Two glutathione S-transferase (GST) isozymes, A1/A1 and B1/B2, were purified from etiolated, O-1,3-dioxolan-2-yl-methyl-2,2,2,-trifluoro-4 -chloroacetophenone-oxime-treated sorghum (Sorghum bicolor L. Moench) shoots. GST A1/A1, a constitutively expressed homodimer, had a subunit molecular mass of 26 kD and an isoelectric point of 4.9. GST A1/A1 exhibited high activity with 1-chloro-2, 4,dinitrobenzene (CDNB) but low activity with the chloroacetanilide herbicide metolachlor. For GST A1/A1, the random, rapidequilibrium bireactant kinetic model provided a good description of the kinetic data for the substrates CDNB and glutathione (GSH). GST B1/B2 was a heterodimer with subunit molecular masses of 26 kD (designated the B1 subunit) and 28 kD (designated the B2 subunit) and a native isoelectric point of 4.8. GST B1/B2 exhibited low activity with CDNB and high activity with metolachlor as the substrate. The kinetics of GST B1/B2 activity with GSH and metolachlor fit a model describing a multisite enzyme having two binding sites with different affinities for these substrates. Both GST A1/A1 and GST B1/B2 exhibited GSH-conjugating activity with ethacrynic acid and GSH peroxidase activity with cumene hydroperoxide, 9-hydroperoxy-trans-10,cis-12-octadecadienoic acid and 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid. Both GST A1/A1 and GST B1/B2 are glycoproteins, as indicated by their binding of concanavalin A. Polyclonal antibodies raised against GST A1/A1 exhibited cross-reactivity with the B1 subunit of GST B1/B2. Comparisons of the N-terminal amino acid sequences of the GST A1, B1, and B2 subunits with other type I -GSTs indicated a high degree of homology with the maize GST I subunit and a sugarcane GST.GSTs (EC 2.5.1.18) are dimeric enzymes found in mammals, insects, plants, and microbes that catalyze nucleophilic attack by the thiolate anion of GSH at electrophilic centers of hydrophobic molecules (Mannervik and Danielson, 1988). In addition to catalyzing GSH conjugation, GSTs also exhibit GSH peroxidase activity and ligandbinding functions (Mannervik and Danielson, 1988;Marrs, 1996). Mammalian GSTs compose a multigene family; in rat liver at least 13 different cytosolic GST subunits are found as either heterodimers or homodimers . Mammalian cytosolic GSTs have been divided into four classes (␣, , , and ) based on immunological, biochemical, and sequence similarities (Buetler and Eaton, 1992). It is well established that mammalian GSTs play an important role in the detoxification of electrophilic xenobiotics (Mannervik and Danielson, 1988). Although endogenous substrates for mammalian GSTs have not been clearly defined, there is evidence that ␣-GSTs protect against oxidative stress by detoxifying reactive products generated by lipid peroxidation (Ålin et al., 1985;Ketterer and Coles, 1991;Singhal et al., 1992).In general, plant GSTs have not been as well characterized as mammalian GSTs. Plant cytosolic GSTs belong to the archaic class of GSTs (Meyer et al., 1991;Marrs, 1996). This class, which is very heterog...

No abstract

Growth chamber and field experiments were conducted to assess the potential of Pseudomonas syringae pv. tagetis (Pst) as a biocontrol agent for Canada thistle. Silwet L-77, an organosilicone surfactant, was required to facilitate Pst penetration into Canada thistle leaves. Growth chamber experiments indicated that maximum Pst populations inside leaves were obtained with a Silwet L-77 concentration of 0.3% (v/v) or greater. High Pst populations (109 colony-forming units [cfu] per gram fresh weight) were found in leaves 48 h after treatment with 108 or 109 cfu ml−1 Pst plus Silwet L-77 (0.3%, v/v). In growth chamber experiments, foliar application of Pst (109 cfu ml−1) plus Silwet L-77 (0.3%, v/v) on 4- to 5-wk-old Canada thistle reduced shoot dry weight by 52% (measured 14 d after treatment) and chlorophyll content of emerging leaves by 92% (measured 10 d after treatment). In field trials conducted in 1999 and 2000, Pst (109 cfu ml−1) plus Silwet L-77 (0.3%, v/v) were applied at 700 L ha−1, and the method of application (paint gun, backpack sprayer, boom) and the number of applications (one or two separated by 14 d) were examined. Averaged over 2 yr, two applications with a backpack sprayer resulted in 67% disease incidence (apical chlorosis) of treated plants measured 4 wk after the initial treatment (WAIT). At the time of flower bud formation (8 WAIT), there was little or no disease incidence, 31% reduction in plant height, 81% reduction in number of flower buds, and 20% reduction in shoot survival during 1999 but no effect on survival in 2000.

During soybean [Glycine max (L.) Merrill] embryo development, cell wall polysaccharides (CWPs) derived from UDP-glucuronic acid (UDP-GlcA) (uronic acids, arabinose, xylose) exhibited a linear increase during the period of 25-45 days after flowering (daf). At embryo maturity, CWPs derived from UDP-GlcA accounted for 39% of total CWPs. To ascertain the relative importance of the nucleotide sugar oxidation (NSO) and the myo-inositol oxidation (MIO) pathways to UDP-GlcA biosynthesis, UDP-glucose (UDPGlc) dehydrogenase (UDP-Glc DH, EC 1.1.1.22) and UDP-glucuronic acid pyrophosphorylase (UDP-GlcA PPase, EC 2.7.7.44) activities, respectively, were measured in desalted extracts of developing embryos. UDP-Glc DH and UDP-GlcA PPase activities, expressed on a per seed basis, increased 3.5-and 3.9-fold, respectively, during the period of 25-45 daf. However, UDP-GlcA PPase activity was 35-50-fold greater than UDP-Glc DH activity. The soybean UDP-sugar pyrophosphorylase gene (USP1), a homolog of pea USP, and a candidate gene for UDP-GlcA PPase, was cloned and the recombinant enzyme characterized. Recombinant soybean USP1 (71 kDa) exhibited high activity with glucuronic acid 1-phosphate (GlcA-1-P), glucose 1-phosphate (Glc-1-P) and galactose 1-phosphate (Gal-1-P), but low activity with mannose 1-phosphate (Man-1-P), N-acetylglucosamine 1-phosphate and Glc-6-P. Determination of kinetic constants indicated that USP1 has a higher affinity for GlcA-1-P (K m ¼ 0.14 AE 0.02 mM) than for Glc-1-P (K m ¼ 0.23 AE 0.02 mM). Semiquantitative RT-PCR was used to measure transcript levels of the UDPglucose DH (UGD) and USP gene families in developing soybean embryos. Transcript levels, normalized to the 18S rRNA controls, were greater for UGD than USP throughout embryo development. The possibility that USP serves as UDP-GlcA PPase, the terminal enzyme of the MIO pathway, is discussed.

The effects of the fungal protein Nep1 and Pseudomonas syringae pv. tagetis (Pst) applied separately or in combination on Canada thistle, common ragweed, and common dandelion were examined in growth chamber experiments. Experiments examined five treatments: (1) untreated control, (2) Silwet L-77 (0.3%, v/v) control, (3) Nep1 (5 μg ml−1) plus Silwet L-77 (0.3%, v/v), (4) Pst (109 colony-forming units [cfu] ml−1) plus Silwet L-77 (0.3%, v/v), and (5) Pst (109 cfu ml−1) and Nep1 (5 μg ml−1) plus Silwet L-77 (0.3%, v/v). Foliar treatments were applied at 28, 26, and 21 d after planting for Canada thistle, common dandelion, and common ragweed, respectively. For all three species, foliar application of Nep1 alone or in combination with Pst caused rapid desiccation and necrosis of leaves, with the greatest effect on recent, fully expanded (RFE) leaves. Within 4 to 8 h after treatment (HAT), 60 to 80% of RFE leaves of all three species were necrotic. Measured 72 HAT, Pst populations in Canada thistle leaves treated with Nep1 plus Pst were approximately 105 cfu cm−2 compared with 107 cfu cm−2 for leaves treated with Pst alone. Measured 2 wk after treatment, foliar application of Nep1 reduced shoot dry weight of the three weeds by 30 to 41%. Treatment with Pst reduced shoot growth of common ragweed, Canada thistle, and common dandelion by 82, 31, and 41%, respectively. The large suppression of common ragweed shoot growth caused by Pst treatment was associated with a high percentage (60%) of leaf area exhibiting chlorosis. Treatment with Pst plus Nep1 did not result in significant decreases in shoot dry weight for Canada thistle and common dandelion compared with either treatment alone. For common ragweed, shoot growth reduction caused by applying Pst and Nep1 together was not greater than that caused by Pst alone.