phenolic compounds under this treatment,there were vanillic acid,syringic acid appearing in leaves,not appearing in leaves of other flowering plants.Phenolic compounds are closely related to flower formation.更多还原