Absorption, translocation, and metabolism of14C-clomazone in soybean (Glycine max) and threeAmaranthus weed species

The patterns of clomazone (2-[(2-chlorophenyl) methyl-4,4-dimethyl-3-isoxazolidinone) absorption, translocation, and metabolism and their contribution to the plant selectivity of this herbicide were studied in tolerant soybean [Glycine max (L.) Merr.] andAmaranthus hybridus and in susceptibleA. retroflexus andA. lividus. Differential root absorption appeared to play a significant role in the differential response of these four plant species to clomazone. Absorption of root-applied¹⁴C-clomazone was greater by the two sensitiveAmaranthus weeds than by the tolerant soybean andA. hybri-dus. Following application of¹⁴C-clomazone to roots, most of the absorbed radioactivity was translocated to the leaves of all four species. Approximately 50% of the absorbed¹⁴C-clomazone was metabolized by all four plant species as early as 12 h after treatment. Thin layer Chromatographic (TLC) analysis of plant tissue extracts from all four species revealed the formation of two major metabolites of clomazone. These unidentified metabolites had Rf values of 0.4 and 0.8, respectively, in a butanolacetic acidwater (1235, vol/vol/vol) developing system. The Rf value of unaltered clomazone in this system was 0.95. Differential metabolism or differential rate of metabolism of clomazone was not observed in this study and did not seem to account for the tolerance of soybean andA. hybridus or the suceptibility ofA. retroflexus andA. lividus to this herbicide.Plant Pathology, Physiology, and Weed Science Department, Contribution No. 600.     

Effects of sethoxydim on the metabolism of isolated leaf cells of soybean [Glycine max (L.) Merr.]

The effects of the herbicide sethoxydim {2-[1-ethoxyimino)-butyl]-5-[2-(ethylthio)-propyl]-3-hydroxy-2-cyclohexen-1-one} on selected metabolic processes of enzymatically isolated leaf cells from soybeans [Glycine max (L.) Merr., cv. Essex] were studied. Photosynthesis, protein, ribonucleic acid (RNA), and lipid synthesis were measured by the incorporation of NaH¹⁴CO₃, [¹⁴C]leucine, [¹⁴C]uracil, and [¹⁴C]acetic acid into the isolated soybean cells, respectively. Time-course and concentration studies included incubation times of 30, 60, and 120 min and concentrations of 0.1, 1, 10, and 100 M of sethoxydim. Lipid synthesis was the most sensitive and first metabolic process inhibited by the lowest concentration of sethoxydim. Photosynthesis was not affected significantly by sethoxydim and did not appear to be a target site involved in its herbicidal action. RNA and protein syntheses were inhibited significantly but only by the high concentrations of sethoxydim. It is suggested that sethoxydim exhibits its phytotoxic action by altering or modifying the lipid composition of plant membranes.Abbreviations MES [2-(N-morpholino)-ethanesulfonic acid] - HEPES [N-2-hydroxyethyl piperazine-N-2-ethane sulfonic acid]     

Inhibition of photosynthetic electron transport in isolated spinach chloroplasts by two 1,3,4-thiadiazolyl derivatives

Buthidazole (3-[5-(1,1-dimethylethyl)-1,3,4-thiadiazol-2-yl]-4-hydroxy-1-methyl-2-imidazolidinone) and tebuthiuron (N-[5-(1,1-dimethylethyl)-1,3,4-thiadiazol-2-yl]-N,N′-dimethylurea) are two new promising herbicides for selective weed control in corn (Zea mays L.) and sugarcane (Saccharum officinarum L.), respectively. The effects of these two compounds on various photochemical reactions of isolated spinach (Spinacia oleracea L.) chloroplasts were studied at concentrations of 0, 0.05, 0.5, 5, and 500 micromolar. Buthidazole and tebuthiuron at concentrations higher than 0.5 micromolar inhibited uncoupled electron transport from water to ferricyanide or to methyl viologen very strongly. Photosystem II-mediated transfer of electrons from water to oxidized diamonodurene, with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) blocking photosystem I, was inhibited 34 and 37% by buthidazole and tebuthiuron, respectively, at 0.05 micromolar. Inhibition of photosystem I-mediated transfer of electrons from diaminodurene to methyl viologen with 3,4-dichlorophenyl-1,1-dimethylurea (DCMU) blocking photosystem II was insignificant with either herbicide at all concentrations tested. Transfer of electrons from catechol to methyl viologen in hydroxylamine-washed chloroplasts was inhibited 50 and 47% by buthidazole and tebuthiuron, respectively, at 0.5 micromolar. The data indicate that the inhibition of electron transport by both herbicides is primarily at the reducing side of photosystem II. However, since catechol is an electron donor at the oxidizing side of photosystem II, between water and chlorophyll a₆₈₀, and lower inhibition levels were observed in the last study (catechol to methyl viologen), it may be that there is also a small inhibition of the mechanism of water oxidation by both herbicides.     

Expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in maize tissues

The activity of the enzyme 3-hydroxy-3-methlglutaryl-coenzyme A reductase (HMGR, EC 1.1.1.34) is highly expressed in 4-day-old etiolated seedlings of normal (cv. DeKalb XL72AA), dwarf ( d ₅) and albino ( lw ₃) maize ( Zea mays L.). HMGR activity of maize seedlings appeared to be exclusively associated with the microsomal rather than the plastidic fraction of maize cells. Maize tissues with high meristematic activity such as germinating seeds, leaf bases, root tips and the site of origin of lateral roots contained high levels of microsomal HMGR activity. The activity of HMGR extracted from leaf tips of normal, dwarf and albino maize seedlings is regulated by light. Microsomal HMGR activity from leaf tips of 4-day-old maize seedlings was inhibited significantly following exposure to strong light (600 μmol m⁻² s⁻¹) for more than 10 h. By comparison, microsomal HMGR activity from leaf bases and root tips of maize was not inhibited by exposure to strong light. These results suggest that the microsomal HMGR which is highly expressed in maize may be related to sterol biosynthesis and membrane biogenesis rather than plastidic-associated isoprenoid synthesis and that light may regulate HMGR activity indirectly by increasing cell differentiation.     

Inhibition of the photosynthetic electron transport of isolated thylakoids by hemolyzed rabbit sera. Evidence for the potential involvement of parallel electron transport in photosystem I Mehler reactions

The inhibition patterns of rabbit sera (RS1 & RS2) from two different rabbits on the photosynthetic electron transport of isolated spinach thylakoids were studied. Fifty l of RSI were required for 100% inhibition of a H₂O MV/O₂ reaction, while only 10 l of a 1:10 dilution of RS2 were needed for 100% inhibition. The RS2 serum was greatly hemolyzed. The -globulin fraction from purified rabbit serum (RS1) did not inhibit photosynthetic electron transport, indicating that the antibody fraction of the rabbit serum does not contain the inhibitor. It appears that the inhibitor is from the hemolyzed red blood cells. Rabbit sera added prior to chloroplast illumination caused no inhibition, while addition of rabbit sera during illumination inhibited a H₂O MV/O₂ reaction within 1–3s. Aminotriazole, a catalase inhibitor, did not affect the efficacy of the rabbit sera indicating that the unknown rabbit serum inhibitor is not catalase. Various Hill reactions were employed to determine the site of inhibition. Rabbit sera inhibited the following reactions: DHQ/DCMU MV/O₂, DAD/Asc/DBMIB MV/O₂, and DCIP/Asc/DBMIB MV/O₂. Rabbit sera did not inhibit a H₂O DAD_ox reaction indicating that inhibition is on the reducing side of PSI. However, a H₂O Fd/NADP⁺ reaction was not inhibited by rabbit sera. NADP did not interfere with the ability of RS2 to inhibit a MV-mediated Mehler reaction. In simultaneously measured assays of Fd-mediated O₂ and NADP⁺ reductions, RS2 serum inhibited the reduction of O₂ by ferredoxin without inhibiting the reduction of NADP⁺. These results indicate the potential involvement of parallel (branched) electron transport of the reducing side of PSI in the reduction of oxygen.Abbreviations RS1 and RS2 Rabbit serum 1 and 2 - MV methylviologen - DCMU 3,4-dichlorophenyl-N,N-dimethylurea - KFeCN potassium ferricyanide - DCIP dichlorophenolindolphenol - DAD 2,3,5,6-tetramethyl-p-phenylenediamine - DHQ tetramethyl-p-hydroquinone (durohydroquinone) - MES [2-(N-morpholino)-esthanesulfonic acid] - HEPES [N-2-hydroxyethyl piperazine-N-2-ethanesulfonic acid] - DBMIB dibromothymoquinone - PSI and PSII photosystem I and II - Fd ferredoxin - Chl chlorophyll - Asc ascorbate - SOD superoxide dismutase     

Comparative effects of CGA-92194, cyometrinil,and flurazole on selected metabolic processes of isolated soybean leaf cells

The potential adverse phytotoxic effects of the herbicide safeners CGA-92194 {-[1,3-dioxolan-2-yl-methoxy)imino]benzeneacetonitrile}, cyometrinil [-(cyanomethoxy)imino-benzeneacetonitrile] and flurazole [phenylmethyl 2-chloro-4-(trifluoromethyl)-5-thiazole-carboxylate] on selected metabolic processes of enzymatically isolated leaf cells of soybean [Glycine max (L.) Merr.] were compared in time- and concentration-course studies. CO₂ fixation, protein synthesis, RNA synthesis, DNA synthesis, and lipid synthesis were assayed by the incorporation of NaH¹⁴CO₃, [¹⁴C]-leucine, [¹⁴C]-uracil, [³H]thymidine, and [¹⁴C]-acetate, respectively, into the isolated cells. CGA-92194 and cyometrinil behaved similarly, and at low concentrations (0.1, 1, and 10 M) they stimulated rather than inhibited the five metabolic processes assayed, following incubation periods of up to 2 h. At the highest concentration of 100 M, both safeners inhibited all metabolic processes of the soybean leaf cells but neither compound exhibited rapid and distinct inhibitions as might be expected in the case of inhibition of a primary target site by a potent inhibitor. At low concentrations and early incubation periods (30 and 60 min), flurazole effects on all metabolic processes were also stimulatory rather than inhibitory. However, the stimulation of CO₂ fixation by 0.1 and 1.0 M was highly significant. At 100 M flurazole was extremely potent on all metabolic processes of soybean leaf cells examined. At the 2-h incubation period, flurazole also inhibited all metabolic processes at concentrations lower than 100 M. The sensitivity of the five metabolic processes to flurazole decreased in the following order: photosynthesis = lipid synthesis >DNA synthesis>protein synthesis>RNA synthesis.Contribution No. 534, Department of Plant Pathology, Physiology and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg VA 24061 USA.     

PapA3 Is an Acyltransferase Required for Polyacyltrehalose Biosynthesis in Mycobacterium tuberculosis

Mycobacterium tuberculosis possesses an unusual cell wall that is replete with virulence-enhancing lipids. One cell wall molecule unique to pathogenic M. tuberculosis is polyacyltrehalose (PAT), a pentaacylated, trehalose-based glycolipid. Little is known about the biosynthesis of PAT, although its biosynthetic gene cluster has been identified and found to resemble that of the better studied M. tuberculosis cell wall component sulfolipid-1. In this study, we sought to elucidate the function of papA3, a gene from the PAT locus encoding a putative acyltransferase. To determine whether PapA3 participates in PAT assembly, we expressed the protein heterologously and evaluated its acyltransferase activity in vitro. The purified enzyme catalyzed the sequential esterification of trehalose with two palmitoyl groups, generating a diacylated product similar to the 2,3-diacyltrehalose glycolipids of M. tuberculosis. Notably, PapA3 was selective for trehalose; no activity was observed with other structurally related disaccharides. Disruption of the papA3 gene from M. tuberculosis resulted in the loss of PAT from bacterial lipid extracts. Complementation of the mutant strain restored PAT production, demonstrating that PapA3 is essential for the biosynthesis of this glycolipid in vivo. Furthermore, we determined that the PAT biosynthetic machinery has no cross-talk with that for sulfolipid-1 despite their related structures.Mycobacterium tuberculosis, the bacterium that causes tuberculosis in humans, has a complex cell wall that contains a number of unique glycolipids intimately linked to mycobacterial pathogenesis (1, 2). The biosynthesis of many of these virulence factors, including the trehalose mycolates, phenolic glycolipids, and sulfolipid-1 (SL-1),³ is largely understood (3–5). In contrast, relatively little is known about the biosynthesis of other prominent M. tuberculosis glycolipids, such as di-, tri-, and polyacyltrehaloses. These acyltrehaloses are located in the outer surface of the cell wall and contain di- and tri-methyl branched fatty acids that are only found in pathogenic species of mycobacteria (6, 7). Previous studies suggest a role for these glycolipids in anchoring the bacterial capsule, which impedes phagocytosis by host cells (6).The major polyacyltrehalose (PAT) of M. tuberculosis, also referred to as pentaacyl or polyphthienoyl trehalose, consists of five acyl chains, four mycolipenic (phthienoic) acids and one fully saturated fatty acid, linked to trehalose (Fig. 1A) (8). The mycolipenic acid side chains of PAT are products of the polyketide synthase gene pks3/4 (7). Disruption of pks3/4 (also referred to as msl3 (7)) abolishes PAT biosynthesis and causes cell aggregation. At present, the remaining proteins required for PAT assembly have not been characterized.Open in a separate windowFIGURE 1.PAT and SL-1 share related structures and biosynthetic gene clusters. A, structure of PAT. B, structure of SL-1. C, genomic arrangement of the PAT and SL-1 biosynthetic gene clusters.Interestingly, the PAT biosynthetic gene cluster strongly resembles that of SL-1, which is a structurally similar trehalose-based glycolipid unique to pathogenic mycobacteria (Fig. 1B) (9). Both gene clusters contain polyketide synthase (pks), acyltransferase (pap), and lipid transport (mmpL) genes in a similar genomic arrangement (Fig. 1C). The SL-1 locus encodes two acyltransferase genes, papA1 and papA2, which are required for SL-1 biosynthesis (5, 10). These proteins belong to the mycobacterium-specific polyketide-associated protein (Pap) family of acyltransferases, which share a conserved HX₃DX₁₄Y motif that is required for activity (11). The PapA2 enzyme catalyzes the esterification of the 2′-position of trehalose 2-sulfate with a saturated fatty acid. PapA1 mediates the subsequent esterification of this intermediate with a hydroxyphthioceranoyl group produced by Pks2 (5). Interestingly, the PAT locus contains a gene, Rv1182, that is homologous to both papA1 and papA2 (55 and 53% amino acid identity, respectively). This gene is annotated as papA3 in the genome and was previously shown to encode a protein bearing the signature Pap motif (11).Here we demonstrate that papA3 encodes an acyltransferase essential for the biosynthesis of PAT. Deletion of the papA3 gene resulted in loss of the glycolipid from M. tuberculosis lipid extracts, as determined by high resolution mass spectrometry. Moreover, the purified enzyme was shown to selectively and sequentially acylate trehalose in vitro, generating a diacylated product similar to the 2,3-diacyltrehaloses of M. tuberculosis. Together, these data confirm that PapA3 plays a crucial role in PAT biosynthesis and highlight its potential involvement in the biosynthesis of related M. tuberculosis acyltrehaloses.     

The Mycobacterium tuberculosis CysQ phosphatase modulates the biosynthesis of sulfated glycolipids and bacterial growth

CysQ is a 3'-phosphoadenosine-5'-phosphatase that dephosphorylates intermediates from the sulfate assimilation pathway of Mycobacterium tuberculosis (Mtb). Here, we demonstrate that cysQ disruption attenuates Mtb growth in vitro and decreases the biosynthesis of sulfated glycolipids but not major thiols, suggesting that the encoded enzyme specifically regulates mycobacterial sulfation.