Indole acetonitrile-sensitivity of transgenic tobacco containing Arabidopsis thaliana nitrilase genes

 The Arabidopsis thaliana genome has four nitrilase (nitrile aminohydrolase, EC 3.5.5.1) genes (NIT1 to NIT4). These nitrilases catalyze hydrolysis of indole-3-acetonitrile (IAN) to indole-3-acetic acid (IAA). Growth of A. thaliana is inhibited by IAN probably due to hydrolysis of IAN to IAA, while the tobacco (Nicotiana tabacum) genome has only NIT4 homologs and is resistant to IAN. In this study, we introduced A. thaliana NIT1 to NIT4 into tobacco. Introduction of NIT1, NIT2 or NIT3 into tobacco conferred growth inhibition by IAN. NIT2 transgenic plants were highly sensitive to IAN, and NIT1 and NIT3 transgenic plants were moderately sensitive. On the other hand, NIT4 transgenic plants were less sensitive to IAN, although some morphological changes in the roots were observed as the wild-type tobacco. These findings suggest that the ability of transgenic tobacco to convert IAN to IAA in vivo is markedly different among transgenes of NIT1 to NIT4. Received: 22 November 1999 / Revision received: 28 January 2000 / Accepted: 4 February 2000     

The Nitrilase ZmNIT2 converts indole-3-acetonitrile to indole-3-acetic acid

We isolated two nitrilase genes, ZmNIT1 and ZmNIT2, from maize (Zea mays) that share 75% sequence identity on the amino acid level. Despite the relatively high homology to Arabidopsis NIT4, ZmNIT2 shows no activity toward beta-cyano-alanine, the substrate of Arabidopsis NIT4, but instead hydrolyzes indole-3-acetonitrile (IAN) to indole-3-acetic acid (IAA). ZmNIT2 converts IAN to IAA at least seven to 20 times more efficiently than AtNIT1/2/3. Quantitative real-time polymerase chain reaction revealed the gene expression of both nitrilases in maize kernels where high concentrations of IAA are synthesized tryptophan dependently. Nitrilase protein and endogenous nitrilase activity are present in maize kernels together with the substrate IAN. These results suggest a role for ZmNIT2 in auxin biosynthesis.     

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.     

Auxin biosynthesis in maize

For the biosynthesis of the phytohormone indole-3-acetic acid (IAA), a number of tryptophan-dependent and -independent pathways have been discussed. Maize is an appropriate model system to analyze IAA biosynthesis particularly because high quantities of IAA conjugates are stored in the endosperm. This allowed precursor feeding experiments in a kernel culture system followed by retrobiosynthetic NMR analysis, which strongly suggested that tryptophan-dependent IAA synthesis is the predominant route for auxin biosynthesis in the maize kernel. Two nitrilases ZmNIT1 and ZmNIT2 are expressed in seeds. ZmNIT2 efficiently hydrolyzes indole-3-acetonitrile (IAN) to IAA and thus could be involved in auxin biosynthesis. Redundant pathways, e.g., via indole-3-acetaldehyde could imply that multiple mutants will be necessary to obtain IAA-deficient plants and to conclusively identify relevant genes for IAA biosynthesis.     

Enzymatic characterization of the recombinant Arabidopsis thaliana nitrilase subfamily encoded by the NIT2/NIT1/NIT3-gene cluster

Three of the nitrilase isoenzymes of Arabidopsis thaliana (L.) Heynh. are located on chromosome III in tandem and these genes (NIT2/NIT1/NIT3 in the 5′→3′ direction) encode highly similar polypeptides. Copy DNAs encompassing the entire coding sequences for all three nitrilases were expressed in Escherichia coli as fusion proteins containing a C-terminal hexahistidine extension. All three nitrilases were obtained as enzymatically active proteins, and their characteristics were determined, including a detailed comparative analysis of their substrate preferences. All three nitrilases converted indole-3-acetonitrile (IAN) to indole-3-acetic acid (IAA), albeit, compared to the most effective substrates found, phenylpropionitrile (PPN), allylcyanide, (phenylthio)acetonitrile and (methylthio)acetonitrile, with low affinity and velocity. The preferred substrates are either naturally occurring substrates, which may originate from glucosinolate breakdown, or they are close relatives of these. Thus, a major function of NIT1, NIT2 and NIT3 is assigned to be the conversion to carboxylic acids of nitriles from glucosinolate turnover or degradation. While all nitrilases exhibit a similar pH optimum around neutral, and NIT1 and NIT3 exhibit a similar temperature optimum around 30 °C independent of the substrate analyzed (IAN, PPN), NIT2 showed a remarkably different temperature optimum for IAN (15 °C) and PPN (35–40 °C). A potential role for NIT2 in breaking seed dormancy in A. thaliana by low temperatures (stratification), however, was ruled out, although NIT2 was the predominantly expressed nitrilase isoform in developing embryos and in germinating seeds, as judged from an analysis of β-glucuronidase reporter gene expression under the control of the promoters of the four isogenes. It is possible that NIT2 is involved in supplying IAA during seed development rather than during stratification. Received: 13 May 2000 / Accepted: 14 August 2000     

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.     

Characterization of a Nitrilase and a Nitrile Hydratase from Pseudomonas sp. Strain UW4 That Converts Indole-3-Acetonitrile to Indole-3-Acetic Acid

Indole-3-acetic acid (IAA) is a fundamental phytohormone with the ability to control many aspects of plant growth and development. Pseudomonas sp. strain UW4 is a rhizospheric plant growth-promoting bacterium that produces and secretes IAA. While several putative IAA biosynthetic genes have been reported in this bacterium, the pathways leading to the production of IAA in strain UW4 are unclear. Here, the presence of the indole-3-acetamide (IAM) and indole-3-acetaldoxime/indole-3-acetonitrile (IAOx/IAN) pathways of IAA biosynthesis is described, and the specific role of two of the enzymes (nitrilase and nitrile hydratase) that mediate these pathways is assessed. The genes encoding these two enzymes were expressed in Escherichia coli, and the enzymes were isolated and characterized. Substrate-feeding assays indicate that the nitrilase produces both IAM and IAA from the IAN substrate, while the nitrile hydratase only produces IAM. The two nitrile-hydrolyzing enzymes have very different temperature and pH optimums. Nitrilase prefers a temperature of 50°C and a pH of 6, while nitrile hydratase prefers 4°C and a pH of 7.5. Based on multiple sequence alignments and motif analyses, physicochemical properties and enzyme assays, it is concluded that the UW4 nitrilase has an aromatic substrate specificity. The nitrile hydratase is identified as an iron-type metalloenzyme that does not require the help of a P47K activator protein to be active. These data are interpreted in terms of a preliminary model for the biosynthesis of IAA in this bacterium.     

Redirection of tryptophan metabolism in tobacco by ectopic expression of an Arabidopsis indolic glucosinolate biosynthetic gene

Indole-3-acetaldoxime (IAOx) is a branch point compound of tryptophan (Trp) metabolism in glucosinolate-producing species such as Arabidopsis, serving as a precursor to indole-glucosinolates (IGs), the defense compound camalexin, indole-3-acetonitrile (IAN) and indole-3-acetic acid (IAA). We synthesized [²H₅] and [¹³C₁₀¹⁵N₂]IAOx and [¹³C₆], [²H₅] and [2′,2′-²H₂]IAN in order to quantify endogenous IAOx and IAN in Arabidopsis and tobacco, a non-IG producing species. We found that side chain-labeled [2′,2′-²H₂]IAN overestimated the amount of IAN by 2-fold compared to when [²H₅]IAN was used as internal standard, presumably due to protium-deuterium exchange within the internal standard during extraction of plant tissue. We also determined that [¹³C₁]IAN underestimated the amount of IAN when the ratio of [¹³C₁]IAN standard to endogenous IAN was greater than five to one, whereas either [²H₅]IAN or [¹³C₆]IAN showed a linear relationship with endogenous IAN over a broader range of concentrations. Transgenic tobacco vector control lines did not have detectable levels of IAOx or IAN (limit of detection ∼ 100 pg/g fr. wt), while lines expressing either the IAOx-producing CYP79B2 or CYP79B3 genes from Arabidopsis under CaMV 35S promoter control accumulated IAOx in the range of 1-9 μg/g fr. wt. IAN levels in these lines ranged from 0.6 to 6.7 μg/g fr. wt, and IAA levels were ∼9-14-fold above levels in control lines. An Arabidopsis line expressing the same CYP79B2 overexpression construct accumulated IAOx in two of three lines measured (∼200 and 400 ng/g fr. wt) and accumulated IAN in all three lines. IAN is proposed to be a metabolite of IAOx or an enzymatic breakdown product of IGs induced upon tissue damage. Since tobacco does not produce detectable IGs, the tobacco data are consistent with IAN being a metabolite of IAOx. IAOx and IAN were also examined in the Arabidopsis activation tagged yucca mutant, and no accumulation of IAOx was found above the limits of detection but accumulation of IAN (3-fold above wt) occurred. The latter was surprising in light of recent reports that rule out IAOx and IAN as intermediates in YUCCA-mediated IAA synthesis.     

Arabidopsis Acetyl-Amido Synthetase GH3.5 Involvement in Camalexin Biosynthesis through Conjugation of Indole-3-Carboxylic Acid and Cysteine and Upregulation of Camalexin Biosynthesis Genes

Camalexin (3-thiazol-2 -yl-indole) is the major phytoalexin found in Arabidopsis thaliana. Several key intermediates and corresponding enzymes have been identified in camalexin biosynthesis through mutant screening and biochemical experiments. Camalexin is formed when indole-3-acetonitrile (IAN) is catalyzed by the cytochrome P450 monooxygenase CYP71A13. Here, we demonstrate that the Ara- bidopsis GH3.5 protein, a multifunctional acetyl-amido synthetase, is involved in camalexin biosynthesis via conjugating indole-3-carboxylic acid (ICA) and cysteine (Cys) and regulating camalexin biosynthesis genes. Camalexin levels were increased in the activation-tagged mutant gh3.5-1D in both Col-0 and cyp71A13-2 mutant backgrounds after pathogen infection. The recombinant GH3.5 protein catalyzed the conjugation of ICA and Cys to form a possible intermediate indole-3-acyl-cysteinate (ICA(Cys)) in vitro. In support of the in vitro reaction, feeding with ICA and Cys increased camalexin levels in Col-0 and gh3.5-1D. Dihydrocamalexic acid (DHCA), the precursor of camalexin and the substrate for PAD3, was accumulated in gh3.5-1D/pad3-1, suggesting that ICA(Cys) could be an additional precursor of DHCA for camalexin biosynthesis. Furthermore, expression of the major camalexin biosynthesis genes CYP79B2, CYP71A12, CYP71A13 and PAD3 was strongly induced in gh3.5-1D. Our study suggests that GH3.5 is involved in camalexin biosynthesis through direct catalyzation of the formation of ICA(Cys), and upregulation of the major biosynthetic pathway genes.     

Metabolism of [14C]-indole-3-acetic acid by soybean callus and hypocotyl sections

Metabolism of indole-3-acetic acid in soybean [ Glycine max (L.) Merr.] was investigated with [1-¹⁴C]- and [2-¹⁴C]-indole-3-acetic acid (IAA) applied by injection into soybean hypocotyl sections and by incubation with soybean callus. Free IAA and its metabolites were extracted with 80% methanol and separated by high performance liquid chromatography with [³H]-IAA as an internal standard. Metabolism of IAA in soybean callus was much greater than that in tobacco ( Nicotiana tabacum L.) callus used for comparison. High performance liquid chromatography of soybean extracts showed at least 10 metabolite peaks including both decarboxylated and undecarboxylated products. A major unstable decarboxylated metabolite was purified. [¹⁴C]-indole-3-methanol (IM) was three times more efficient than [2-¹⁴C]-IAA as substrate for producing this metabolite. It was hydrolyzable by β-glucosidase (EC 3.2.1.21), yielding an indole and D-glucose. The indole possessed characteristics of authentic IM. Thus, the metabolite is tentatively identified as indole-3-methanol-β-D-glucopyranoside. The results suggest that soybean tissues are capable of oxidizing IAA via the decarboxylative pathway with indole-3-methanol-glucoside as a major product. The high rate of metabolism of IAA may be related to the observed growth of soybean callus with high concentrations of IAA in the culture medium.     

A mutation affecting the synthesis of 4-chloroindole-3-acetic acid

Traditionally, schemes depicting auxin biosynthesis in plants have been notoriously complex. They have involved up to four possible pathways by which the amino acid tryptophan might be converted to the main active auxin, indole-3-acetic acid (IAA), while another pathway was suggested to bypass tryptophan altogether. It was also postulated that different plants use different pathways, further adding to the complexity. In 2011, however, it was suggested that one of the four tryptophan-dependent pathways, via indole-3-pyruvic acid (IPyA), is the main pathway in Arabidopsis thaliana,¹ although concurrent operation of one or more other pathways has not been excluded. We recently showed that, for seeds of Pisum sativum (pea), it is possible to go one step further.² Our new evidence indicates that the IPyA pathway is the only tryptophan-dependent IAA synthesis pathway operating in pea seeds. We also demonstrated that the main auxin in developing pea seeds, 4-chloroindole-3-acetic acid (4-Cl-IAA), which accumulates to levels far exceeding those of IAA, is synthesized via a chlorinated version of the IPyA pathway.     

ILR2, a novel gene regulating IAA conjugate sensitivity and metal transport in Arabidopsis thaliana

Plants can regulate levels of the auxin indole-3-acetic acid (IAA) by conjugation to amino acids or sugars, and subsequent hydrolysis of these conjugates to release active IAA. These less active auxin conjugates constitute the majority of IAA in plants. We isolated the Arabidopsis ilr2-1 mutant as a recessive IAA-leucine resistant mutant that retains wild-type sensitivity to free IAA. ilr2-1 is also defective in lateral root formation and primary root elongation. In addition, ilr2-1 is resistant to manganese- and cobalt-mediated inhibition of root elongation, and microsomal preparations from the ilr2-1 mutant exhibit enhanced ATP-dependent manganese transport. We used a map-based positional approach to clone the ILR2 gene, which encodes a novel protein with no predicted membrane-spanning domains that is polymorphic among Arabidopsis accessions. Our results demonstrate that ILR2 modulates a metal transporter, providing a novel link between auxin conjugate metabolism and metal homeostasis.     

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。     

Induction of auxin biosynthetic enzymes by jasmonic acid and in clubroot diseased Chinese cabbage plants

Nitrilase (NIT) and myrosinase are important enzymes for auxin biosynthesis in Brassicaceae, which is increased during clubroot disease. Therefore, NIT and myrosinase levels during club development and possible regulation mechanisms were investigated. In addition, the occurrence of different nitrilase isoforms in Chinese cabbage has been shown. Nitrilase activity was enhanced in infected roots during later stages of club development (35–42 days after inoculation). However, no differences in nitrilase mRNA levels between infected and healthy roots were found during symptom development. Myrosinase expression was increased in clubbed roots at slightly earlier time points (28 days after inoculation) and also at later time points during infection. The activities of tryptophan oxidizing enzyme (TrpOxE), which catalyzes the first step in tryptophan-dependent auxin biosynthesis in Brassicaceae, and nitrilase were enhanced after treatment with jasmonic acid (JA) and methyl jasmonate. Similarly, the amount of myrosinase mRNA was increased by JA. During clubroot disease the endogenous concentration of JA increased in infected roots 3–5 weeks after inoculation. From our results it can be concluded that: (1) de novo indole-3-acetic acid (IAA) biosynthesis plays a role for symptom development of clubroot disease in Brassicaceae during later developmental stages; and (2) JA which increased during club development, may be involved in the up-regulation of three enzymes important for IAA synthesis.     

Many roads lead to "auxin": of nitrilases, synthases, and amidases

Recent progress in understanding the biosynthesis of the auxin, indole-3-acetic acid (IAA) in Arabidopsis thaliana is reviewed. The current situation is characterized by considerable progress in identifying, at the molecular level and in functional terms, individual reactions of several possible pathways. It is still too early to piece together a complete picture, but it becomes obvious that A. thaliana has multiple pathways of IAA biosynthesis, not all of which may operate at the same time and some only in particular physiological situations. There is growing evidence for the presence of an indoleacetamide pathway to IAA in A. thaliana, hitherto known only from certain plant-associated bacteria, among them the phytopathogen Agrobacterium tumefaciens.