Arabidopsis thaliana contains two salicylic acid (SA) glucosyltransferase enzymes designated UGT74F1 and UGT74F2. UGT74F1 forms only SA 2-O-beta-D-glucose (SAG), while UGT74F2 forms both SAG and the SA glucose ester (SGE). In an attempt to determine the in vivo role of each SA glucosyltransferase (SAGT), the metabolism of SA in ugt74f1 and ugt74f2 mutants was examined and compared with that of the wild-type. The three major metabolites formed in wild-type Arabidopsis included SAG, SGE, and 2,5-dihydroxbenzoic acid 2-O-beta-D-glucose (DHB2G). This is the first description of DHB2G as a major metabolite of SA in plants. The major metabolites of SA formed in ugt74f1 mutants were SGE, SAG and 2,5-dihydroxybenzoic acid 5-O-beta-D-glucose (DHB5G). DHB5G was not formed in the wild-type plants. SAG and DHB2G were the main metabolites of SA in ugt74f2 mutants. The ugt74f2 mutant was unable to form SGE. Only SGE could be detected during in vitro SAGT assays of untreated wild-type and ugt74f1 mutants. This activity was because of constitutive UGT74F2 activity. Both SGE and SAG could be formed during in vitro assays of SA-pretreated wild-type and ugt74f1 leaves. Neither SAG nor SGE could be detected during the in vitro SAGT assays of untreated ugt74f2 leaves. Only SAG was formed during the in vitro SAGT assays of SA-pretreated ugt74f2 leaves. The SAG formation was a result of the UGT74F1 activity. This work demonstrates that changes in the activity of either SAGT enzyme can have a dramatic effect on the metabolism of exogenously supplied SA in Arabidopsis.

The metabolism of salicylic acid (SA) in tobacco (Nicotiana tabacum L. cv. KY 14) cell suspension cultures was examined by adding [7-14C]SA to the cell cultures for 24 h and identifying the metabolites through high performance liquid chromatography analysis. The three major metabolites of SA were SA 2-O-beta-D: -glucose (SAG), methylsalicylate 2-O-beta-D: -glucose (MeSAG) and methylsalicylate. Studies on the intracellular localization of the metabolites revealed that all of the SAG associated with tobacco protoplasts was localized in the vacuole. However, the majority of the MeSAG was located outside the vacuole. The tobacco cells contained an SA inducible SA glucosyltransferase (SAGT) enzyme that formed SAG. The SAGT enzyme was not associated with the vacuole and appeared to be a cytoplasmic enzyme. The vacuolar transport of SAG was characterized by measuring the uptake of [14C]SAG into tonoplast vesicles isolated from tobacco cell cultures. SAG uptake was stimulated eightfold by the addition of MgATP. The ATP-dependent uptake of SAG was inhibited by bafilomycin A1 (a specific inhibitor of the vacuolar H(+)-ATPase) and dissipation of the transtonoplast H(+)-electrochemical gradient. Vanadate was not an inhibitor of SAG uptake. Several beta-glucose conjugates were strong inhibitors of SAG uptake, whereas glutathione and glucuronide conjugates were only marginally inhibitory. The SAG uptake exhibited Michaelis-Menten type saturation kinetics with a K(m) and V(max) value of 11 microM and 205 pmol min-1 mg-1, respectively, for SAG. Based on the transport characteristics it appears as if the vacuolar uptake of SAG in tobacco cells occurs through an H(+)-antiport-type mechanism.

Flavonoids have important developmental, physiological, and ecological roles in plants and are primarily stored in the large central vacuole. Here we show that both an ATP-binding cassette (ABC) transporter(s) and an H+-antiporter(s) are involved in the uptake of cyanidin 3-O-glucoside (C3G) by Arabidopsis vacuolar membrane-enriched vesicles. We also demonstrate that vesicles isolated from yeast expressing the ABC protein AtABCC2 are capable of MgATP-dependent uptake of C3G and other anthocyanins. The uptake of C3G by AtABCC2 depended on the co-transport of glutathione (GSH). C3G was not altered during transport and a GSH conjugate was not formed. Vesicles from yeast expressing AtABCC2 also transported flavone and flavonol glucosides. We performed ligand docking studies to a homology model of AtABCC2 and probed the putative binding sites of C3G and GSH through site-directed mutagenesis and functional studies. These studies identified residues important for substrate recognition and transport activity in AtABCC2, and suggest that C3G and GSH bind closely, mutually enhancing each other’s binding. In conclusion, we suggest that AtABCC2 along with possibly other ABCC proteins are involved in the vacuolar transport of anthocyanins and other flavonoids in the vegetative tissue of Arabidopsis.

A two-step purification protocol was used in an attempt to separate the constitutive NAD(P)H-nitrate reductase INAD(P)H-NR, pH 6.5; EC 1.6.6.21 activity from the nitric oxide and nitrogen dioxide (NO(X)) evolution activity extracted from soybean (Glycine max [L.] Merr.) leaflets.Both of these activities were eluted with NADPH from Blue Sepharose columns loaded with extracts from either wild-type or LNR-5 and LNR-6 (lack constitutive NADH-NR [pH 6.51) mutant soybean plants regardless of nutrient growth conditions. Fast protein liquid chromatographyanion exchange (Mono Q column) chromatography following Blue Sepharose affinity chromatography was also unable to separate the two activities. These data provide strong evidence that the constitutive NAD(P)H-NR (pH 6.5) in soybean is the enzyme responsible for NO(X) formation. The Blue Sepharose-purified soybean enzyme has a pH optimum of 6.75, an apparent K. for nitrite of 0.49 millimolar, and an apparent K. for NADPH and NADH of 7.2 and 7.4 micromolar, respectively, for the NO(X) evolution activity. In addition to NAD(P)H, reduced flavin mononucleotide (FMNH2) and reduced methyl viologen (MV) can serve as electron donors for NO(X) evolution activity. The NADPH-, FMNH2-, and reduced MV-NO(,) evolution activities were all inhibited by cyanide. The NADPH activity was also inhibited by p-hydroxymercuribenzoate, whereas, the FMNH2 and MV activities were relatively insensitive to inhibition. These data indicate that the terminal molybdenum-containing portion of the enzyme is involved in the reduction of nitrite to NO(X). NADPH eluted both NR and NO(X) evolution activities from Blue Sepharose columns loaded with extracts of either nitrate-or zero N-grown winged bean (Psophocarpus tetragonolobus [L.]), whereas NADH did not elute either type of activity. Winged bean appears to contain only one type of NR enzyme that is similar to the constitutive NAD(P)H-NR (pH 6.5) enzyme of soybean.It has been shown that the predominant compound evolved from soybean leaves during the purged in vivo NR3 assay is nitric oxide (NO) with trace amounts of nitrous oxide (N20) and 'Supported by an American Soybean Association research grant, project number 84953.

This study was conducted to determine by gas chromatography (GC) and mass spectrometry (MS) the identity and the quantity of volatile N products produced during the helium-purged in vivo NR assay of soybean (Glycine max [L.] Merr. cv Williams) and winged bean (Psophocarpus tetragonolobus [L.] DC. cv Lunita) leaflets. Gaseous material for identification and quantitation was collected by cryogenic trapping of volatile compounds carried in the He-purge gas stream. As opposed to an earlier report, acetaldehyde oxime production was not detected by our GC method, and acetaldehyde oxime was shown to be much more soluble in water than the compound(s) evolved from soybean leaflets. Nitric oxide (NO) and nitrous oxide (N20) were identified by GC and GC/MS as the main N products formed. NO and N20 produced from soybean leaflets were both labeled with 5N when "5N-nitrate was used in the assay medium, demonstrating that both were produced from nitrate during nitrate reduction. Other compounds co-trapped with NO and N20 were identified as air (N2, 02), C02, methanol, acetaldehyde, and ethanol. Leaves of winged bean, subjected to the purged in vivo NR assay, evolved greater quantities of NO and N20 (13.9 and 0.37 micromole per gram fresh weight per 30 minutes, respectively) than did the soybean cv Williams (1.67 and 0.09 micromole per gram fresh weight per 30 minutes, respectively). In both species NO production was dominant. In contrast, with similar assays, NO and N20 were not evolved from leaves of the nrl soybean mutant which lacks the constitutive NR enzymes. In addition to soybean cv Williams, six other Glycine sp. examined evolved significant quantities of NO(x) (NO and NO2). Other species including Neonotonia wightii (Arn.) Lackey comb. nov., Pueraria montana (Lour.) Meff., and Pueraria thunbergiana Benth. evolved lower levels of NO(,).Klepper (5) demonstrated that herbicide treated soybean leaves form and release NO(X3 (thought to be predominately NO). He suggested that NO and NO2 were formed by a chemical reaction of NO2-(accumulated due to herbicide treatments) with plant metabolites, forming NO at low NO2-levels and NO and NO2 at higher NO2 levels. It was later shown (4) that N2 gas purging during the in vivo NR assay of soybean leaflets also resulted in the formation of NO(X) derived from NO2-accumulated during the assay. Harper's work (4) also indicated that the predominate ' Supported by an American Soybean Association research grant, project number 84953.2 Grateful recipient of a Wright Fellowship from the College of Agriculture, University of Illinois.3 Abbreviations: NO(X), refers collectively to nitric oxide (NO) and nitrogen dioxide (NO2); NR, nitrate reductase; DAP, days after planting; N20, nitrous oxide; m/z, mass to charge ratio; bp, boiling point. compound evolved was NO, but based on results with boiled leaf discs he implied that an enzymic reaction was responsible for the NO(x) evolution. Additional evidence for an enzymic reaction was provided by the isolation of a mutant soybean line (nr...

The metabolism and intracellular localization of salicylic acid (SA) was investigated in soybean (Glycine max[L.] cv Williams 82) cell suspension cultures. [7–14C]SA was added to the cell cultures, the metabolites were extracted from the cells at various time points and analysed by TLC and HPLC. The [7–14C]SA was taken up rapidly from the culture media and converted primarily to SA 2‐O‐β‐d‐glucose (SAG). Lower levels of glucosylated 2,5‐dihydroxbenzoic acid (gentisic acid) and methyl salicylate 2‐O‐β‐d‐glucose were also formed. Examination of the intracellular localization of the glucose conjugates revealed that all of the conjugates associated with the protoplasts were found in the vacuoles. An SA glucosyltransferase (SAGT) that could catalyse the formation of SAG from SA and UDP‐glucose could be extracted from soybean cells and assayed in vitro. Increasing concentrations of SA added to the culture media induced the SAGT activity. The highest levels of SAGT activity were observed in cells treated with 0.5 mM SA. The SAGT activity in these cells was 88‐fold greater than the SAGT activity in the untreated cells. The intracellular localization of the SAGT activity was also examined and it was determined that the majority of the SAGT activity in the protoplasts was located outside the vacuole. Therefore, it appears as if SAG is formed from SA outside the vacuole, presumably in the cytoplasm, and then subsequently transported into the vacuole where it accumulates.

Salicylic acid (SA) is a signaling molecule utilized by plants in response to various stresses. Through conjugation with small organic molecules such as glucose, an inactive form of SA is generated which can be transported into and stored in plant vacuoles. In the model organism Arabidopsis thaliana, SA glucose conjugates are formed by two homologous enzymes (UGT74F1 and UGT74F2) that transfer glucose from UDP-glucose to SA. Despite being 77% identical and with conserved active site residues, these enzymes catalyze the formation of different products: UGT74F1 forms salicylic acid glucoside (SAG), while UGT74F2 forms primarily salicylic acid glucose ester (SGE). The position of the glucose on the aglycone determines how SA is stored, further metabolized, and contributes to a defense response. We determined the crystal structures of the UGT74F2 wild-type and T15S mutant enzymes, in different substrate/product complexes. On the basis of the crystal structures and the effect on enzyme activity of mutations in the SA binding site, we propose the catalytic mechanism of SGE and SAG formation and that SA binds to the active site in two conformations, with each enzyme selecting a certain binding mode of SA. Additionally, we show that two threonines are key determinants of product specificity.

In soybean (Glycine max L.), salicylic acid (SA) is converted primarily to SA 2-O-beta-d-glucose (SAG) in the cytoplasm and then accumulates exclusively in the vacuole. However, the mechanism involved in the vacuolar transport of SAG has not been investigated. The vacuolar transport of SAG was characterized by measuring the uptake of [(14)C]SAG into tonoplast vesicles isolated from etiolated soybean hypocotyls. The uptake of SAG was stimulated about six-fold when MgATP was included in the assay media. In contrast, the uptake of SA was only stimulated 1.25-fold by the addition of MgATP and was 2.2-fold less than the uptake of SAG providing an indication that the vacuolar uptake of SA is promoted by glucosylation. The ATP-dependent uptake of SAG was inhibited by increasing concentrations of vanadate (64% inhibition in the presence of 500 microM) but was not very sensitive to inhibition by bafilomycin A(1) (a specific inhibitor of vacuolar H(+)-ATPase; EC 3.6.1.3), and dissipation of the transtonoplast H(+)-electrochemical gradient. The SAG uptake exhibited Michaelis-Menten-type saturation kinetics with a K(m) value of 90 microM for SAG. SAG uptake was inhibited 60% by beta-estradiol 17-(beta-d-glucuronide), but glutathione conjugates and uncharged glucose conjugates were only slightly inhibitory. Based on the characteristics of SAG uptake into soybean tonoplast vesicles it is likely that this uptake occurs through an ATP-binding cassette transporter-type mechanism. However, this vacuolar uptake mechanism is not universal since the uptake of SAG by red beet (Beta vulgaris L) tonoplast vesicles appears to involve an H(+)-antiport mechanism.