Inhibition of enzymic oxidation of indole-3-acetic acid by metabolites of the insecticide carbofuran

Metabolites of carbofuran, a carbamate insecticide, inhibit the enzymic oxidation of indole-3-acetic acid. The metabolites differ in stability and effectiveness. 2,2-Dimethyl-7-hydroxy-2,3-dihydrobenzofuran represents one type which is broken down in the IAA oxidation reaction; thus the induced inhibition is limited by depletion of the the inhibitor. 2,2-Dimethyl-3-keto-7-hydroxy-2,3-dihydrobenzofuran represents the other type which is stable in the reaction; thus the inhibition is persistent. With both types of inhibitors the inhibition is reversible by higher substrate concentrations, but the Lineweaver-Burk plot is curvilinear suggesting the complex nature of competitive inhibition.     

Role of Phenolic Inhibitors in Peroxidase-mediated Degradation of Indole-3-acetic Acid

7-Hydroxy-2,3-dihydrobenzofuran derivatives, metabolites of a carbamate insecticide carbofuran, and five other phenolic inhibitors of indoleacetic acid (IAA) oxidase interfered with IAA-induced spectral change in the Soret band of horseradish peroxidase (HRP). The onset of IAA degradation required transformed HRP intermediates. The inhibitors, when added before IAA, protected HRP from reacting with IAA, thus preventing formation of highly reactive enzyme intermediates, and consequently, IAA degradation. When added after IAA, the inhibitors quickly reversed the IAA-induced spectral change of HRP and inhibited further IAA degradation.     

Nitrogen fixation and indole acetic acid production by Azospi-rillum sp. as influenced by an insecticide, carbofuran

Both indole acetic acid (IAA) accumulation and nitrogen fixation were increased in Azospirillum cultures isolated from rice roots and soils by carbofuran (2, 3-dihydro-2, 2-dimethyl-7-benzofuranyl-N-methyl carbamate), an insecticide widely used in rice cultivation. Addition of carbofuran at 5 and 10 parts/10₆ significantly stimulated nitrogen fixation in Azospirillum. Indole acetic acid accumulation by Azospirillum cultures was more pronounced at a lower level (250 g/50 ml) of carbofuran. Evidence is provided for carbofuran degradation by Azospirillum cultures. The 7-benzofuranol (2, 3-dihydro-2–2-dimethyl-7 benzofuranol), a major degradation product of carbofuran, however, did not enhance the IAA accumulation. The higher accumulation of IAA in Azospirillum in the presence of carbofuran is probably related to the increased growth due to fixed N present in the insecticide. Results indicate the involvement of parent compound carbofuran and/or compounds other than the 7-benzofuranol in the higher accumulation of IAA by Azospirillum sp.     

New phenolic inhibitors of the peroxidse-catalysed oxidation of indole-3-acetic acid

Previously we reported two metabolites of the insecticide carbofuran as persistent inhibitors of the peroxidase-catalysed oxidtion ofindole-3-acetic acid. In searching for more active inhibitors of this type, we have found that 5-hydroxy-2,2-dimethylchromene (β-tubanol), 2′,6′-dihydroxycetophenone oxime, 5-hydroxy-2,2-dimethylchroman, 2′,6′-dihydroxyacetophenone and 2,6-dihydroxybenzoic acid methyl ester were more active than the carbofuran metabolite 7-hydroxy-2,2-dimethyl-3-oxo-2,3-dihydrobenzofuran. Resorcinol, 5-hydroxy-2,2-dimethylchroman-4-one, 3-hydroxy-5-methoxy-2,2-dimethylchroman-4-one and 5-hydroxy-2-methylchrom-4-one were also inhibitory but with less activity. The new inhibitors differed from the well-known phenolic inhibitors such as caffeic acid in inhibition kinetics as demonstrated by the rate of disappearance of indole-3-acetic acid, the rate of formation of the oxidation products, and the transient spectral change in the enzyme.     

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.     

A method for profiling classes of plant hormones and their metabolites using liquid chromatography-electrospray ionization tandem mass spectrometry: an analysis of hormone regulation of thermodormancy of lettuce (Lactuca sativa L.) seeds

A highly selective and sensitive method for the simultaneous analysis of several plant hormones and their metabolites is described. The method combines high-performance liquid chromatography (HPLC) with positive and negative electrospray ionization-tandem mass spectrometry (ESI-MS/MS) to quantify a broad range of chemically and structurally diverse compounds. The addition of deuterium-labeled analogs for these compounds prior to sample extraction permits accurate quantification by multiple reaction monitoring (MRM). Endogenous levels of abscisic acid (ABA), abscisic acid glucose ester (ABA-GE), 7'-hydroxy-abscisic acid (7'-OH-ABA), phaseic acid (PA), dihydrophaseic acid (DPA), indole-3-acetic acid (IAA), indole-3-aspartate (IAAsp), zeatin (Z), zeatin riboside (ZR), isopentenyladenine (2iP), isopentenyladenosine (IPA), and gibberellins (GA)1, GA3, GA4, and GA7 were determined simultaneously in a single run. Detection limits ranged from 0.682 fmol for Z to 1.53 pmol for ABA. The method was applied to the analysis of plant hormones and hormonal metabolites associated with seed dormancy and germination in lettuce (Lactuca sativa L. cv. Grand Rapids), using extracts from only 50 to 100 mg DW of seed. Thermodormancy was induced by incubating seeds at 33 degrees C instead of 23 degrees C. Germinating seeds transiently accumulated high levels of ABA-GE. In contrast, thermodormant seeds transiently accumulated high levels of DPA after 7 days at 33 degrees C. GA1 and GA3 were detected during germination, and levels of GA1 increased during early post-germinative growth. After several days of incubation, thermodormant seeds exhibited a striking transient accumulation of IAA, which did not occur in seeds germinating at 23 degrees C. We conclude that hormone metabolism in thermodormant seeds is surprisingly active and is significantly different from that of germinating seeds.     

DDT降解菌株Chryseobacterium sp. PYR2对小麦的促生作用及其机理

【背景】前期结果表明,DDT降解菌株Chryseobacterium sp. PYR2可高效去除土壤中的DDT等污染物,具有潜在的应用价值,但该菌对植物的影响尚不清楚。【目的】探讨菌株Chryseobacterium sp. PYR2对植物的促生作用及其机理,为后续开发DDT降解及植物促生双效功能菌剂提供理论依据。【方法】配制该菌株的不同梯度稀释菌悬液,用纸卷发芽法和盆栽法研究菌悬液对小麦种子萌发和植株生长的影响;Salkowski法测定PYR2合成吲哚-3-乙酸(Indole-3-acetic acid,IAA)量;单因素实验研究不同培养条件对菌株生长及IAA合成的影响;液相色谱-串联质谱-多反应监测(LC-MS/MS-MRM)方法分析IAA在PYR2菌体内的生物合成途径。【结果】PYR2菌悬液可明显提高小麦种子萌发率并促进小麦植株的生长,小麦的侧根数、株高、鲜重、干重等指标均明显提高。该作用是由于菌株PYR2可以合成植物生长激素IAA。最适IAA合成条件:温度30°C,pH 7.0-8.0,盐浓度0.5%,L-色氨酸50mg/L。代谢液中检测到色醇、色胺和吲哚-3-乙酰胺3种中间代谢产物,推测PYR2体内存在3条IAA合成途径,分别为吲哚-3-丙酮酸(IPy A)、TAM和IAM途径。【结论】菌株PYR2对小麦具有明显的促生效果,是由于其具有多条高效合成IAA的代谢途径,表明其在农药污染土壤的生物修复及作物种植中具有潜在的应用前景。     

Exogenous application of IAA alleviates effects of supplemental ultraviolet‐B radiation in the medicinal plant Withania somnifera Dunal

• Supplemental (s)‐UV‐B radiation has adverse effects on the majority of plants. The present study was conducted to evaluate the effects of exogenous application of the growth hormone indole acetic acid (IAA) on various morphological, physiological and biochemical characteristics of Withania somnifera, an indigenous medicinal plant, subjected to s‐UV‐B.
• The s‐UV‐B‐treated plants received ambient + 3.6 kJm^?2·day^?1 biologically effective UV‐B, and IAA was applied at two doses (200 and 400 ppm) to s‐UV‐B‐exposed plants.
• The plant was forced to compromise its growth, development and photosynthetic patterns to survive under s‐UV‐B by increasing concentrations of secondary metabolites and antioxidants (thiol, proline, ascorbic acid, α‐tocopherol, ascorbate peroxidase, catalase, glutathione reductase, peroxidase, polyphenol oxidase, superoxide dismutase) to counteract oxidative stress. Increases in secondary metabolites were evidenced as increased activity of phenylpropanoid pathway enzymes: phenylalanine ammonia lyase, cinnamyl alcohol dehydrogenase, 4‐coumarate CoA ligase, chalcone isomerase and dihydroflavonol reductase. Application of different IAA doses reversed the detrimental effects of s‐UV‐B on W. somnifera by improving growth and photosynthesis and reducing concentrations of secondary metabolites and non‐enzymatic antioxidants. Antioxidant enzymes, however, had a synergistic effect on s‐UV‐B treatment and IAA application.
• The effects of s‐UV‐B on W. somnifera are ameliorated to varying degrees upon exogenous IAA application, and synergistic enhancement of antioxidant enzymes under s‐UV‐B+IAA treatment might be responsible for the partial recuperation of growth and plant protein content, as a UV‐B‐exposed plant is forced to allocate most of its photosynthate towards production of enzymes related to antioxidant defence.

     

Studies on plant growth substances,IAA metabolism and nitrogenase activity in root nodules of phaseolus aureus Roxb. var.mungo

The mature nodules ofPhaseolus aureus Roxb. var.mungo possessed, in comparison with young and old nodules, higher activities of nitrogenase (N_2ase), and indol-3-ylacetic acid (IAA) metabolic enzymes like IAA oxidase, methylene oxindole reductase and peroxidase; higher levels of IAA-like, gibberellic acid-like (GA), and cytokinin-like (CK) substances and tryptophan, and lower level of phenol. The abscisic acid-like (ABA) substance level was higher in the old nodules. The N_2ase activity in the mature nodules changed parallelly with IAA and OK. butoppositely with GA and ABA. The changes in tryptophan level, IAA oxidizing enzymes, and phenol metabolism controlled the IAA level in the nodules. The nodules developed similarly throughout the year, but they had variable hormone levels in different seasons. This indicated that the formation and growth of the nodules was controlled not only by the nodular hormones.     

硼对吲哚乙酸在植物体内运输的影响

以绿豆为指示作物,研究缺硼对侧芽生长及³H-吲哚乙酸（IAA）在完整植株体内运输的影响.结果表明：缺硼诱导侧芽生长,导致³H-IAA移动峰靠近植株顶端,茎中³H-IAA的放射性活度也低于供硼充分的植株,说明缺硼抑制了³H-IAA在植株体内的极性运输;无论缺硼与否侧芽中均未检测到³H-IAA,所以侧芽的生长与³H-IAA在其中的积累没有关系,表明硼并不是通过调节IAA在侧芽中的积累,而是通过调节IAA在主茎的移动流调控侧芽生长;给缺硼植株供硼24 h能够恢复IAA在植株体内的极性运输能力.     

Induction of carbofuran oxidation to 4-hydroxycarbofuran by Pseudomonas sp. 50432

Pseudomonas sp. 50432 biotransformed a highly toxic pesticide, carbofuran (2,3-dihydro-2,2-dimethylbenzofuran-7-yl methylcarbamate) to 7-phenol (2,3-dihydro-2,2-dimethyl-7-hydroxy benzofuran) and several unknown metabolites. One of the unknown metabolites identified by gas chromatography/mass spectroscopy was 4-hydroxycarbofuran (2,3-dihydro-2,2-dimethyl-4-hydroxybenzofuran-7-yl methylcarbamate). It had a mass (237) similar to 3-hydroxycarbofuran and 5-hydroxycarbofuran but different fragmentation patterns. This is the first report in which an inducible oxidative enzyme, hydroxylase, mediated the conversion of carbofuran to 4-hydroxycarbofuran. A second constitutively synthesized enzyme hyrolase transformed carbofuran to 7-phenol.     

Growth and metabolism of dark septate endophytes and their stimulatory effects on plant growth

Dark septate endophytes (DSE) colonize plant roots extensively and increase host plant growth and nutrition. However, the development of DSE-produced metabolites as plant biostimulants has been largely ignored. DSE growth curves and extracellular metabolite components were analyzed and the growth-promoting effects of DSE extracellular metabolites on alfalfa (Medicago sativa L.) grown for 4, 8 12, 16 and 20 days were evaluated. The growth curve of the DSE strain Alternaria sp. shows days 0-8 in the growth phase, days 8-16 in the stable phase, and days 16-20 in the senescent phase. The extracellular metabolite components of DSE were significantly different at different growth stages. The biomass of alfalfa was increased significantly by DSE extracellular metabolites (P < 0.05). Biomass of alfalfa inoculated with DSE extracellular metabolites more than doubled after growth for 8 days and nutrient availability also increased significantly compared with the uninoculated control. Six DSE extracellular metabolites, calycosin 7-galactoside, 1-[(5-amino-5-carboxypentyl)amino]-1-deoxyfructose, N2-fructopyranosylarginine, 2-(4-methyl-5-thiazolyl)ethyl hexanoate, kenposide B, and medinoside E, were significantly positively correlated with alfalfa biomass (P < 0.01). This study combines the DSE extracellular metabolites with plant and soil traits to provide a theoretical basis for the use of DSE metabolites in the product development of plant biostimulants.     

Micropropagation of the medicinal plant, Scilla natalensis Planch.

Primary bulb explants of Scilla natalensis were cultured in vitro on modified MS medium. Some of these explants initiated shoots, which provided a sterile source of secondary leaf and bulb explants. The secondary explants responded similarly to various combinations of plant growth regulators. Shoots were initiated spontaneously on medium containing no plant growth regulators. The number of shoots initiated was increased by the addition of kinetin or thidiazuron (TDZ) alone, but was reduced by the addition of indole-3-acetic acid (IAA) or naphthalene acetic acid (NAA) alone. Optimal shoot initiation occurred on medium containing 1 to 2 mg l^–1 kinetin and 1 to 2 mg l^–1 IAA. These shoots were rooted on medium containing 1 mg l^–1 IAA. The plantlets were successfully acclimatised in the misthouse/shadehouse.     

Screening of free-living rhizospheric bacteria for their multiple plant growth promoting activities

Plant growth promoting rhizobacteria (PGPR) are known to influence plant growth by various direct or indirect mechanisms. In search of efficient PGPR strains with multiple activities, a total of 72 bacterial isolates belonging to Azotobacter, fluorescent Pseudomonas, Mesorhizobium and Bacillus were isolated from different rhizospheric soil and plant root nodules in the vicinity of Aligarh. These test isolates were biochemically characterized. These isolates were screened in vitro for their plant growth promoting traits like production of indoleacetic acid (IAA), ammonia (NH(3)), hydrogen cyanide (HCN), siderophore, phosphate solubilization and antifungal activity. More than 80% of the isolates of Azotobacter, fluorescent Pseudomonas and Mesorhizobium ciceri produced IAA, whereas only 20% of Bacillus isolates was IAA producer. Solubilization of phosphate was commonly detected in the isolates of Bacillus (80%) followed by Azotobacter (74.47%), Pseudomonas (55.56%) and Mesorhizobium (16.67%). All test isolates could produce ammonia but none of the isolates hydrolyzed chitin. Siderophore production and antifungal activity of these isolates except Mesorhizobium were exhibited by 10-12.77% isolates. HCN production was more common trait of Pseudomonas (88.89%) and Bacillus (50%). On the basis of multiple plant growth promoting activities, eleven bacterial isolates (seven Azotobacter, three Pseudomonas and one Bacillus) were evaluated for their quantitative IAA production, and broad-spectrum (active against three test fungi) antifungal activity. Almost at all concentration of tryptophan (50-500 microg/ml), IAA production was highest in the Pseudomonas followed by Azotobacter and Bacillus isolates. Azotobacter isolates (AZT(3), AZT(13), AZT(23)), Pseudomonas (Ps(5)) and Bacillus (B(1)) showed broad-spectrum antifungal activity on Muller-Hinton medium against Aspergillus, one or more species of Fusarium and Rhizoctonia bataticola. Further evaluation of the isolates exhibiting multiple plant growth promoting (PGP) traits on soil-plant system is needed to uncover their efficacy as effective PGPR.     

Metabolism of carbosulfan. I. Species differences in the in vitro biotransformation by mammalian hepatic microsomes including human

The in vitro metabolism of carbosulfan, a widely used carbamate insecticide, by hepatic microsomes from human, rat, mouse, dog, rabbit, minipig, and monkey was studied. Altogether eight (8) phase I metabolites were detected by LC–MS; phase II metabolites were not found in human homogenates fortified with appropriate cofactors. The primary metabolic pathways were the initial oxidation of sulfur to carbosulfan sulfinamide (‘sulfur oxidation pathway’) and the cleavage of the nitrogen sulfur bond (N–S) to give carbofuran and dibutylamine (‘carbofuran pathway’). Carbofuran was further hydroxylated to 3-hydroxycarbofuran and/or 7-phenolcarbofuran, which were further oxidized to 3-ketocarbofuran or 3-hydroxy-7-phenolcarbofuran, respectively, and finally to 3-keto-7-phenolcarbofuran. 3-Hydroxycarbofuran was the main metabolite in all species, but otherwise there were some qualitative interspecies differences in carbofuran pathway metabolites. Only rabbit liver microsomes were able to metabolize carbofuran via hydroxylation to 7-phenolcarbofuran. Carbofuran was not detected in dog liver microsomes due to rapid further metabolism. In general, liver microsomes from all seven species produced more toxic products (carbofuran, 3-hydroxy-carbofuran, 3-ketocarbofuran) more rapidly than a detoxification product (carbosulfan sulfinamide). Differences in intrinsic hepatic clearances (CL_int) between the lowest and highest species were moderate; 2-fold for the carbofuran pathway, 2.7-fold for carbosulfan sulfinamide and 6.2-fold for dibutylamine. Our studies, although restricted to in vitro metabolic data from human and animal hepatic preparations, provide valuable quantitative carbosulfan-specific data for risk assessment, which suggest that interspecies differences, for carbosulfan active chemical moiety, in toxicokinetics are within the standard applied factor for species extrapolation in toxicokinetics. These results will be valuable in further defining the risks associated with exposure to carbosulfan.     

Seasonal changes and metabolism of plant hormones in root nodules ofLens sp.

The mature nodules ofLens esculenta Moench. contained higher levels of indolyl acetic acid (IAA), cytokinins (CK), gibberellic acid (GA)-like substances and more active in nitrogenase (N₂-ase) activity than young or old ones. Synthesis of IAA and its metabolism was found to be controlled by tryptophan (tryp) and phenol metabolism, respectively, in nodules of different ages. An abscisic acid (ABA)-like substance being a ‘late growth phase’ hormone, was highest in old nodules. IAA and CK were highest in winter when N₂-ase activity was also highest but then GA and ABA were low. The IAA metabolic pattern of both roots and nodules was regulated by phenols. The hormones hardly changed seasonally in the roots and showed higher activities of IAA oxidase, MeOx reductase, peroxidase and polyphenol oxidase than nodules. The nodular anabolic changes are more pronounced in winter as lentil is a winter crop. The size of nodules at a particular age was the same in different seasons, even though their hormone content varied with the season showing that the nodular hormones were not solely utilized for nodule development and growth.     

Temperature-Sensitive Plant Cells with Shunted Indole-3-Acetic Acid Conjugation

Cells of henbane (Hyoscyamus muticus L.) grow indefinitely in culture without exogenous auxin. Cells of its temperature-sensitive variant XIIB2 grow like the wild type at 26[deg]C but die rapidly at 33[deg]C unless auxin is added to the medium. Despite this temperature-sensitive auxin auxotrophy, XIIB2 produces wild-type amounts of indole-3-acetic acid (IAA). IAA is the predominant auxin and is important for plant growth and development. Since the IAA production of the variant is functional, we investigated whether the synthesis or degradation of IAA metabolites, possibly active auxins themselves, is altered. The IAA metabolites were IAA-aspartate (IAAsp) and IAA-glucose. The wild type converted IAA mainly to IAAsp, whereas the variant produced mainly IAA-glucose. Exogenous auxin corrected the shunted IAA metabolism of the variant. The half-life of labeled IAAsp in the variant was reduced 21-fold, but in the presence of exogenous auxin it was not different from the wild type. The temperature sensitivity of XIIB2 was also corrected by supplying IAAsp. Pulse-chase experiments revealed that henbane rapidly metabolizes IAAsp to compounds not identical to IAA. The data show that the variant XIIB2 is a useful tool to study the function of IAA conjugates to challenge the popular hypothesis that IAA conjugates are merely slow-release storage forms of IAA.     

Auxin redistributes upwards in graviresponding gynophores of the peanut plant

The peanut (Arachis hypogaea L.) produces flowers aerially, but buries the recently fertilized ovules into the soil, where fruit and seed development occur. The young seeds are carried down into the soil at the tip of a specialized organ called the gynophore. Although the gynophore has a typical shoot anatomy, it responds positively to gravity like a root. In this study, we explore the role of the plant growth regulator indole-3-acetic acid (IAA) in the growth and the gravitropic response of the peanut gynophore. With an immunolocalization technique using an IAA monoclonal antibody, we localized IAA within the tissues of vertically oriented and gravistimulated gynophores. We found that in vertically oriented gynophores, IAA labeling occurs in the periphery of the gynophore, in the entire cortex and epidermis. Within 20 min of horizontal reorientation, the IAA signal gradually increases in the upper cortex/epidermis and diminishes in the lower cortex/epidermis. At 1.5 h after gravistimulation, all of the IAA immunolocalization signal is detected in the upper cortex and epidermis – none is detected in the lower side. Growth rate measurements also indicate that after 1–2 h of reorientation, the growth rate maximum on the upper side corresponds temporally and spatially to the growth rate minimum on the lower side. Experiments using radioactively labeled IAA corroborate an upper-side redistribution of this hormone upon horizontal reorientation. These results are analyzed with respect to the current theories of plant gravitropic response, and a model for a possible gravity-induced IAA redistribution from the lower to the upper side of the peanut gynophore is proposed. Received: 25 January 1999 / Accepted: 24 February 1999