陆地棉GhPYR1基因的克隆和功能分析

植物激素脱落酸(Abscisic acid,ABA)在植物应对干旱、盐碱等逆境胁迫以及植物种子萌发、根伸长、芽休眠等阶段发挥重要作用。PYR/PYL/RCAR蛋白家族是ABA受体,与ABA结合后能够启动ABA信号传导通路,诱导ABA应答基因的表达。利用电子克隆和RT-PCR技术从陆地棉中克隆了Gh PYR1基因,其编码的Gh PYR1蛋白与拟南芥中At PYR1蛋白相似度为73%。将Gh PYR1蛋白序列与拟南芥14个PYR/PYL/RCAR家族成员蛋白序列进行比对并构建进化树,发现它与拟南芥PYR/PYL/RCAR蛋白亚家族III亲缘关系最近。过表达Gh PYR1基因的T3代拟南芥在外源ABA处理下,其种子萌发和初期根生长均滞后于野生型,表现出对ABA更加敏感;高盐和干旱胁迫对转基因种子的萌发抑制更强烈,但苗期胁迫处理下转基因拟南芥的长势却明显优于野生型;同时在外源ABA诱导条件下ABA应答基因RD29A、RAB18的表达量较野生型有明显提高。以上结果说明Gh PYR1基因编码的蛋白是ABA的受体,过表达该基因能够提高植物对ABA的敏感性和增强应对逆境胁迫的能力。     

拟南芥RabGAP7基因对ABA反应的功能分析

酶母的GYP能够加速小G蛋白YPT内在的GTPase的活性,是因为其基因所编码的氨基酸序列中具有保守的TBC区域,拟南芥AtGAPs的氨基酸序列中也具有此保守区,但对于它们的生物学功能,特别对胁迫的反应却研究不多。我们鉴定得到了RabGAP7基因的纯合突变体。通过根伸长和失水实验发现,与野生型相比,突变体幼苗对ABA和脱水不敏感。另外,表达分析表明,该基因在转录水平上对GPA1和Rab7起到负调控作用。这些结果暗示着RabGAP7可能通过调节G蛋白来参与了ABA反应。     

拟南芥prr5突变体对ABA的响应

以拟南芥为材料,统计PRRs （pseudo-response regulators）突变体 prr5及其野生型经ABA处理后的萌发率、根长和NaCl处理后的萌发率,并采用实时定量PCR方法,对不同浓度ABA处理的拟南芥幼苗中的PRR5基因表达进行分析.结果表明：prr5突变体对ABA弱敏感,其种子萌发率比野生型显著或极显著增高,主根比野生型长,且PRR5基因表达受ABA抑制.同时,NaCl处理后,prr5的萌发率比野生型极显著增高.因此,推测prr5可能为ABA信号通路相关基因.     

拟南芥RabGAP7基因对ABA反应的功能分析

酶母的GYP能够加速小G蛋白YPT内在的GTPase的活性,是因为其基因所编码的氨基酸序列中具有保守的TBC区域,拟南芥AtGAPs的氨基酸序列中也具有此保守区,但对于它们的生物学功能,特别对胁迫的反应却研究不多。我们鉴定得到了RabGAP7基因的纯合突变体。通过根伸长和失水实验发现,与野生型相比,突变体幼苗对ABA和脱水不敏感。另外,表达分析表明,该基因在转录水平上对GPA1和Rab7起到负调控作用。这些结果暗示着RabGAP7可能通过调节G蛋白来参与了ABA反应。     

H_2S信号和WRKY在ABA调节根生长和气孔运动中的关系

气体信号分子硫化氢(H_2S)和植物激素脱落酸(ABA)都可以调节植物的种子萌发、根生长、气孔运动、逆境响应等生理过程,但两者之间的关系了解非常有限。转录因子WRKY18、WRKY40和WRKY60参与拟南芥对ABA信号的响应。本文以拟南芥野生型(WT),lcd(L型半胱氨酸脱巯基酶编码基因缺失突变体),wrky18wrky40wrky60(WRKY18、WRKY40和WRKY60编码基因缺失三突变体)为实验材料,研究在幼苗根生长和气孔运动过程中,H_2S与ABA信号及WRKY之间的相互作用。实验结果表明:ABA处理可诱导WT的LCD基因表达量和内源H_2S含量升高;外源H_2S处理WT,WRKY40表达量下降,WRKY60表达量升高;lcd中WRKY18和WRKY40表达量升高,WRKY60表达量下降。幼苗根生长和气孔运动实验表明:与WT相比,lcd根生长和气孔运动受ABA影响作用较小;H_2S对wrky18wrky40wrky60根生长的促进作用明显,并促进wrky18wrky40wrky60和WT气孔关闭,但两者间无显著差异。     

拟南芥IQM1涉及主根生长对茉莉酸甲酯的反应

目的:进一步找出拟南芥钙调素结合蛋白IQM1介导茉莉酸信号转导的证据。方法:比较分析IQM1基因的功能缺失突变体iqm1-1及其野生型幼苗在茉莉酸甲酯(Me JA)处理后的主根长度和JAZ(jasmonate ZIM-domain)家族基因的表达。结果:Me JA抑制iqm1-1及其野生型植株的主根生长,但iqm1-1对Me JA反应的敏感度比野生型弱;与此结果一致,iqm1-1幼苗中几个JAZ基因表达上调。结论:IQM1介导茉莉酸信号转导,参与对植物根生长的调节。     

拟南芥ABA敏感突变体asm1的分离与鉴定

以拟南芥(Arabidopsis thaliana)为研究材料,从T-DNA突变体库中筛选分离得到1株脱落酸(ABA)敏感突变体asm1(ABA sensitive mutant 1,asm1),在含有ABA的培养基中,与野生型相比,asm1突变体的根伸长明显受到抑制,且其种子萌发结果显示asm1对ABA同样表现出敏感特性。在生长发育方面,asm1突变体抽苔时间提前,植株矮化,并且荚果长度明显小于野生型。利用远红外成像系统分析发现,在干旱胁迫下asm1突变体叶面温度高于野生型;失水率分析显示突变体失水率降低以及水分散失减少。遗传学分析表明,asm1是单基因隐性突变且与一个T-DNA插入共分离;通过图位克隆成功获得候选基因ASM1。RT-PCR结果显示,在突变体中ASM1的表达受到抑制,并且能够调控多种ABA信号通路和胁迫应答基因的表达水平。研究结果表明,ASM1可能参与调控ABA信号转导并应答干旱胁迫。     

拟南芥类受体蛋白激酶基因CRK45对外源钙离子的响应

以拟南芥野生型和类受体蛋白激酶基因CRK45的T-DNA插入突变体crk45为材料,采用差异基因表达筛选技术检测ABA处理后野生型和crk45中基因表达的差异。结果显示:(1)crk45突变体中有1个基因的表达比野生型高约4倍。(2)NCBI数据库检索表明,该基因编码的蛋白具有EF手型结构,蛋白序列全长为130个氨基酸,是典型的Ca2+结合蛋白,故命名为CRK45抑制的钙离子结合蛋白(CICBP)。(3)Northern blotting分析结果显示,ABA处理后crk45突变体中CICBP的表达明显升高,证明CICBP基因的确受ABA诱导,且其表达受CRK45的抑制。(4)外源75mmol/L的Ca2+处理后,crk45突变体的萌发率(30.8%)显著高于野生型(17.16%),说明在Ca2+介导下CRK45的功能是抑制种子萌发。(5)qRT-PCR检测显示,野生型中CRK45的表达受Ca2+诱导明显升高,而crk45突变体中的表达一直保持很低,说明crk45突变体是一个基因敲除突变体;Ca2+处理后crk45突变体中CICBP基因表达上调,而野生型中CICBP的表达反而降低,说明Ca2+处理下CRK45抑制CICBP基因的表达。研究表明,ABA或Ca2+处理后,CRK45通过负调控CICBP基因的表达,从而抑制拟南芥种子萌发。     

拟南芥中一个类似钙调素蛋白的基因结构及其缺磷、缺钾诱导表达

通过拟南芥芯片杂交分析发现推测的钙调素基因(GenBank accession No.Atlg76650)与低磷胁迫有关.对该基因的结构研究确认了该基因编码一个含有三个EF-hand结构域的类似钙调素的蛋白,Northern检测表明该基因在缺钾、缺磷条件下诱导表达,但在缺氮、高盐等胁迫条件下不受影响.经RT-PCR和启动子融合GUS转基因植株的组织化学染色分析,表明该基因在拟南芥中为全株表达,但各器官中表达丰度不尽相同.     

从拟南芥表达文库中筛选钙调素结合蛋白cDNA克隆

以生物素标记的钙调素为探针,筛选拟南芥λZAPⅡ表达文库,分离得到9个阳性克隆。融合蛋白的Westem印迹分析表明,这些阳性克隆确是编码钙调素结合蛋白的(图2,3),并且其中Y9等8个克隆编码的融合蛋白与钙调素有依赖于钙离子的结合(图3B);而唯独Y7编码的融合蛋白与钙调素的结合,与CaMBP-10一样,不依赖于钙离子的存在(图3A)。     

A cDNA clone encoding Brassica calmodulin

A 834 bp cDNA encoding calmodulin (CaM) has been isolated from Brassica juncea. On Northern analysis this cDNA hybridises this cDNA to mRNAs of about 0.9 kb in leaf, silique and peduncle. Genomic Southern analysis indicates the presence of a CaM multigene family in Brassica juncea. Comparison of the predicted amino acid sequence of Brassica CaM with that of Arabidopsis CaM ACaM-2 and ACaM-3 showed 100% homology, which is not unusual, since both plants belong to the family Cruciferae. In situ hybridisation studies on Brassica seedlings using a digoxigenin-labelled RNA probe showed that high levels of CaM mRNA were detected in the leaf primordia and the shoot apical meristem, and to a lesser degree, in the zone of root elongation of the root tip. The occurrence of a higher rate of cell division and growth in these regions than its surrounding tissue may possibly be related to higher levels of CaM mRNA.     

1,8-Cineole inhibits root growth and DNA synthesis in the root apical meristem ofBrassica campestris L.

1,8-cineole is a volatile growth inhibitor produced bySalvia species. We examined the effect of this allelopathic compound on the growth of other plants usingBrassica campestris as the test plant. Cineole inhibited germination and growth ofB. campestris in a dosedependent manner. WhenB. campestris was grown for 5 days with various concentrations of cineole, the length of the roots was found to be shorter as the concentration of cineole increased, whereas the length of the hypocotyl remained constant up to 400 μM cineole, indicating that cineole specifically inhibited growth of the root. The mitotic index in the root apical meristem of 3-day-old seedlings decreased from 5.6% to 1.6% when exposed to 400 μM cineole, showing that cineole inhibits the proliferation of root cells. We then examined the effect of cineole on DNA synthesis by indirect immunofluorescence microscopy using antibody raised against 5-bromo-2′-deoxyuridine (BrdU, an analogue of thymidine) in thin sections of samples embedded in Technovit 7100 resin. The results clearly demonstrated that cineole inhibits DNA synthesis in both cell nuclei and organelles in root apical meristem, suggesting that cineole may interfere with the growth of other plant species by inhibiting DNA synthesis in the root apical meristem.     

Effect of abscisic acid on the cell cycle in the growing maize root

The mechanism by which the rate of cell proliferation is regulated in different regions of the root apical meristem is unknown. The cell populations comprising the root cap and meristem cycle at different rates, proliferation being particularly slow in the quiescent centre. In an attempt to detect the control points in the cell cycle of the root apical meristem of Zea mays L. (cv. LG 11), quiescent-centre cells were stimulated to synthesise DNA and to enter mitosis either by decapping or by immersing intact roots in an aqueous 3,3-dimethyl-glutaric acid buffer solution. From microdensitometric and flow-cytometric data, we conclude that, upon immersion, the G₂ phase of the cell cycle of intact roots was shortened. However, when 50 M abscisic acid (ABA) was added to the immersion buffer, parameters of the cell cycle were restored to those characteristic of intact roots held in a moist atmosphere. On the other hand, decapping of primary roots preferentially shortened the G₁ phase of the cell cycle in the quiescent centre. When supplied to decapped roots, ABA reversed this effect. Therefore, in our model, applied ABA retarded the completion of the cell cycle and acted upon the exit from either the G₁ or the G₂ phase. Immersion of roots in buffer alone seems to trigger cells to more rapid cycling and may do so by depleting the root of some ABA-like factor.Abbreviations ABA cis-abscisic acid - DGA 3,3-dimethyl-glutaric acid - DAPI 4,6-diamidino-2-phenylindole - LI labelling index We thank Pierre Zaech of the Ludwig Institute, Epalinges, Switzerland, for expert assistance in flow cytometry and Dr. Jean-Marcel Ribaut of our Institute for providing data on exodiffusion and metabolism of ABA.     

Calcium and calmodulin inhibitor effects on the growth of carrot (Daucus carota L.) and radish (Raphanus sativus L.) in nutrient culture

Growth of carrot and radish seedlings in nutrient culture was inhibited by pretreatment with three calmodulin inhibitors. There was little selective effect on specific organs, shoots, tap root and fibrous roots over a range of concentrations. Although pretreatment with CaCl₂ (0.5 mM) did not affect growth of untreated seedlings, it partially reduced the inhibitory effects of trifluoperazine over the concentration range 0.01–0.05 mM. Trifluoperazine reduced the growth of GA₃-treated seedlings but did not overcome the modifying effect of GA₃ in favouring shoot/root ratio; ABA exacerbated its inhibitory effect on overall seedling growth and particularly on tap root development.Abbreviations GA₃ gibberellic acid - ABA abscisic acid - CaCl₂ calcium chloride - GAs gibberellins - Tfp trifluoperazine     

WGA reduces the level of oxidative stress in wheat seedlings under salinity

Protective effect of exogenous wheat germ agglutinin (WGA) on wheat seedling (Triticum aestivum L.) during salinity stress was studied. In particular, we examined the state of pro- and antioxidant systems as well as the level of peroxide oxidation of lipids and electrolyte leakage under control conditions and when stressed with NaCl. Generation of superoxide anions and activity of both superoxide dismutase (SOD) and peroxidase increased during saline stress. Accumulation of O₂ ^·− resulted in peroxide oxidation of lipids and electrolyte leakage in response to stress. The injurious effect of salinity on root growth of seedlings was manifested by a decreased mitotic index (MI) in apical root meristem. This study show that WGA pretreatment decreased salt-induced superoxide anion generation, SOD and peroxidase activities, levels of lipid peroxidation and electrolytes leakage as well as correlating with a reduction in the inhibition of root apical meristem mitotic activity in salt-treated plants. This suggests that exogenous WGA reduced the detrimental effects of salinity-induced oxidative stress in wheat seedlings. Thus WGA effects on a balance of reactive oxygen species (ROS) and activities of antioxidant enzymes may provide an important contribution to a range of the defense reactions induced by this lectin in wheat plants.     

脱落酸激素诱导拟南芥幼苗中花青素的合成

脱落酸(abscisic acid,ABA)激素是一类重要的生长调节物质,参与调控植物的多种生理过程。花青素(anthocyanins)是植物次生代谢产生的类黄酮化合物,对植物的生长发育和逆境胁迫响应有重要作用。该文以拟南芥(Arabidopsis thaliana)为研究对象,探讨ABA信号对花青素生物合成的调控功能和作用机制。结果表明:外源施加ABA显著提高野生型幼苗茎尖中花青素的积累。相一致的是,ABA能诱导某些与花青素合成相关的转录因子及合成酶基因的表达。遗传学分析发现,ABA诱导花青素合成部分依赖于MBW复合体中的核心转录因子,如TTG1、TT8及MYB75等。初步机制研究揭示,ABA信号途径中的bZIP类转录因子ABI5能与TTG1、TT8及MYB75等相互作用形成蛋白复合物。综上结果认为,ABA信号诱导拟南芥幼苗中花青素的积累,并可能通过ABI5与MBW复合体协同作用调控花青素的合成。     

Cross-talks between Ca<Superscript>2+</Superscript>/CaM and H<Subscript>2</Subscript>O<Subscript>2</Subscript> in abscisic acid-induced antioxidant defense in leaves of maize plants exposed to water stress

Here we examined whether Ca²⁺/Calmodulin (CaM) is involved in abscisic acid (ABA)-induced antioxidant defense and the possible relationship between CaM and H₂O₂ in ABA signaling in leaves of maize (Zea mays L.) plants exposed to water stress. An ABA-deficient mutant vp5 and its wild type were used for the experimentation. We found that water stress enhanced significantly the contents of CaM and H₂O₂, and the activities of chloroplastic and cytosolic superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), and the gene expressions of the CaM1, cAPX, GR1 and SOD4 in leaves of wild-type maize. However, the increases mentioned above were almost arrested in vp5 plants and in the wild-type plants pretreated with ABA biosynthesis inhibitor tungstate (T), suggesting that ABA is required for water stress-induced H₂O₂ production, the enhancement of CaM content and antioxidant defense. Besides, we showed that the up-regulation of water stress-induced antioxidant defense was almost completely blocked by pretreatment with Ca²⁺ inhibitors, CaM antagonists and reactive oxygen (ROS) manipulators. Moreover, the analysis of time course of CaM and H₂O₂ production under water stress showed that the increase in CaM content preceded that of H₂O₂. These results suggested that Ca²⁺/CaM and H₂O₂ were involved in the ABA-induced antioxidant defense under water stress, and the increases of Ca²⁺/CaM contents triggered H₂O₂ production, which inversely affected the contents of CaM. Thus, a cross-talk between Ca²⁺/CaM and H₂O₂ may play a pivotal role in the ABA signaling.     

Phloem transport and differential unloading in pea seedlings after source and sink manipulations

Phloem transport was investigated in pea seedlings after application of [¹⁴C]sucrose to the cotyledons. The accumulation of the label in segments of young seedlings shows a differential unloading along the plant axis. Shoot and root exhibit tip-to-base gradients of sink strength. In the primary root, the sink-strength profiles reflect not only the importance of the apical meristem, but show also the starting points of cambial activity and production of secondary vascular elements. Experiments including partial removal of the source and manipulations of the sink strength indicate that translocation of pea seedlings is sink-regulated and responds rapidly to changed apoplastic conditions in the apical root region. Here, a lowered water potential leads to an increase of phloem unloading that is suggested to supply the assimilate demand for the short-term osmoregulation of affected cells via the symplasmic pathway.Abbreviation PCMBS parachloromercuribenzenesulfonic acid Discussions with Prof. R. Kollmann, Botanisches Institut, Universität Kiel, and financial support from the Deutsche Forschungsgemeinschaft are gratefully acknowledged.     

Plant meristems: <Emphasis Type="Italic">CLAVATA3/ESR</Emphasis>-related signaling in the shoot apical meristem and the root apical meristem

The plant meristems, shoot apical meristem (SAM) and root apical meristem (RAM), are unique structures made up of a self-renewing population of undifferentiated pluripotent stem cells. The SAM produces all aerial parts of postembryonic organs, and the RAM promotes the continuous growth of roots. Even though the structures of the SAM and RAM differ, the signaling components required for stem cell maintenance seem to be relatively conserved. Both meristems utilize cell-to-cell communication to maintain proper meristematic activities and meristem organization and to coordinate new organ formation. In SAM, an essential regulatory mechanism for meristem organization is a regulatory loop between WUSCHEL (WUS) and CLAVATA (CLV), which functions in a non-cell-autonomous manner. This intercellular signaling network coordinates the development of the organization center, organ boundaries and distant organs. The CLAVATA3/ESR (CLE)-related genes produce signal peptides, which act non-cell-autonomously in the meristem regulation in SAM. In RAM, it has been suggested that a similar mechanism can regulate meristem maintenance, but these functions are largely unknown. Here, we overview the WUS–CLV signaling network for stem cell maintenance in SAM and a related mechanism in RAM maintenance. We also discuss conservation of the regulatory system for stem cells in various plant species. S. Sawa is the recipient of the BSJ Award for Young Scientist, 2007.     

Organ identity of the thalloid plant body of Griffithella hookeriana and Polypleurum stylosum – Podostemoideae (Podostemaceae)

The morphological nature of the thalloid plant body of podostemads has remained controversial for long. The present investigation was carried out on two members of the Podostemoideae i.e. Griffithella hookeriana and Polypleurum stylosum to understand their organ identity. The origin of the plant body was traced from the embryo by germinating the seeds under aseptic conditions. Mature embryo of both species does not show an identifiable shoot apical meristem (SAM) and root apical meristem (RAM). Upon germination, the radicular pole does not form a primary root but differentiates adhesive hairs. At the cotyledonary junction, SAM is initiated that differentiates 6–9 leaves apically (primary axis) and a primordium laterally. This primordium subsequently emerges from the hypocotyl and develops into a thalloid plant. The latter has been interpreted as a flattened stem because it not only shows tunica-corpus like organization at the tip but also originates endogenously from the same SAM that forms the `primary axis'.