拟南芥MeIAA抗性突变体的筛选和初步图位克隆分析

生长素是最重要的植物激素之一,参与了植物生长发育的各个方面。植物体内游离的IAA是生长素的主要活性形式,在IAA甲基转移酶1（IAMT1）的作用下,IAA可以转变为IAA甲酯（MelAA）。MelAA本身没有活性,在植物体内的MelAA酯解酶作用下可以重新转变为IAA。MelAA是非极性分子,能够在植物体内自由扩散。利用MelAA的这种特殊性质筛选突变体,可以分离到MelAA代谢途径或者IAA途径中新的成分。我们对拟南芥种子进行EMS诱变,通过观察黑暗下下胚轴的生长情况,筛选MelAA的抗性突变体。我们成功分离到了8株可能的抗性突变体,并对其中的一个Methyl-JAAresistant1（mir1）突变体进行了深入分析。MelAA抗性突变体的筛选将为进一步了解MelAA的代谢、IAA稳态调控和响应机理提供新的材料。     

拟南芥MelAA抗性突变体的筛选和初步图位克隆分析

生长素是最重要的植物激素之一,参与了植物生长发育的各个方面。植物体内游离的IAA是生长素的主要活性形式,在IAA甲基转移酶1（IAMT1）的作用下,IAA可以转变为IAA甲酯（MelAA）。MelAA本身没有活性,在植物体内的MelAA酯解酶作用下可以重新转变为IAA。MelAA是非极性分子,能够在植物体内自由扩散。利用MelAA的这种特殊性质筛选突变体,可以分离到MelAA代谢途径或者IAA途径中新的成分。我们对拟南芥种子进行EMS诱变,通过观察黑暗下下胚轴的生长情况,筛选MelAA的抗性突变体。我们成功分离到了8株可能的抗性突变体,并对其中的一个Methyl-JAAresistant1（mir1）突变体进行了深入分析。MelAA抗性突变体的筛选将为进一步了解MelAA的代谢、IAA稳态调控和响应机理提供新的材料。     

重金属对植物吲哚乙酸合成与分解影响研究进展

生长素在调节植物生长和抗重金属胁迫中具有重要作用。重金属胁迫下植物为维持自身生长,必须维持生长素的内稳态和自身代谢平衡。生长素的内稳态受到生物合成、生长素结合以及水解、代谢失活等生理活动的严格控制。一些涉及生长素合成与分解的相关酶系和基因已被识别或克隆,然而重金属胁迫下与生长素合成与分解有关基因的上调或下调以及相关酶系的激活或失活却研究尚少。揭示植物遭受重金属胁迫后生长素合成与分解变化的机理,可为植物修复实践中合理使用植物生长调节剂提供理论依据。本文以生长素的主要代表物吲哚乙酸(IAA)为例,讨论重金属胁迫下,植物体内IAA合成、分解机制及其赋存形态等方面的研究进展,并从重金属胁迫下植物IAA合成途径的相对重要性、IAA形态变化和作用以及激素间的交互作用等方面探讨了该领域的研究方向。     

生长素信号转导途径与植物胁迫反应相互作用的证据(英)

生长素影响植物多种生理过程 ,有报道显示生长素可能影响植物对逆境胁迫的反应。我们利用cDNA阵列技术鉴定拟南芥 (Arabidopsisthaliana (L .)Heynh .)的生长素应答基因 ,发现多个胁迫应答基因受生长素抑制 ,包括ArabidopsishomologofMEKkinase1(ATMEKK1) ,RelA/SpoThomolog 3(At_RSH3) ,Catalase 1(Cat1)和Ferritin 1(Fer1) ,说明生长素可调节胁迫应答基因的表达。此外 ,我们还证明吲哚乙酸 (IAA)合成途径中的腈水解酶基因nitrilase 1(NIT1)和nitrilase 2 (NIT2 )受盐胁迫诱导 ,提示在逆境条件下IAA的合成可能随之增加。我们利用生长素不敏感突变体研究生长素与逆境反应相互作用的信号转导 ,发现胁迫应答基因在野生型和生长素不敏感突变体auxinresistant2 (axr2 )中可被盐胁迫诱导 ,而在auxinresistant1_3(axr1_3)中则不被诱导 ,说明生长素与逆境胁迫反应的相互作用可能发生在泛素途径。     

拟南芥突变体在生长素信号传导中的研究进展

近年来,在植物激素的信号传导研究上已取得突破性进展.生长素的信号传导通路研究除了在生长素结合蛋白(ABP)上有所进展外,在生长素应答基因(Aux IAA),生长素调节因子(ARF)以及感应突变体的研究上也取得较大进展.对生长素运输通路及PIN1蛋白的功能和其抑制剂的研究也使对生长素信号传导的认识更清楚.生长素应答基因(Aux IAA)是生长素处理后快速诱导的基因.Aux IAA蛋白具有组织特异性(例如SAU蛋白)可以用来研究外源激素对植物生长发育的影响.生长素调节因子(ARF)与生长素应答基因的启动子序列具有特异性结合,Aux IAA蛋白与生长素调节因子(ARF)相互作用,并引发一系列蛋白质降解.使用转基因的拟南芥突变体,能有效地研究生长素在植物体内的特异性分布.借助运输载体抑制剂,可以对生长素的极性运输有更深入的了解.已经证明PIN蛋白参与生长素运输并与肌动蛋白有关.而且生长素参与了赤霉素介导的植物伸长反应.     

生长素信号转导途径与植物胁迫反应相互作用的证据

生长素影响植物多种生理过程,有报道显示生长素可能影响植物对逆境胁迫的反应.我们利用cDNA阵列技术鉴定拟南芥(Arabidopsis thaliana (L.) Heynh.)的生长素应答基因,发现多个胁迫应答基因受生长素抑制,包括Arabidopsis homolog of MEK kinase1 (ATMEKK1),RelA/SpoT homolog 3 (At-RSH3),Catalase 1 (Cat1) 和Ferritin 1 (Fer1),说明生长素可调节胁迫应答基因的表达.此外,我们还证明吲哚乙酸(IAA)合成途径中的腈水解酶基因nitrilase 1 (NIT1) 和nitrilase 2 (NIT2) 受盐胁迫诱导,提示在逆境条件下IAA的合成可能随之增加.我们利用生长素不敏感突变体研究生长素与逆境反应相互作用的信号转导,发现胁迫应答基因在野生型和生长素不敏感突变体auxin resistant 2 (axr2) 中可被盐胁迫诱导,而在auxin resistant 1-3 (axr1-3)中则不被诱导,说明生长素与逆境胁迫反应的相互作用可能发生在泛素途径.     

泛素降解途径与生长素的调节

就近几年来泛素降解途径在生长素调节中的作用作了介绍,主要是3个蛋白家族突变体的一系列分子分析研究,即生长素应答因子(auxin responsefactors,ARFs)、生长素/吲哚乙酸(Aux/IAA)家族和泛素蛋白酶解组分.ARFs可以直接与DNA结合,介导生长素调节的基因表达;Aux/IAA通过与ARFs形成异源二聚体阻碍ARFs执行功能;泛素降解途径包括泛素激活酶El、泛素连接酶E2、泛素连接酶E3及26S蛋白酶体.生长素通过促进Aux/IAA与E3-SCFTIR1的相互作用降解Aux/IAA蛋白,释放出的ARFs与DNA结合,调节生长素相关基因表达.COP9(constitutive photomorphogenic locus 9)信号体也通过调节SCFTIl活性参与此过程.     

生长素合成途径的研究进展

生长素是一类含有一个不饱和芳香族环和一个乙酸侧链的内源激素, 参与植物生长发育的许多过程。植物和一些侵染植物的病原微生物都可以通过改变生长素的合成来调节植株的生长。吲哚-3-乙酸(IAA)是天然植物生长素的主要活性成分。近年来, 随着IAA生物合成过程中一些关键调控基因的克隆和功能分析, 人们对IAA的生物合成途径有了更加深入的认识。IAA的生物合成有依赖色氨酸和非依赖色氨酸两条途径。依据IAA合成的中间产物不同, 依赖色氨酸的生物合成过程通常又划分成4条支路: 吲哚乙醛肟途径、吲哚丙酮酸途径、色胺途径和吲哚乙酰胺途径。该文综述了近几年在IAA生物合成方面取得的新进展。     

吲哚乙酸跨界信号调节植物与细菌互作

吲哚-3-乙酸(indole-3-acetic acid,IAA)作为植物体内普遍存在的内源生长素参与调节植物生命活动的诸多方面。研究发现,自然界中不仅植物可以合成IAA,许多微生物(包括植物病原菌或益生菌)同样具有分泌IAA的能力,可以诱发植物病害,或促进植物生长。有趣的是IAA不仅作为细菌的次生代谢物干扰寄主植物的激素稳态,也作为信号分子影响细菌基因表达和生理活动,通过整合进入细菌复杂代谢网络,调节植物与细菌的相互作用。通过讨论植物相关细菌IAA的生物合成途径及其调控,以及参与调节细菌基因表达、影响细菌生理和行为及其与寄主植物的互作等,概述该领域的研究动态与进展,揭示IAA不仅调节植物生长发育和防御,也作为跨界信号在调控植物与微生物互作中发挥重要作用,旨在为深入研究和更好地了解IAA跨界信号机制,通过遗传操纵细菌IAA信号通路以改善植物生长发育及其胁迫耐力提供新思路。     

木醋液的植物生长调节剂特性的分子机理研究

木醋液(wood vinegar,WV)是一种农业生产上广泛应用的植物生长调节剂类似物,但对其分子水平调节机制研究的缺乏严重限制了它的更进一步应用。试验以模式生物拟南芥为材料,通过半定量PCR(Semi-quantitative PCR,SQ-PCR)和条件控制培养,研究了不同浓度、不同时间的木醋液、生长素吲哚乙酸(IAA)处理对生长素诱导基因表达和形态学变化的影响。结果表明,木醋液可调节拟南芥生长素诱导基因Aux/IAA1、Aux/IAA5、Aux/IAA19、ARF19和SAUR-AC1的表达。通过Aux/IAA和ARF蛋白相互作用,木醋液调节途径中存在与生长素类似的负反馈。在形态学方面,木醋液、生长素IAA均可抑制叶片数量和叶片伸展,促进主根伸长和侧根形成。这表明木醋液不但以与生长素IAA相似的途径促进植物生长,且以自身的调节方式促进植物生长发育。     

Inactive methyl indole-3-acetic acid ester can be hydrolyzed and activated by several esterases belonging to the AtMES esterase family of Arabidopsis

The plant hormone auxin (indole-3-acetic acid [IAA]) is found both free and conjugated to a variety of carbohydrates, amino acids, and peptides. We have recently shown that IAA could be converted to its methyl ester (MeIAA) by the Arabidopsis (Arabidopsis thaliana) enzyme IAA carboxyl methyltransferase 1. However, the presence and function of MeIAA in vivo remains unclear. Recently, it has been shown that the tobacco (Nicotiana tabacum) protein SABP2 (salicylic acid binding protein 2) hydrolyzes methyl salicylate to salicylic acid. There are 20 homologs of SABP2 in the genome of Arabidopsis, which we have named AtMES (for methyl esterases). We tested 15 of the proteins encoded by these genes in biochemical assays with various substrates and identified several candidate MeIAA esterases that could hydrolyze MeIAA. MeIAA, like IAA, exerts inhibitory activity on the growth of wild-type roots when applied exogenously. However, the roots of Arabidopsis plants carrying T-DNA insertions in the putative MeIAA esterase gene AtMES17 (At3g10870) displayed significantly decreased sensitivity to MeIAA compared with wild-type roots while remaining as sensitive to free IAA as wild-type roots. Incubating seedlings in the presence of [(14)C]MeIAA for 30 min revealed that mes17 mutants hydrolyzed only 40% of the [(14)C]MeIAA taken up by plants, whereas wild-type plants hydrolyzed 100% of absorbed [(14)C]MeIAA. Roots of Arabidopsis plants overexpressing AtMES17 showed increased sensitivity to MeIAA but not to IAA. Additionally, mes17 plants have longer hypocotyls and display increased expression of the auxin-responsive DR5:beta-glucuronidase reporter gene, suggesting a perturbation in IAA homeostasis and/or transport. mes17-1/axr1-3 double mutant plants have the same phenotype as axr1-3, suggesting MES17 acts upstream of AXR1. The protein encoded by AtMES17 had a K(m) value of 13 microm and a K(cat) value of 0.18 s(-1) for MeIAA. AtMES17 was expressed at the highest levels in shoot apex, stem, and root of Arabidopsis. Our results demonstrate that MeIAA is an inactive form of IAA, and the manifestations of MeIAA in vivo activity are due to the action of free IAA that is generated from MeIAA upon hydrolysis by one or more plant esterases.     

The possible action mechanisms of indole-3-acetic acid methyl ester in <Emphasis Type="Italic">Arabidopsis</Emphasis>

We previously reported that Arabidopsis indole-3-acetic acid (IAA)-methyltransferase-1 (IAMT1) catalyzes the conversion of IAA, an essential phytohormone, to methyl-IAA (MeIAA) and that IAMT1 plays an important role in leaf development. Here, we present the possible mechanisms of action of MeIAA in Arabidopsis. We showed that MeIAA was more potent than IAA in the inhibition of hypocotyl elongation and that MeIAA and naphthalene-acetic acid (NAA), but not IAA, rescued the hypocotyl gravitropic defects in dark-grown aux1. However, MeIAA was less potent than IAA in the inhibition of primary root elongation in light-grown seedlings, and could not rescue the agravitropic root phenotype of aux1. MeIAA had a stronger capacity to induce lateral roots than both IAA and NAA and rescued the defective lateral root phenotype of aux1 seedlings. However, its capacity to induce root hairs was weaker than IAA and NAA and did not rescue the defective root hair phenotype of aux1 seedlings. These data indicate that MeIAA is an inactive form of IAA. The different sensitivities to MeIAA among different organs probably resulted from different expression localization and capacities of a putative MeIAA esterase to convert MeIAA to IAA.     

An indole-3-acetic acid carboxyl methyltransferase regulates Arabidopsis leaf development

Auxin is central to many aspects of plant development; accordingly, plants have evolved several mechanisms to regulate auxin levels, including de novo auxin biosynthesis, degradation, and conjugation to sugars and amino acids. Here, we report the characterization of an Arabidopsis thaliana mutant, IAA carboxyl methyltransferase1-dominant (iamt1-D), which displayed dramatic hyponastic leaf phenotypes caused by increased expression levels of the IAMT1 gene. IAMT1 encodes an indole-3-acetic acid (IAA) carboxyl methyltransferase that converts IAA to methyl-IAA ester (MeIAA) in vitro, suggesting that methylation of IAA plays an important role in regulating plant development and auxin homeostasis. Whereas both exogenous IAA and MeIAA inhibited primary root and hypocotyl elongation, MeIAA was much more potent than IAA in a hypocotyl elongation assay, indicating that IAA activities could be effectively regulated by methylation. IAMT1 was spatially and temporally regulated during the development of both rosette and cauline leaves. Changing expression patterns and/or levels of IAMT1 often led to dramatic leaf curvature phenotypes. In iamt1-D, the decreased expression levels of TCP genes, which are known to regulate leaf curvature, may partially account for the curly leaf phenotype. The identification of IAMT1 and the elucidation of its role in Arabidopsis leaf development have broad implications for auxin-regulated developmental process.     

Arabidopsis mutants resistant to the auxin effects of indole-3-acetonitrile are defective in the nitrilase encoded by the NIT1 gene.

Indole-3-acetonitrile (IAN) is a candidate precursor of the plant growth hormone indole-3-acetic acid (IAA). We demonstrated that IAN has auxinlike effects on Arabidopsis seedlings and that exogenous IAN is converted to IAA in vivo. We isolated mutants with reduced sensitivity to IAN that remained sensitive to IAA. These mutants were recessive and fell into a single complementation group that mapped to chromosome 3, within 0.5 centimorgans of a cluster of three nitrilase-encoding genes, NIT1, NIT2, and NIT3. Each of the three mutants contained a single base change in the coding region of the NIT1 gene, and the expression pattern of NIT1 is consistent with the IAN insensitivity observed in the nit1 mutant alleles. The half-life of IAN and levels of IAA and IAN were unchanged in the nit1 mutant, confirming that Arabidopsis has other functional nitrilases. Overexpressing NIT2 in transgenic Arabidopsis caused increased sensitivity to IAN and faster turnover of exogenous IAN in vivo.     

Expression of AMIDASE1 (AMI1) is suppressed during the first two days after germination

The regulation of cellular auxin levels is a critical factor in determining plant growth and architecture, as indole-3-acetic acid (IAA) gradients along the plant axis and local IAA maxima are known to initiate numerous plant growth responses. The regulation of auxin homeostasis is mediated in part by transport, conjugation and deconjugation, as well as by de novo biosynthesis. However, the pathways of IAA biosynthesis are yet not entirely characterized at the molecular and biochemical level. It is suggested that several biosynthetic routes for the formation of IAA have evolved. One such pathway proceeds via the intermediate indole-3-acetamide (IAM), which is converted into IAA by the activity of specific IAM hydrolases, such as Arabidopsis AMIDASE1 (AMI1). In this article we present evidence to support the argument that AMI1-dependent IAA synthesis is likely not to be used during the first two days of seedling development.Key words: Arabidopsis thaliana, auxin biosynthesis, AMIDASE1, indole-3-acetic acid, indole-3-acetamide, LEAFY COTYLEDON1, seed developmentAuxins are versatile plant hormones that play diverse roles in regulating many aspects of plant growth and development.¹ To enable auxins to develop their activity, a tight spatiotemporal control of cellular indole-3-acetic acid (IAA) contents is absolutely necessary since it is well-documented that auxin action is dose dependent, and that high IAA levels can have inhibitory effects on plant growth.² To achieve this goal, plants have evolved a set of different mechanisms to control cellular hormone levels. On the one hand, plants possess several pathways that contribute to the de novo synthesis of IAA. This multiplicity of biosynthetic routes presumably facilitates fine-tuning of the IAA production. On the other hand, plants are equipped with a variety of enzymes that are used to conjugate free auxin to either sugars, amino acids or peptides and small proteins, respectively, or on the contrary, that act as IAA-conjugate hydrolases, releasing free IAA from corresponding conjugates. IAA-conjugates serve as a physiologically inactive storage form of IAA from which the active hormone can be quickly released on demand. Alternatively, conjugation of IAA can mark the first step of IAA catabolism. In general, conjugation and deconjugation of free IAA are ways to positively or negatively affect active hormone levels, which adds another level of complexity to the system. Additionally, IAA can be transported from cell to cell in a polar manner, which is dependent on the action of several transport proteins. All together, these means are used to form auxin gradients and local maxima that are essential to initiate plant growth processes, such as root or leaf primordia formation.³     

Meloidogyne javanica chorismate mutase 1 alters plant cell development

Root-knot nematodes are obligate plant parasites that alter plant cell growth and development by inducing the formation of giant cells for feeding. Nematodes inject secretions from their esophageal glands through their stylet and into plant cells to induce giant cell formation. Meloidogyne javanica chorismate mutase 1 (MjCM-1) is one such esophageal gland protein likely to be secreted from the nematode as giant cells form. MjCM-1 has two domains, an N-terminal chorismate mutase (CM) domain and a C-terminal region of unknown function. It is the N-terminal CM domain of the protein that is the predominant form produced in root-knot nematodes. Transgenic expression of MjCM-1 in soybean hairy roots results in a phenotype of reduced and aborted lateral roots. Histological studies demonstrate the absence of vascular tissue in hairy roots expressing MjCM-1. The phenotype of MjCM-1 expressed at low levels can be rescued by the addition of indole-3-acetic acid (IAA), indicating MjCM-1 overexpression reduces IAA biosynthesis. We propose MjCM-1 lowers IAA by causing a competition for chorismate, resulting in an alteration of chorismate-derived metabolites and, ultimately, in plant cell development. Therefore, we hypothesize that MjCM-1 is involved in allowing nematodes to establish a parasitic relationship with the host plant.     

Identification and characterization of Arabidopsis indole-3-butyric acid response mutants defective in novel peroxisomal enzymes

Genetic evidence suggests that indole-3-butyric acid (IBA) is converted to the active auxin indole-3-acetic acid (IAA) by removal of two side-chain methylene units in a process similar to fatty acid beta-oxidation. Previous studies implicate peroxisomes as the site of IBA metabolism, although the enzymes that act in this process are still being identified. Here, we describe two IBA-response mutants, ibr1 and ibr10. Like the previously described ibr3 mutant, which disrupts a putative peroxisomal acyl-CoA oxidase/dehydrogenase, ibr1 and ibr10 display normal IAA responses and defective IBA responses. These defects include reduced root elongation inhibition, decreased lateral root initiation, and reduced IBA-responsive gene expression. However, peroxisomal energy-generating pathways necessary during early seedling development are unaffected in the mutants. Positional cloning of the genes responsible for the mutant defects reveals that IBR1 encodes a member of the short-chain dehydrogenase/reductase family and that IBR10 resembles enoyl-CoA hydratases/isomerases. Both enzymes contain C-terminal peroxisomal-targeting signals, consistent with IBA metabolism occurring in peroxisomes. We present a model in which IBR3, IBR10, and IBR1 may act sequentially in peroxisomal IBA beta-oxidation to IAA.     

Quantification of free plus conjugated indoleacetic acid in arabidopsis requires correction for the nonenzymatic conversion of indolic nitriles.

The genetic advantages to the use of Arabidopsis thaliana mutants for the study of auxin metabolism previously have been partially offset by the complexity of indolic metabolism in this plant and by the lack of proper methods. To address some of these problems, we developed isotopic labeling methods to determine amounts and examine the metabolism of indolic compounds in Arabidopsis. Isolation and indentification of endogenous indole-3-acetonitrile (IAN; a possible precursor of the auxin indole-3-acetic acid [IAA]) was carried out under mild conditions, thus proving its natural occurrence. We describe here the synthesis of 13C1-labeled IAN and its utility in the gas chromatography-mass spectrometry quantification of endogenous IAN levels. We also quantified the nonenzymatic conversion of IAN to IAA under conditions used to hydrolyze IAA conjugates. 13C1-Labeled IAN was used to assess the contribution of IAN to measured IAA following hydrolysis of IAA conjugates. We studied the stability and breakdown of the indolic glucosinolate glucobrassicin, which is known to be present in Arabidopsis. This is potentially an important concern when using Arabidopsis for studies of indolic biochemistry, since the levels of indolic auxins and auxin precursors are well below the levels of the indolic glucosinolates. We found that under conditions of extraction and base hydrolysis, formation of IAA from glucobrassicin was negligible.