NO和茉莉酸甲酯对黄芩悬浮细胞生长及黄芩苷合成的影响

以硝普钠(sodium nitroprusside,SNP)为一氧化氮(nitric oxide,NO)的供体,向黄芩(Scutellaria baicalensis)悬浮培养细胞系中添加SNP和茉莉酸甲酯(methyl jasmonate,MJ),考察这两种诱导子在不同的添加时间、添加浓度及混合配比使用对黄芩悬浮细胞系生长和黄芩苷含量的影响。研究结果表明：低浓度的外源NO有利于细胞的生长,但对黄芩苷积累无作用,而MJ有利于黄芩苷的合成,但抑制细胞生长,且两者的适用浓度范围和添加时间存在差异。在细胞培养初期(0天)添加0.05 mmol·L~(-1)SNP,而在细胞生长对数中期(8天)添加10μmol·L~(-1)的MJ,细胞鲜重可达到对照的1.2倍,黄芩苷总量达到对照的2.96倍。     

茉莉酸甲酯、水杨酸和一氧化氮诱导怀槐悬浮细胞合成异黄酮及细胞结构变化

本文对比研究了茉莉酸甲酯(MeJA)、水杨酸(SA)和一氧化氮(NO)三种激发子对怀槐悬浮培养物异黄酮合成及细胞结构变化的影响。结果表明,在三种激发子的作用下怀槐细胞异黄酮合成量显著提高:200μmol/L MeJA、100μmol/L SA及50μmol／L SNP处理培养细胞9d后,异黄酮含量分别为同期对照的417．18％、185．45％和222．45％。同时细胞内发现染色很深的电子致密小体(EDB),其数量随着异黄酮含量的升高而增加,亦在第九天达到最多,与异黄酮积累呈现正相关性。推测激发子可能诱导植物细胞结构变化来响应次生代谢产物的合成。     

茉莉酸甲酯与二氢茉莉酮酸甲酯对悬浮培养的甘草细胞生长和黄酮积累的影响

建立了稳定的甘草细胞悬浮培养体系,在一个培养周期内,细胞的生长曲线呈"S"型,培养21 d的干重、鲜重和黄酮产量都达到最高值.甘草细胞悬浮培养体系中分別添加100 μmol·L-1 二氢茉莉酮酸甲酯和茉莉酸甲酯时,虽然对细胞生长有一定程度的抑制,但细胞中甘草黄酮产量仍有提高.添加二氢茉莉酮酸甲酯和茉莉酸甲酯的最适时间分别为细胞培养后的第5天和第10天.     

茉莉酸甲酯对茶条槭悬浮细胞没食子酸合成的影响

为了研究茶条槭悬浮细胞中没食子酸的合成,该研究进行了茉莉酸甲酯诱导试验。通过添加茉莉酸甲酯,利用HPLC检测诱导后细胞中没食子酸的含量变化情况,同时利用电导仪、酸度计、分光光度计法和激光共聚焦显微镜对培养液和细胞进行电导率、pH值、苯丙氨酸解氨酶(PAL)活性以及细胞形态等进行分析。结果表明:(1)相对于正常培养的细胞,添加100μmol·L~(-1)的茉莉酸甲酯诱导24 h时,没食子酸的含量达到最高为12.49 mg·g~(-1),其含量是对照的2倍左右。(2)茉莉酸甲酯的添加导致细胞培养液的pH值和电导率成波动趋势,细胞膜受损,通透性增大,细胞核分散,出现多个细胞核现象。(3)细胞内可溶性蛋白含量在诱导24 h、72 h和5 d时达到高峰,其含量分别是对照的1.4、1.67、2.07倍左右。(4)苯丙氨酸解氨酶活性在诱导24 h和5 d时分别出现一次高峰,其活性分别是对照的2倍和3.75倍。研究认为,茉莉酸甲酯处理短时间内促进了茶条槭细胞内没食子酸含量的积累,细胞内PAL活性和可溶性蛋白含量有所增加,对细胞液中的pH值和电导率影响不显著。     

茉莉酸甲酯对水稻幼苗生长的影响

２．５×１０＾－７ｍｏｌ·Ｌ＾－１茉莉酸甲酯促进水稻幼苗生长,而高于２．５×１０＾－５ｍｏｌ·Ｌ＾－１则抑制。不同水稻品种对茉莉酸甲酯的反应不同。     

外源茉莉酸和茉莉酸甲酯诱导植物抗虫作用及其机理

综述了茉莉酸(jasmonic acid, JA)和茉莉酸甲酯(methyl jasmo nate, MJA)的分子结构和应用其诱导的植物抗虫作用及其机制。植物受外源茉莉酸或茉莉酸甲酯刺激后,一条反应途径是由硬脂酸途径激活防御基因,另一条途径是直接激活防御基因。防御基因激活后导致代谢途径重新配置,并可能诱导植物产生下列4种效应：（1）直接防御,即植物产生对害虫有毒的物质、抗营养和抗消化的酶类,或具驱避性和妨碍行为作用的化合物;（2）间接防御,即产生吸引天敌的挥发物;（3）不防御,即无防御反应;（4）负防御,即产生吸引害虫的挥发物。     

水杨酸、茉莉酸甲酯和乙烯利对野葛细胞悬浮培养生产葛根素的影响

野葛〔Ｐｕｅｒａｒｉａｌｏｂａｔａ(Ｗｉｌｌｄ.)Ｏｈｗｉ〕是我国及其他东亚国家传统的药用资源,其主要药用成分为葛根素,广泛用于治疗多种心血管疾病[1]。由于近年来生态环境的人为破坏,野生野葛资源已不能满足人们对葛根素药物的需求,通过细胞工程可以实现野葛资源的可持续性开发。本项目组已经建立了野葛组织培养和植株再生[2]及细胞悬浮培养体系[3],研究结果表明,悬浮细胞培养物能够生产葛根素,但产量仍有待于提高[3]。已知水杨酸(ｓａｌｉｃｙｌｉｃａｃｉｄ,ＳＡ)、乙烯和茉莉酸类化合物(ｊａｓｍｏｎａｔｅｓ,ＪＡｓ)能够在高…     

茉莉酸甲酯与真菌诱导物、水杨酸组合对红豆杉细胞中紫杉醇含量的影响

比较了茉莉酸甲酯与真菌诱导物、水杨酸组合对红豆杉细胞几个抗病相关指标（POD、CAT活力、H2O2含量）及紫杉醇含量的影响,3种信号分子的组合对POD、CAT、H2O2及紫杉醇含量的影响是不一致的,MJ单独添加,MJ与SA联合作用以及MJ与F5联合作用都可使POD活力增加,且12h后H2O2含量均升高,约在48h达到高峰,为对照的2倍左右,但72h后,MJ单独添加和MJ与SA联合作用组中H2O2含量变化不大,F5与MJ联合作用则使H2O2含量持续比对照高。MJ单独添加使CAT酶活在144h后才较对照低,F5、SA的加入都可使CAT酶活下降,SA的作用更显著。说明三者的诱导途径并不完全一样,以SA和MJ联合添加对紫杉醇合成的促进作用最大,含量达到细胞干重的0．04％。     

茉莉酸甲酯对花生幼苗生长和抗旱性的影响

经过茉莉酸甲酯处理的花生幼苗,在形态、解剖和生理上都发生变化,其中以１２５ｍｇ／Ｌ处理最显著。处理后的植株幼苗矮化,叶小而厚,叶片贮水细胞大、蒸腾减弱、内源脱落酸和脯氨酸含量增多、过氧化物酶活性加大。由于水分的丧失减少,叶片水分的贮存增加,从而提高幼苗的抗旱性。     

茉莉酸甲酯处理雷公藤悬浮细胞的基因差异表达分析

以不同浓度茉莉酸甲酯(MeJA)为诱导子对雷公藤悬浮细胞进行处理,采用cDNA-AFLP技术对差异表达基因进行研究。结果表明,茉莉酸甲酯在50～400μmol/L浓度范围内对雷公藤悬浮细胞总碱的积累呈抑制作用。茉莉酸甲酯处理后,分析筛选出了19个雷公藤悬浮细胞内差异表达的基因。通过与NCBI蛋白质数据库比对,7个片段的功能得以预测,涉及植物细胞的信号转导、转录调控和能量代谢等。这些结果对今后利用生物技术手段提高雷公藤生物碱含量奠定了一定基础。     

光质对悬浮培养黄岑细胞生长及黄岑苷积累的影响

研究了不同光质、光强和光期对悬浮培养黄芩(Scutellaria baicalensis)细胞的生长、细胞中苯丙氨酸氨基裂解酶(PAL)活性及黄芩苷含量的影响。白光、紫外光、红光和蓝光对悬浮培养黄芩细胞PAL活性和黄芩苷含量有不同的影响。白光、紫外光和蓝光对PAL活性和黄芩苷的积累具有诱导促进作用,其中紫外光的促进作用最强,白光和蓝光的促进作用较弱,而红光照射对PAL活性和黄芩苷的积累有一定的抑制作用。在0-60μmolm-2s-1紫外光照射强度范围内,PAL活性和黄芩苷含量随着紫外光照射强度的增加而显著提高。实验结果同时表明,红光照射显著促进黄芩细胞的生长,紫外光抑制黄芩细胞的生长,而蓝光和白光对悬浮培养黄芩细胞生长的促进作用不明显。与黑暗(对照)相比,每天8h光照强度为60μmolm-2s-1的紫外光、16h光照强度为50μmolm-2s-1的红光交替处理,在培养12d后,细胞的鲜重和黄芩苷含量为对照的1.16和3.2倍,黄芩苷的产量达到439mgL-1,是对照的3.8倍。     

Effects of Methyl Jasmonate on Shikonin and Dihydroechinofuran Production in Lithospermum Cell Cultures

Methyl jasmonate, when administered to Lithospermum erythrorhizoncell suspension cultures, was found to induce the productionof shikonin derivatives (the red naph-thoquinone pigments ofthe root) and dihydroechinofuran (an abnormal metabolite ofgeranylhydroquinone). Culture experiments showed that methyljasmonate caused a rapid increase in the activities of enzymesinvolved in the biosynthesis of shikonin such as p-hydroxybenzoategeran-yltransferase, which was followed by the rapid accumulationof dihydroechinofuran and the delayed production of shikonin.The induction patterns observed were similar to those elicitedby oligogalacturonides in Lithospermum cells, suggesting thatjasmonic acid or its derivative may act as a signaling moleculein the elicitation of shikonin biosynthesis. Interestingly,however, the copper ion, which is essential for inducing shikoninbiosynthesis by oligogalacturonides, was not required for shikonininduction by methyl jasmonate ¹Present address: Laboratory of Molecular & Cellular Biology,Department of Agricultural Chemistry, Kyoto University, Kitashirakawa,Kyoto, 606-01 Japan     

NO对金丝桃悬浮细胞生长及金丝桃素生物合成的促进作用研究

一氧化氮 (NO)是近年来发现的一种新型植物信号分子。以硝普钠 (Sodiumnitroprusside ,SNP)为一氧化氮 (NO)的供体 ,研究外源NO对金丝桃悬浮细胞生长及金丝桃素生物合成的影响。试验结果表明 ,金丝桃悬浮细胞在含 0 5和 15 0mmol LSNP的培养基中培养 2 0d后 ,细胞的干重分别为对照组的 140%和50% ;细胞中金丝桃素的含量分别为对照组的 98%和210%。试验结果表明 ,低浓度SNP处理有利于金丝桃悬浮细胞生长 ,而高浓度SNP可以促进金丝桃素的合成。在细胞培养初期 (0d)加入 0.5mmol LSNP并在指数生长后期 (14d)加入15.0mmol LSNP的金丝桃悬浮细胞在培养 2.5d后 ,细胞的干重和金丝桃素的含量分别为对照组的1.4和1.8倍 ,金丝桃素的产量达15.2mg/L ,比对照高3.2倍。SNP对金丝桃悬浮细胞生长及金丝桃素含量的影响可以被NO专一性淬灭剂CPITO(2-4-carboxyphenyl-4 ,4 ,5 ,5-tetramethylimidazoline-1-oxyl-3-oxide)所抑制,说明SNP是通过其分解产物NO影响细胞生长和金丝桃素的合成。试验结果同时表明,在15.0mmol/L的SNP处理下,金丝桃悬浮细胞中的苯丙氨酸解氨酶(PAL)的活性显著升高,推测NO可能通过触发金丝桃悬浮细胞的防卫反应,激活了细胞中金丝桃素的生物合成途径。     

NO对银杏悬浮细胞生长及黄酮类物质合成的影响

以硝普钠(sodium nitroprusside,SNP)为一氧化氮(NO)的供体,向银杏悬浮细胞培养液中加入不同浓度的SNP,研究外源NO对银杏悬浮细胞生长状况、过氧化氢酶(CAT)活性、苯丙氨酸解氨酶(PAL)活性和黄酮类物质生物合成的影响.结果表明,低浓度SNP有利于银杏悬浮细胞生长,而高浓度SNP可以促进黄酮类物质的合成.银杏悬浮细胞在添加0.5和10 mmol/L SNP的培养基中培养16 d时,细胞干重分别为对照组的134%和73%;在添加10 mmol/L SNP的培养基中培养20 d时,细胞中黄酮类物质的含量为对照组的136%.同时,10 mmol/L SNP促进银杏悬浮细胞PAL和CAT活性显著升高.NO专一性淬灭剂c-PITO(carboxyl phenyltetramethylimidazoleoxide)抑制SNP对银杏悬浮细胞生长、CAT活性、PAL活性和黄酮类物质含量的促进作用,说明SNP是通过其分解产物NO影响细胞生长和黄酮类物质的合成.根据这些结果推测,NO可能通过触发银杏悬浮细胞的防卫反应,激活了细胞中黄酮类物质的生物合成途径.     

几种影响黄芩愈伤组织中黄芩苷含量的因素

研究碳源、生长调节物质、氮源、磷酸盐以及有机添加物对黄芩愈伤组织中黄芩苷含量和产量影响的结果表明,在(25±1)℃暗培养条件下,黄芩苷积累的最佳培养基为MS基本培养基,其中氮源浓度为60mmol·L-1(NH4+:NO3-为1:1),KH2PO4浓度为1.5mmol·L-1,附加80g·L-1蔗糖、0.3mg·L-1IAA、2mg·L-16-BA和200mg·L-1蛋白胨。在上述培养基中,黄芩愈伤组织培养40d后的黄芩苷含量达到167.4mg·g-1,产量达到4.8g·L-1,分别是对照的5.54倍和18.2倍。     

荣莉酸甲酯对丹参培养细胞中迷迭香酸生物合成及相关酶活性的影响

茉莉酸甲酯是植物细胞响应外界刺激产生的重要信号分子,与植物次生代谢物的生物合成有关。本研究考察了茉莉酸甲酯（methyl jasmonate,MeJA）对丹参培养细胞中迷迭香酸（rosmarinic acid,RA）生物合成的影响。结果显示,诱导24h后可显著提高丹参愈伤细胞中RA的积累量及其相关酶（PAL、TAT）的活性,在48h时RA积累量和酶活性达到最大。布洛芬（IBu）处理可抑制MeJA对RA积累量和相关酶活性的促进作用,外源施加MeJA可部分解除IBU对RA合成及其相关酶活性的抑制作用。说明MeJA可以显著促进丹参培养细胞中RA的生物合成,IBU抑制了MeJA合成、PAL和TAT活性,从而导致了RA合成受阻。     

茉莉酸甲酯与水杨酸对肉苁蓉悬浮细胞中苯乙醇甙合成的影响

向肉苁蓉悬浮细胞培养系中添加茉莉酸甲酯(MJ)和水杨酸(SA) ,分别考察了这两种诱导子的添加浓度及添加时间对肉苁蓉悬浮细胞系中苯乙醇甙含量的影响。研究结果表明:MJ和SA能够促进肉苁蓉悬浮细胞系中苯乙醇甙(PeG)和松果菊甙(Echinacoside)的合成,但两者的适用的浓度范围和最佳添加时间存在差异。与未经诱导子处理的细胞培养结果相比,MJ在对数生长初期(培养14d) ,添加浓度为5 μmol L条件下,可使肉苁蓉悬浮细胞系中PeG含量提高2 5 9倍,Echin含量提高3 82倍;而SA在对数生长后期(培养2 8d) ,添加浓度为5 0 μmol L条件下,可使PeG含量提高2 71倍,Echin含量提高3 16倍。     

Nitric Oxide Is Involved in Cadmium-Induced Programmed Cell Death in Arabidopsis Suspension Cultures

Exposure to cadmium (Cd²⁺) can result in cell death, but the molecular mechanisms of Cd²⁺ cytotoxicity in plants are not fully understood. Here, we show that Arabidopsis (Arabidopsis thaliana) cell suspension cultures underwent a process of programmed cell death when exposed to 100 and 150 μm CdCl₂ and that this process resembled an accelerated senescence, as suggested by the expression of the marker senescence-associated gene12 (SAG12). CdCl₂ treatment was accompanied by a rapid increase in nitric oxide (NO) and phytochelatin synthesis, which continued to be high as long as cells remained viable. Hydrogen peroxide production was a later event and preceded the rise of cell death by about 24 h. Inhibition of NO synthesis by N^G-monomethyl-arginine monoacetate resulted in partial prevention of hydrogen peroxide increase, SAG12 expression, and mortality, indicating that NO is actually required for Cd²⁺-induced cell death. NO also modulated the extent of phytochelatin content, and possibly their function, by S-nitrosylation. These results shed light on the signaling events controlling Cd²⁺ cytotoxicity in plants.Cadmium (Cd²⁺) is a heavy metal with a long biological half-life, and its presence as a pollutant in agricultural soil is due mainly to anthropogenic activities. It is rapidly taken up by roots and enters the food chain, resulting in toxicity for both plants and animals (for review, see Sanità di Toppi and Gabbrielli, 1999). Cd²⁺ inhibits seed germination, decreases plant growth and photosynthesis, and impairs the distribution of nutrients. Overall, the symptoms of chronic exposure to sublethal amounts of Cd²⁺ mimic premature senescence (Rascio et al., 1993; McCarthy et al., 2001; Sandalio et al., 2001; Rodriguez-Serrano et al., 2006). Depending on the concentration, Cd²⁺ treatment of tobacco (Nicotiana tabacum) cell cultures and onion (Allium cepa) roots eventually triggers either necrosis or programmed cell death (PCD; Fojtovà and Kovařik, 2000; Behboodi and Samadi, 2004).Although Cd²⁺ is an environmental threat, the mechanisms by which it exerts its toxic effects in plants are not fully understood. In plant cells, Cd²⁺ is believed to enter through Fe²⁺, Ca²⁺, and Zn²⁺ transporters/channels (Clemens, 2006). Once in the cytosol, Cd²⁺ stimulates the production of phytochelatins (PCs), a glutathione-derived class of peptides containing repeated units of Glu and Cys, which bind the metal ions and transport them into the vacuole (Sanità di Toppi and Gabbrielli, 1999). Strong evidence exists that high (millimolar) concentrations of Cd²⁺ induce reactive oxygen species (ROS) bursts in plants, which might have a role in signaling and/or degenerative steps leading to cell death (Piqueras et al., 1999; Olmos et al., 2003; Cho and Seo, 2005; Garnier et al., 2006). Treatment with a lower, nontoxic Cd²⁺ concentration also caused increase in ROS production in pea (Pisum sativum) leaves and roots (Sandalio et al., 2001; Romero-Puertas et al., 2004; Rodriguez-Serrano et al., 2006) and Arabidopsis (Arabidopsis thaliana) cell cultures (Horemans et al., 2007).Nitric oxide (NO) is a gaseous reactive molecule with a pivotal signaling role in many developmental and response processes (for review, see Neill et al., 2003; Besson-Bard et al., 2008). In plants, it can be synthesized via several routes, either enzymatically or by chemical reduction of nitrite. Nitrate reductase and a root-specific plasma membrane nitrite-NO reductase also utilize nitrite as substrate. In animals, nitric oxide synthase (NOS) converts l-Arg into NO and l-citrulline. Although no plant NOS has been unambiguously identified yet, activity assays and pharmacological evidence suggests the existence of a NOS-like counterpart in plants. Depending on its concentration and possibly on the timing and localization of its production, NO can either act as an antioxidant or promote PCD, often in concert with ROS (Delledonne et al., 2001; Beligni et al., 2002; de Pinto et al., 2006). Extensive research has shown that NO plays a fundamental role in the hypersensitive response, but its involvement in other types of PCD, such as that resulting from mechanical stress and natural and cytokinin-induced senescence of cell cultures, has also been demonstrated (Garcês et al., 2001; Carimi et al., 2005). Because of its participation in numerous biotic and abiotic responses, NO has been proposed as a general stress molecule (Gould et al., 2003). However, the mechanisms by which NO determines its effects are far from being completely elucidated, and a number of downstream signaling pathways, involving Ca²⁺, cyclic GMP, and cyclic ADP-Rib, are involved (Neill et al., 2003; Besson-Bard et al., 2008). NO can also modulate biological responses by direct modification of proteins, reacting with Cys residues (S-nitrosylation), Tyr residues (nitration), or iron and zinc in metalloproteins (metal nitrosylation; Besson-Bard et al., 2008).The aim of this work is to study the plant responses to various concentrations of Cd²⁺ and, in particular, the role of ROS and NO in the signaling events leading to cell death. Cell cultures of the model plant Arabidopsis were chosen as an experimental system because the homogeneity and undifferentiated state of the cells, combined with the uniform delivery of the treatments, allow a clear and reproducible response. The results point to NO as a master regulator of Cd²⁺-induced cell death. Possible mechanisms that explain this evidence will be discussed.