拟南芥色氨酸与吲哚乙酸生物合成的研究进展

拟南芥色氨酸生物合成途径的研究已逐成为植物分析生物学家了解植物基因结构和表达调控最主要的模式系统之一。到目前为止,编码拟南芥色氨酸合成途径的七种酶蛋白的基因已经全部被克隆,并进行了不同程度的分子生物学研究。长期以来,色氨酸一直被认为是生长素吲哚乙酸乙酸（ＩＡＡ）生物合成（从头合成）的前体物,但近年来人们发现生长素的非色氨酸途径可能是其在植物中生物合成的主要途径,植物在不同的发育阶段可能采用不同的方     

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。     

拟南芥吲哚—3—甘油磷酸合酶免疫分析

吲哚３甘油磷酸合酶（ＩＧＳ,ｉｎｄｏｌｅ３ｇｌｙｃｅｒｏｌｐｈｏｓｐｈａｔｅｓｙｎｔｈａｓｅ,ＥＣ４．１．１．４８）在色氨酸与吲哚乙酸的生物合成途径中,催化生成吲哚３甘油磷酸。研究该基因的表达调控,对于阐明高等植物是如何调控色氨酸及生长素合成是十分重要的。利用已克隆的ＩＧＳｃＤＮＡ,构建了谷胱甘肽转移酶（ＧＳＴ,ｇｌｕｔａｔｈｉｏｎｅＳｔｒａｎｓｆｅｒａｓｅ,ＥＣ２．５．１．１８）与吲哚３甘油磷酸合酶融合蛋白的表达质粒,并将其导入到在异丙基βＤ硫代半乳糖苷（ＩＰＴＧ）诱导下能高效表达的ＩＧＳ基因缺陷菌株ｔｒｐＣ９８００λＫＣ大肠杆菌中。高表达的融合蛋白通过谷胱甘肽琼脂糖（ｇｌｕｔａｔｈｉｏｎｅａｇａｒｏｓｅ）亲和层析和ＳＤＳ聚丙烯酰胺凝胶电泳纯化后,用以免疫兔子制备抗血清。免疫印迹法分析表明拟南芥（Ａｒａｂｉｄｏｐｓｉｓｔｈａｌｉａｎａ（Ｌ．）Ｈｅｙｎｈ．）四种常用生态型只合成一种分子量约为４０ｋＤ的吲哚３甘油磷酸合酶蛋白。在Ａｇ＋、紫外线等逆境条件下,ＩＧＳ含量都有较大幅度的增加,这说明ＩＧＳ可能与植物的防御反应紧密相关。     

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（Arabiopsis 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 kinasel（ATMEKK1）,RelA/SpoT homolog 3(At-RSH3),Catalase 1(Cat1)和Ferriitn 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（LAA）合成途径中的腈水解酶基因nitrilase 1(NIT1)和nitrilae 2（NIT2）受盐胁迫诱导,提示在逆境条件下1AA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 resitant1-3（axl-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。     

独脚金内酯调控植物侧枝发育的分子机制及其与生长素交互作用的研究进展

对独脚金内酯（strigolactones,SLs）调控植物侧枝发育的分子机制及其与生长素相互作用的相关研究结果进行了总结和归纳,在此基础上提出今后的重点研究方向。相关的研究结果显示：在拟南芥[Arabidops~thaliana（Linn．）Heynh．]、豌豆（Pisum sativum Linn．）和水稻（Oryza sativa Linn．）等植物多枝突变体中SLs作为可转导信号参与侧枝发育的分子调控,从这些植物中已克隆获得参与SLs生物合成及信号应答途径的一些基因。作为一种植物激素,SLs在侧枝发育调控网络中与生长素相互作用;腋芽发育与其中生长素的输出密切相关,SLs通过调控芽中生长素的输出间接抑制腋芽发育和侧枝生长,而生长素则在SLs生物合成中起调节作用。     

植物GH3基因家族的功能研究概况

植物GH3基因是一种典型的植物生长素原初反应基因,此类基因与植物的生长发育密切相关。GH3基因在植物生长素信号途径、光信号途径以及植物的防卫反应中起着重要作用。植物GH3蛋白具有植物生长素氨基酸化合成酶活性,这有助于维持植物生长素的动态平衡。该文介绍拟南芥等植物中GH3基因的生物学功能研究概况和最新进展,为植物GH3基因家族的进一步研究提供参考。     

植物GH3 基因家族的功能研究概况

植物GH3基因是一种典型的植物生长素原初反应基因, 此类基因与植物的生长发育密切相关。GH3基因在植物生长素信号途径、光信号途径以及植物的防卫反应中起着重要作用。植物GH3蛋白具有植物生长素氨基酸化合成酶活性, 这有助 于维持植物生长素的动态平衡。该文介绍拟南芥等植物中GH3基因的生物学功能研究概况和最新进展, 为植物GH3基因家族的进一步研究提供参考。     

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。     

植物茎分枝的分子调控

植物茎分枝结构决定了不同植物的不同形态结构.本文从腋生分生组织的发生、腋芽的生长两个方面综述了近年来植物分枝发生发育相关的分子机理研究及其进展.发现在不同植物中腋分生组织形成的基本机制是相似的,LS（lateral suppressor）及其同源基因在不同植物中都参与腋生分生组织的形成,而BL(blind)及其同源基因也参与调控腋生分生组织的形成.腋生分生组织的形成可能也是受激素调控的.目前,对腋芽生长的分子调控机制的认识主要集中于生长素通过二级信使的作用调控腋芽的生长.而生长素调控腋芽生长的机制已经较为清楚的有两条途径：一是生长素通过抑制细胞分裂素合成来调控腋芽的生长;另一途径是一种类胡萝卜素衍生的信号物质参与生长素的运输调控(MAX途径)来调控腋芽的生长.最新研究表明,TB1的拟南芥同源基因在MAX途径的下游负调控腋芽的生长.此外,增强表达OsNAC2也促进腋芽的生长.     

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.     

An Inhibitor of Tryptophan-Dependent Biosynthesis of Indole-3-Acetic Acid Alters Seedling Development in Arabidopsis

Although polar transport and the TIR1-dependent signaling pathway of the plant hormone auxin/indole-3-acetic acid (IAA) are well characterized, understanding of the biosynthetic pathway(s) leading to the production of IAA is still limited. Genetic dissection of IAA biosynthetic pathways has been complicated by the metabolic redundancy caused by the apparent existence of several parallel biosynthetic routes leading to IAA production. Valuable complementary tools for genetic as well as biochemical analysis of auxin biosynthesis would be molecular inhibitors capable of acting in vivo on specific or general components of the pathway(s), which unfortunately have been lacking. Several indole derivatives have been previously identified to inhibit tryptophan-dependent IAA biosynthesis in an in vitro system from maize endosperm. We examined the effect of one of them, 6-fluoroindole, on seedling development of Arabidopsis thaliana and tested its ability to inhibit IAA biosynthesis in feeding experiments in vivo. We demonstrated a correlation of severe developmental defects or growth retardation caused by 6-fluoroindole with significant downregulation of de novo synthesized IAA levels, derived from the stable isotope-labeled tryptophan pool, upon treatment. Hence, 6-fluoroindole shows important features of an inhibitor of tryptophan-dependent IAA biosynthesis both in vitro and in vivo and thus may find use as a promising molecular tool for the identification of novel components of the auxin biosynthetic pathway(s).     

Indole-3-glycerol phosphate, a branchpoint of indole-3-acetic acid biosynthesis from the tryptophan biosynthetic pathway in Arabidopsis thaliana

The phytohormone indole-3-acetic acid (IAA) plays a vital role in plant growth and development as a regulator of numerous biological processes. Its biosynthetic pathways have been studied for decades. Recent genetic and in vitro labeling evidence indicates that IAA in Arabidopsis thaliana and other plants is primarily synthesized from a precursor that is an intermediate in the tryptophan (Trp) biosynthetic pathway. To determine which intermediate(s) acts as the possible branchpoint for the Trp-independent IAA biosynthesis in plants, we took an in vivo approach by generating antisense indole-3-glycerol phosphate synthase (IGS) RNA transgenic plants and using available Arabidopsis Trp biosynthetic pathway mutants trp2-1 and trp3-1. Antisense transgenic plants display some auxin deficient-like phenotypes including small rosettes and reduced fertility. Protein gel blot analysis indicated that IGS expression was greatly reduced in the antisense lines. Quantitative analyses of IAA and Trp content in antisense IGS transgenic plants and Trp biosynthetic mutants revealed striking differences. Compared with wild-type plants, the Trp content in all the transgenic and mutant plants decreased significantly. However, total IAA levels were significantly decreased in antisense IGS transgenic plants, but remarkably increased in trp3-1 and trp2-1 plants. These results suggest that indole-3-glycerol phosphate (IGP) in the Arabidopsis Trp biosynthetic pathway serves as a branchpoint compound in the Trp-independent IAA de novo biosynthetic pathway.     

Arabidopsis indole synthase, a homolog of tryptophan synthase alpha, is an enzyme involved in the Trp-independent indole-containing metabolite biosynthesis

The plant tryptophan (Trp) biosynthetic pathway produces many secondary metabolites with diverse functions.Indole-3-acetic acid (IAA),proposed as a derivative from Trp or its precursors,plays an essential role in plant growth and development.Although the Trp-dependant and Trp-independent IAA biosynthetic pathways have been proposed,the enzymes,reactions and regulatory mechanisms are largely unknown.In Arabidopsis,indole-3-glycerol phosphate (IGP) is suggested to serve as a branchpoint component in the Trp-independent IAA biosynthesis.To address whether other enzymes in addition to Trp synthase α(TSA1) catalyze IGP cleavage,we identified and characterized an indole synthase (INS) gene,a homolog of TSA1 in Arabidopsis.INS exhibits different subcellular localization from TSA1 owing to the lack of chloroplast transit peptide (cTP).In silico data show that the expression levels of INS and TSA1 in all examined organs are quite different.Histochemical staining of INS promoter-GUS transgenic lines indicates that INS is expressed in vascular tissue of cotyledons,hypocotyls,roots and rosette leaves as well as in flowers and siliques.INS is capable of complementing the Trp auxotrophy of Escherichia coil △trpA strain,which is defective in Trp synthesis due to the deletion of TSA.This implies that INS catalyzes the conversion of IGP to indole and may be involved in the biosynthesis of Trp-independent IAA or other secondary metabolites in Arabidopsis.     

Differential inhibition of indole-3-acetic acid and tryptophan biosynthesis by indole analogues. I. Tryptophan dependent IAA biosynthesis

Maize liquid endosperm extracts contain the enzymes necessary for all of the steps of the plant IAA biosynthetic pathway from tryptophan, and provide a means to assay the pathway in vitro. We have analyzed the reactions in the presence of a series of indole and indole-like analogues in order to evaluate the potential of these compounds to act as inhibitors of IAA biosynthesis. Such inhibitors will be useful to investigate the tryptophan to IAA pathway, to determine the precursors and intermediates involved, and to select for mutants in this process. A number of such compounds were tested using in vitro enzyme assays for both the tryptophan dependent IAA biosynthesis pathway and for tryptophan synthase activity. Some compounds showed strong inhibition of IAA biosynthesis while having only a slight effect on the reaction rate of tryptophan synthase . These results: (1) show that IAA biosynthesis can be selectively inhibited relative to tryptophan biosynthesis; (2) suggest potential ways to screen for IAA biosynthetic pathway mutations in plants; and (3) provide additional tools for studies of IAA biosynthesis in plants.     

The pathway of auxin biosynthesis in plants

The plant hormone auxin, which is predominantly represented by indole-3-acetic acid (IAA), is involved in the regulation of plant growth and development. Although IAA was the first plant hormone identified, the biosynthetic pathway at the genetic level has remained unclear. Two major pathways for IAA biosynthesis have been proposed: the tryptophan (Trp)-independent and Trp-dependent pathways. In Trp-dependent IAA biosynthesis, four pathways have been postulated in plants: (i) the indole-3-acetamide (IAM) pathway; (ii) the indole-3-pyruvic acid (IPA) pathway; (iii) the tryptamine (TAM) pathway; and (iv) the indole-3-acetaldoxime (IAOX) pathway. Although different plant species may have unique strategies and modifications to optimize their metabolic pathways, plants would be expected to share evolutionarily conserved core mechanisms for auxin biosynthesis because IAA is a fundamental substance in the plant life cycle. In this review, the genes now known to be involved in auxin biosynthesis are summarized and the major IAA biosynthetic pathway distributed widely in the plant kingdom is discussed on the basis of biochemical and molecular biological findings and bioinformatics studies. Based on evolutionarily conserved core mechanisms, it is thought that the pathway via IAM or IPA is the major route(s) to IAA in plants.     

Yucasin is a potent inhibitor of YUCCA,a key enzyme in auxin biosynthesis

Indole‐3–acetic acid (IAA), an auxin plant hormone, is biosynthesized from tryptophan. The indole‐3–pyruvic acid (IPyA) pathway, involving the tryptophan aminotransferase TAA1 and YUCCA (YUC) enzymes, was recently found to be a major IAA biosynthetic pathway in Arabidopsis. TAA1 catalyzes the conversion of tryptophan to IPyA, and YUC produces IAA from IPyA. Using a chemical biology approach with maize coleoptiles, we identified 5–(4–chlorophenyl)‐4H‐1,2,4–triazole‐3–thiol (yucasin) as a potent inhibitor of IAA biosynthesis in YUC‐expressing coleoptile tips. Enzymatic analysis of recombinant AtYUC1‐His suggested that yucasin strongly inhibited YUC1‐His activity against the substrate IPyA in a competitive manner. Phenotypic analysis of Arabidopsis YUC1 over‐expression lines (35S::YUC1) demonstrated that yucasin acts in IAA biosynthesis catalyzed by YUC. In addition, 35S::YUC1 seedlings showed resistance to yucasin in terms of root growth. A loss‐of‐function mutant of TAA1, sav3–2, was hypersensitive to yucasin in terms of root growth and hypocotyl elongation of etiolated seedlings. Yucasin combined with the TAA1 inhibitor l –kynurenine acted additively in Arabidopsis seedlings, producing a phenotype similar to yucasin‐treated sav3–2 seedlings, indicating the importance of IAA biosynthesis via the IPyA pathway in root growth and leaf vascular development. The present study showed that yucasin is a potent inhibitor of YUC enzymes that offers an effective tool for analyzing the contribution of IAA biosynthesis via the IPyA pathway to plant development and physiological processes.     

Tryptophan Biosynthesis from Indole-3-Acetic Acid by Anaerobic Bacteria from the Rumen

Microbes in ruminal contents incorporated (14)C into cells when they were incubated in vitro in the presence of [(14)C]carboxyl-labeled indole-3-acetic acid (IAA). Most of the cellular (14)C was found to be in tryptophan from the protein fractions of the cells. Pure cultures of several important ruminal species did not incorporate labeled IAA, but all four strains of Ruminococcus albus tested utilized IAA for tryptophan synthesis. R. albus did not incorporate (14)C into tryptophan during growth in medium containing either labeled serine or labeled shikimic acid. The mechanism of tryptophan biosynthesis from IAA is not known but appears to be different from any described biosynthetic pathway. We propose that a reductive carboxylation, perhaps involving a low-potential electron donor such as ferredoxin, is involved.     

Tryptophan auxotroph mutants suppress the superroot2 phenotypes,modulating IAA biosynthesis in arabidopsis

Plant phytohormone, Indole-3-acetic acid (IAA ), is synthesized by tryptophan (trp) dependent and independent pathway. Here we report that tryptophan auxotroph mutants completely suppressed the abnormalities of auxin over production mutant, superroot2. SUR2 is considered to modulate Trp dependent pathway, resulting IAA accumulation in Arabidopsis. Tryptophan auxotroph mutants showed hyper-sensitivity to the auxin polar transport inhibitor, NPA, on the phenotype of reduced gravitropism. These results together with the results of histochemical analyses, tryptophan auxotroph mutants seem to have a complete defect in Trp dependent IAA biosynthesis pathway, and it is also suggested that the Trp dependent pathway is responsible for the normal root gravitropism.Key words: Auxin, Trp dependent pathway, trp mutants, sur2, arabidopsis