Indole-3-acetic acid is synthesized from L-tryptophan in roots of Arabidopsis thaliana

The promoter of the nit1 gene, encoding the predominantly expressed isoform of the Arabidopsis thaliana (L.) Heynh. nitrilase isoenzyme family, fused to the β-glucuronidase gene (uidA) drives β-glucuronidase expression in the root system of transgenic A. thaliana and tobacco plants. This expression pattern was shown to be controlled developmentally, suggesting that the early differentiation zone of root tips and the tissue surrounding the zone of lateral root primordia formation may constitute sites of auxin biosynthesis in plants. The root system of A. thaliana was shown to express functional nitrilase enzyme. When sterile roots were fed [²H]₅-L-tryptophan, they converted this precusor to [²H]₅-indole-3-acetonitrile and [²H]₅-indole-3-acetic acid. This latter metabolite was further metabolized into base-labile conjugates which were the predominant form of [²H]₅-indole-3-acetic acid extracted from roots. When [1-¹³C]-indole-3-acetonitrile was fed to sterile roots, it was converted to [1-¹³C]-indole-3-acetic acid which was further converted to conjugates. The results prove that the A. thaliana root system is an autonomous site of indole-3-acetic acid biosynthesis from L-tryptophan. Received: 3 February 1998 / Accepted: 17 April 1998     

Genes encoding nitrilase-like proteins from tobacco.

Nitrilase (nitrile aminohydrolase, EC 3.5.5.1) catalyzes the hydrolysis of indole-3-acetonitrile (IAN) to indole-3-acetic acid (IAA). Arabidopsis thaliana genome has four nitrilase genes (NIT1, NIT2, NIT3 and NIT4). Three (NIT1, NIT2 and NIT3) of the four genes have high similarity. We have cloned two NIT4 homologs (TNIT4A and TNIT4B) from tobacco (Nicotiana tabacum). Genomic Southern hybridization, among other experiments, strongly suggests that tobacco has NIT4 homologs but not NIT1 to NIT3 homologs. Introduction of Arabidopsis NIT2 into tobacco conferred IAN-mediated growth inhibition, probably due to hydrolysis of IAN to IAA, while ectopic expression of TNIT4A had little effect on the sensitivity of transgenic plants to IAN. Nitrilase activity of TNIT4 proteins is discussed.     

Pseudomonas syringae pv. syringae B728a hydrolyses indole-3-acetonitrile to the plant hormone indole-3-acetic acid

Nitrilase enzymes catalyse the hydrolysis of nitrile compounds to the corresponding carboxylic acid and ammonia, and have been identified in plants, bacteria and fungi. There is mounting evidence to support a role for nitrilases in plant–microbe interactions, but the activity of these enzymes in plant pathogenic bacteria remains unexplored. The genomes of the plant pathogenic bacteria Pseudomonas syringae pv. syringae B728a and Pseudomonas syringae pv. tomato DC3000 contain nitrilase genes with high similarity to characterized bacterial arylacetonitrilases. In this study, we show that the nitrilase of P. syringae pv. syringae B728a is an arylacetonitrilase, which is capable of hydrolysing indole-3-acetonitrile to the plant hormone indole-3-acetic acid, and allows P. syringae pv. syringae B728a to use indole-3-acetonitrile as a nitrogen source. This enzyme may represent an additional mechanism for indole-3-acetic acid biosynthesis by P. syringae pv. syringae B728a, or may be used to degrade and assimilate aldoximes and nitriles produced during plant secondary metabolism. Nitrilase activity was not detected in P. syringae pv. tomato DC3000, despite the presence of a homologous nitrilase gene. This raises the interesting question of why nitrilase activity has been retained in P. syringae pv. syringae B728a and not in P. syringae pv. tomato DC3000.     

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     

Maize nitrilases have a dual role in auxin homeostasis and beta-cyanoalanine hydrolysis

The auxin indole-3-acetic acid (IAA), which is essential for plant growth and development, is suggested to be synthesized via several redundant pathways. In maize (Zea mays), the nitrilase ZmNIT2 is expressed in auxin-synthesizing tissues and efficiently hydrolyses indole-3-acetonitrile to IAA. Zmnit2 transposon insertion mutants were compromised in root growth in young seedlings and sensitivity to indole-3-acetonitrile, and accumulated lower quantities of IAA conjugates in kernels and root tips, suggesting a substantial contribution of ZmNIT2 to total IAA biosynthesis in maize. An additional enzymatic function, turnover of beta-cyanoalanine, is acquired when ZmNIT2 forms heteromers with the homologue ZmNIT1. In plants carrying an insertion mutation in either nitrilase gene this activity was strongly reduced. A dual role for ZmNIT2 in auxin biosynthesis and in cyanide detoxification as a heteromer with ZmNIT1 is therefore proposed.     

Indolic constituents and indole-3-acetic acid biosynthesis in the wild-type and a tryptophan auxotroph mutant of Arabidopsis thaliana

 The tryptophan auxotroph mutant trp3-1 of Arabidopsis thaliana (L.) Heynh., despite having reduced levels of l-tryptophan, accumulates the tryptophan-derived glucosinolate, glucobrassicin and, thus, does not appear to be tryptophan-limited. However, due to the block in tryptophan synthase, the mutant hyperaccumulates the precursor indole-3-glycerophosphate (up to 10 mg per g FW). Instability of indole-3-glycerophosphate leads to release of indole-3-acetic acid (IAA) from this metabolite during standard workup of samples for determination of conjugated IAA. The apparent increase in “conjugated IAA” in trp3-1 mutant plants can be traced back entirely to indole-3-glycerophosphate degradation. Thus, the levels of neither free IAA nor conjugated IAA increase detectably in the trp3-1 mutant compared to wild-type plants. Precursor-feeding experiments to shoots of sterile-grown wild-type plants using [²H]₅-l-tryptophan have shown incorporation of label from this precursor into indole-3-acetonitrile and indole-3-acetic acid with very little isotope dilution. It is concluded that Arabidopsis thaliana shoots synthesize IAA from l-tryptophan and that the non-tryptophan pathway is probably an artifact. Received: 1 March 2000 / Accepted: 10 April 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.     

解淀粉芽胞杆菌HZ-12中ysnE参与吲哚-3-乙酸合成的代谢途径研究

【目的】吲哚-3-乙酸是调控植物生长发育和生理活动的重要激素,吲哚-3-乙酸N-乙酰转移酶YsnE在吲哚-3-乙酸合成中发挥重要作用,本研究拟解析解淀粉芽胞杆菌中YsnE参与吲哚-3-乙酸合成的代谢途径。【方法】通过基因ysnE缺失和强化表达,分析ysnE对吲哚-3-乙酸合成影响,结合吲哚-3-乙酸合成中间物(吲哚丙酮酸、吲哚乙酰胺、色胺和吲哚乙腈)添加和体外酶转化实验,解析ysnE参与吲哚-3-乙酸合成的代谢途径。【结果】明确了YsnE在解淀粉芽胞杆菌HZ-12吲哚-3-乙酸合成中发挥重要作用。发现ysnE缺失菌株中的吲哚丙酮酸、吲哚乙酰胺和吲哚乙腈利用显著降低,揭示了YsnE主要发挥吲哚丙酮酸脱羧酶YclB和吲哚乙酰胺水解酶/腈水解酶/腈水合酶YhcX的功能,并通过参与吲哚丙酮酸、吲哚乙酰胺和吲哚乙腈途径来影响吲哚-3-乙酸合成。【结论】初步揭示了YsnE通过影响吲哚丙酮酸、吲哚乙酰胺和吲哚乙腈途径参与吲哚-3-乙酸合成的代谢机理,为吲哚-3-乙酸合成途径解析和代谢工程育种构建吲哚-3-乙酸高产菌株奠定了基础。     

Indole-3-methylglucosinolate biosynthesis and metabolism in clubroot diseased plants

lndole-3-methylglucosinolate biosynthesis and metabolism in roots of Brassica napus (swede, cv. Danestone II) infected with Plasmodiophora brassicae Wor. were investigated with a pulse feeding technique developed to infiltrate intact tissue segments with labelled substrates. Infected root tissue metabolized [¹⁴C]-L-tryptophan to indole-3-methylglucosinolate, indole-3-acetonitrile, and some other lipophilic indole compounds. The incorporation of radioactivity into these compounds was significantly enhanced in infected tissue compared with control tissue. A time course study showed a high turnover of indole-3-methylglucosinolate and indole-3-acetonitrile in infected tissue. However, thioglucoside glucohydrolase activity was not changed in infected tissue compared with control tissue. Disc electrophoresis revealed the same isoenzyme in both tissues. Control and infected tissues both rapidly hydrolyzed [¹⁴C]-indole-3-acetonitrile in vivo. The possibility of a disease specific biosynthesis of indole-3-acetic acid from indole-3-methylglucosinolate as the result of a changed compartmentation is discussed.     

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.     

Overexpression of Maize IAGLU in Arabidopsis thaliana Alters Plant Growth and Sensitivity to IAA but not IBA and 2,4-D

Overexpression of the IAGLU gene from maize (ZmIAAGLU) in Arabidopsis thaliana, under the control of the CaMV 35S promoter, inhibited root but not hypocotyl growth of seedlings in four different transgenic lines. Although hypocotyl growth of seedlings and inflorescence growth of mature plants was not affected, the leaves of mature plants were smaller and more curled as compared to wild-type and empty vector transformed plants. The rosette diameter in transgenic lines with higher ZmIAGLU expression was also smaller compared to the wild type. Free indole-3-acetic acid (IAA) levels in the transgenic plants were comparable to the wild type, even though a decrease in free IAA levels might be expected from overexpression of an IAA-conjugate–forming enzyme. IAA-glucose levels, however, were increased in transgenic lines compared to the wild type, indicating that the ZmIAGLU gene product is active in these plants. In addition, three different 35SZmIAGLU lines showed less inhibition of root growth when cultivated on increasing concentrations of IAA but not indole-3-butyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4-D). Feeding IAA to transgenic lines resulted in increased IAA-glucose synthesis, whereas the levels of IAA-aspartate and IAA-glutamine formed were reduced compared to the wild type. Our results show that IAA homeostasis can be altered by heterologous overexpression of a conjugate-forming gene from maize.     

Improved method for the transformation of Arabidopsis thaliana with chimeric dihydrofolate reductase constructs which confer methotrexate resistance

Summary A modified root explant transformation method has been developed that is effective in producing transgenic Arabidopsis thaliana plants which are methotrexate resistant due to the integration of T-DNA vectors containing a chimeric dihydrofolate reductase gene. Molecular analysis shows that transformed methotrexate resistant plants contain the expected T-DNA construct with the chimeric gene. This transformation method also works well with other plant selectable markers, including hygromycin phosphotransferase and neomycin phosphotransferase II.Abbreviations DHFR (dihydrofolate reductase) - HPT (hygromycin phosphotransferase) - NPTII (neomycin phosphotransferase) - CaMV (Cauliflower Mosaic Virus) - MES (2-[N-morpholino]ethanesulfonic acid) - BAP (N6-benzylaminopurine) - NAA (naphthaleneacetic acid) - 2,4-D (2,4 dichlorophenoxyacetic acid) - 2iP (6-(dimethylallylamino)-purine) - 2iPAde (6-(dimethylallylamino)-ade-nine) - IAA (indole-3-acetic acid) - IBA (indole-3-butyric acid)     

Indole-3-acetic acid homeostasis in transgenic tobacco plants expressing the Agrobacterium rhizogenes rolB gene

The rolB gene of the plant pathogen Agrobacterium rhizogenes has an important role in the establishment of hairy root disease in infected plant tissues. When expressed as a single gene in transgenic plants the RolB protein gives rise to effects indicative of increased auxin activity. It has been reported that the RolB product is a β-glucosidase and proposed that the physiological and developmental alterations in transgenic plants expressing the rolB gene are the result of this enzyme hydrolysing bound auxins, in particular (indole-3-acetyl)-β-D-glucoside (IAGluc), and thereby bringing about an increase in the intracellular concentration of indole-3-acetic acid (IAA). Using tobacco plants as a test system, this proposal has been investigated in detail. Comparisons have been made between the RolB phenotype and that of IaaM/iaaH transformed plants overproducing IAA. In addition, the levels of IAA and IAA amide and IAA ester conjugates were determined in wild-type and transgenic 35S-rolB tobacco plants and metabolic studies were carried out with [¹³C₆]IAA [2′-¹⁴C]IAA, [¹⁴C]IAGluc, [5-³H]-2-o-(indole-3-acetyl)-myo-inositol and [¹⁴C]indole-3-acetylaspartic acid. The data obtained demonstrate that expression of the rolB encoded protein in transgenic tobacco does not produce a phenotype that resembles that of IAA over producing plants, does not alter the size of the free IAA pool, has no significant effect on the rate of IAA metabolism, and, by implication, appears not to influence the overall rate of IAA biosynthesis. Furthermore, the in vivo hydrolysis of IAGluc, and that of the other IAA conjugates that were tested, is not affected. On the basis of these findings, it is concluded that the RolB phenotype is not the consequence of an increase in the size of the free IAA pool mediated by an enhanced rate of hydrolysis of IAA conjugates.     

Nitrilase activity in clubroot diseased plants

Nitrilase activity was detected in desalted extracts of leaves, hypocotyls and roots of swede ( Brassica napus ) but was considerably higher in leaves than in roots. After inoculation with Plasmodiophora brassicae infected roots and hypocotyls showed an increase in nitrilase activity beginning at the early stages of club development before total protein increased significantly. Enzyme activity of infected tissue was partially purified by DEAE ion exchange chromatography and compared to the enzyme extracted from non infected seedlings. It appears that the increase in nitrilase activity is due to an increase of the plant enzyme which is initially present with lower activity. K_m values for the artificial substrate 3-cyanopyridine and for indole-3-acetonitrile were 2.1 × 10⁻³ M and 6.2 × 10⁻⁴M, respectively. The role of nitrilase activity for IAA biosynthesis is discussed.     

An NMR method for the study of proton transport across phospholipid vesicles

We have identified [1-¹⁴C]-oxindole-3-acetic acid as a catabolic product of [1-¹⁴C]-indole-3-acetic acid metabolism in Zea mays seedlings. The isolation, and chemical and mass spectral characterization of oxindole-3-acetic acid from corn kernel tissue is described together with data suggesting oxindole-3-acetic acid to be a major catabolic product of indole-3-acetic acid.     

Auxin Activity of Glucobrassicin

The indole-glucosinolate, glucobrassicin, which is found in large amounts in members of the genus Brassica, was subjected to tests in the standard Avena curvature bioassay. Both the tetramethylammonium salt of glucobrassicin and the non-crystallizted material induced curvature at a concentration of 5 × l0^?4M. The curvature from the salt was, however, only half of that induced by the acid form. Bioassays of chromatograms of fresh methanolic extracts of savoy cabbage indicated the presence of indole-3-acetic acid as well as indole-3-acetonitrile in small amounts along with large amounts of glucobrassicin. Segments of green hypocotyl tissue of savoy cabbage respond with limited growth to high concentrations of glucobrassicin. Segments of etiolated hypocotyls or green epicotyls give no growth response to glucobrassicin. Pea stem segments which do not respond to indoleacetonitrile responded with similar growth to glucobrassicin and indoleacetic acid. Nitrilase activity was found in savoy hypocotyl and epicotyl tissue by chromatography of incubation media. The growth regulation in savoy cabbage in conjunction with data on the distribution of glucobrassicin in the plant, leads to the conclusion that glucobrassicin is a special, inactive storage and transport form of the active auxin in cabbage.     

Indole glucosinolate and auxin biosynthesis in Arabidopsis thaliana (L.) Heynh. glucosinolate mutants and the development of clubroot disease

Mutants and wild type plants of Arabidopsis thaliana were analysed for differences in glucosinolate accumulation patterns, indole-3-acetic acid (IAA) biosynthesis and phenotype. A previously identified series of mutants, termed TU, with altered glucosinolate patterns was used in this study. Only the line TU8 was affected in shoot phenotype (shorter stems, altered branching pattern). Synthesis of IAA and metabolism were not much affected in the TU8 mutant during seedling development, although the content of free IAA peaked earlier in TU8 during plant development than in the wild type. Indole glucosinolates and IAA may, however, be involved in the development of clubroot disease caused by the obligate biotrophic fungus Plasmodiophora brassicae since the TU3 line had a lower infection rate than the wild type, and lines TU3 and TU8 showed decreased symptom development. The decline in clubroot formation was accompanied by a reduced number of fungal structures within the root cortex and slower development of the fungus. Indole glucosinolates were lower in infected roots of TU3 and TU8 than in control roots of these lines, whereas in wild-type plants the differences were not as prominent. Free IAA and indole-3-acetonitrile (IAN) were increased in infected roots of the wild type and mutants with normal clubroot symptoms, whereas they were reduced in infected roots of mutants TU3 and TU8. These results indicate a role for indole glucosinolates and IAN/IAA in relation to symptom development in clubroot disease. Received: 23 July 1998 / Accepted: 12 January 1999     

The chemical compound ‘Heatin’ stimulates hypocotyl elongation and interferes with the Arabidopsis NIT1-subfamily of nitrilases

Temperature passively affects biological processes involved in plant growth. Therefore, it is challenging to study the dedicated temperature signalling pathways that orchestrate thermomorphogenesis, a suite of elongation growth-based adaptations that enhance leaf-cooling capacity. We screened a chemical library for compounds that restored hypocotyl elongation in the pif4-2–deficient mutant background at warm temperature conditions in Arabidopsis thaliana to identify modulators of thermomorphogenesis. The small aromatic compound ‘Heatin’, containing 1-iminomethyl-2-naphthol as a pharmacophore, was selected as an enhancer of elongation growth. We show that ARABIDOPSIS ALDEHYDE OXIDASES redundantly contribute to Heatin-mediated hypocotyl elongation. Following a chemical proteomics approach, the members of the NITRILASE1-subfamily of auxin biosynthesis enzymes were identified among the molecular targets of Heatin. Our data reveal that nitrilases are involved in promotion of hypocotyl elongation in response to high temperature and Heatin-mediated hypocotyl elongation requires the NITRILASE1-subfamily members, NIT1 and NIT2. Heatin inhibits NIT1-subfamily enzymatic activity in vitro and the application of Heatin accordingly results in the accumulation of NIT1-subfamily substrate indole-3-acetonitrile in vivo. However, levels of the NIT1-subfamily product, bioactive auxin (indole-3-acetic acid), were also significantly increased. It is likely that the stimulation of hypocotyl elongation by Heatin might be independent of its observed interaction with NITRILASE1-subfamily members. However, nitrilases may contribute to the Heatin response by stimulating indole-3-acetic acid biosynthesis in an indirect way. Heatin and its functional analogues present novel chemical entities for studying auxin biology.