Formation of sulfatoglucosides from exogenous aldoximes in plant cell cultures and organs

Plant Science, 66 (1990) 11-20

11

Elsevier Scientific Publishers Ireland Ltd.

FORMATION OF SULFATOGLUCOSIDES PLANT CELL CULTURES AND ORGANS

FROM

EXOGENOUS

ALDOXIMES

IN

J.W.D. G R O O T W A S S I N K , J.J. B A L S E V I C H and A.D. K O L E N O V S K Y

Plant Biotechnology Institute, National Research Counc~ Saskatoon~ Saskatchewan, S7N OW9 (CanadaJ

(ReceivedApril 3rd, 1989) (Revision received August 4th, 1989) (Accepted August 18th, 1989) Feeding of 2-nitrobenzaldoxime to Brasswa species led to the accumulatmn of the artificial 2-nltrophenylglucoslnolate as well as several unknown sulfated side products. Isolation, proton nuclear magnetic resonance spectroscopy and hydrolysis by sulfatase and /3-glucosidase led to the identification of these compounds as E- and Z-2- nitrobenzaldoxime-f~-D-(6-sulfato)glucopyranoside and 2-nitrobenzyl alcohol-0-D-sulfatoglucopyranoside.The alcohol conjugate arose via hydrolysis of the aldoxime and subsequent reduction of the aldehyde. A variety of aryl and aliphatic aldoxlmes were converted to sulfatoglucosides. Screening of a number of unrelated crop plants showed that the ability to form sulfatoglucosides of exogenous aldoximes occurs widely. Among the plants that tested positive were Brasswa spp, Arabulops~s, nasturtium, flax, sorghum, carrot, bromegrass, soybean and sunflower. Key words: aldoximes; glucosylation; glucosinolates; metabolite conjugation; sulfation; sulfate esters; sulfatoglucosides; xeno-

biotics

Introduction Aldoximes d e r i v e d from a series of protein and non-protein amino acids via N - h y d r o x y amino acids are the p r e c u r s o r s of the aglycone m o i e t y of two classes of agriculturally import a n t natural plant products, the glucosinolates and the cyanogenic glucosides [ 1 - 3]. A l t h o u g h the two p r o d u c t groups do not a p p e a r in the same plants (with the e x c e p t i o n of Carica p a p a y a [4]), aldoximes occupy b r a n c h point positions for the formation of n u m e r o u s common side-products [5]. T h e s e include nitriles which are p r e c u r s o r s of cyanogenic glucosides (flax, sorghum) but side-products of glucosinolate formation (crucifers). Aldoximes can be hydrolyzed to a l d e h y d e s for conversion to alcohols and t h e i r glucosides (crucifers) [6] or oxidation to carboxylic acids (many species, including crucifers [7,8]). U n d e r inhibitory conditions aldoximes can be glucosylated directly (flax) [5]. Using s y n t h e t i c aldoximes as p r e c u r s o r s of artificial glucosinolates in B r a s s i c a j u n c e a cell cultures, we d e t e c t e d considerable quantities

of new non-glucosinolate compounds [9]. In this r e p o r t , chemical s t r u c t u r e s of aldoxime and alcohol c o n j u g a t e s are identified t h a t s u g g e s t the p r e s e n c e of novel glucosylation and glucopyranoside sulfation reactions. It is shown t h a t these conjugation reactions occur in a much wider r a n g e of plant species t h a n those producing glucosinolates and cyanogenic glucosides.

Methods In vitro c u l t i v a t i o n

Callus and shake flask cultures of B r a s s i c a spp w e r e grown on K-627-1 m e d i u m d e s c r i b e d by Kao and Michayluk [10]. Stock cultures w e r e t r a n s f e r r e d once per m o n t h in the callus s t a t e and once per week in the suspension state. Cult u r e s w e r e g r o w n at 26°C u n d e r dim light. Shake flask cultures r a n g i n g in size from 10 to 2000 ml w e r e incubated at 150 rev./min on a ~ y r o t a r y s h a k e r (New Brunswick Scientific). L a r g e r scale cultures w e r e g r o w n in a bench top f e r m e n t o r (Microferm, New Brunswick Scientific) u n d e r the following o p e r a t i o n condi-

0168-9452/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

12 tions, 10-1 working volume, 150 rev./min agitation, 100 ml/min aeration and 26 °C temperature control. The medium contained Gamborg B5 medium (Sigma) supplemented with sucrose (2.5%, w/v), N-Z-amine A (casein hydrolysate, 500 rag/l), 2,4-dichlorophenoxyacetic acid (2,4-D) (1 rag/l) and zeatin riboside (0.1 mg/1), and was autoclaved for 2.5 h at 121°C. All liquid media were inoculated with shake flask cultures at a ratio of 20-- 80 ml/1.

Chemical analysis Proton magnetic spectra (1HMR) were obtained with a Bruker 400 spectrometer. Chemical shifts are listed in the o scale relative to tetramethylsilane which was used as an external standard. The number of protons represented by a signal, its multiplicity and the coupling (J) in Hz are listed in brackets after chemical shift. The aromatic resonances are strictly speaking double doublets, however, the coupling constants were so similar that the resonances appeared as triplets and are listed as such for simplicity. Spectra were recorded with sample as solutions in deuterium oxide or deuterobenzene as noted. Preparative thin-layer chromatography (TLC) employed Whatman PLK5F 20 × 20 cm, 1000 ~m, silica gel plates or Whatman PK2F 20 × 20 cm, 1000 ~m, cellulose plates as noted.

Cell extraction, analysis

processing

and metabolite

The procedures outlined below are minor adaptations of those used widely for glucosinolate analysis [11]. Cell cultures were suction filtered using Buchner funnels with fritted discs, the cells weighed and 200-- 1000 mg fresh cells extracted in ~ 4 ml boiling 90% (v/v) ethanol. The cooled extracts were clarified using glass fibre filters and evaporated to dryness. The residues were suspended in 2 ml high pressure liquid chromatography (HPLC) grade (Norganic Cartridge, Millipore), deionized water and applied to diethylaminoethyl (DEAE)-Sephadex A-25 (Pharmacia) columns (0.75 ml bed vol.), which had been prepared and prequilibrated with the same water. The col-

umns were washed twice with 2 ml of water and loaded with 0.5 ml of arylsulfatase (EC 3.1.6.1) solution (Helix pomatia, Type H-l, Sigma). The enzyme was used either directly in deionized water (0.7 mg/ml) or after removal of 13-glucosidase activity by anion-exchange chromatography (0.0225-0.15 M NaCl gradient in 20 mM T r i s - HCI pH 7.5 on a Mono Q HR 16/ 10 column, Fast Protein Liquid Chromatography, Pharmacia) and diafiltration (10 000 tool. wt. cut-off) against water. The sulfatase-loaded DEAE-Sephadex columns were kept overnight at room temperature and the desulfated metabolites eluted with two 0.5 ml volumes of water. The eluate was heated for 3 min in a boiling water bath to inactivate all enzyme activities. 2-Nitrophenol-fl-D-galactoside (0.1 ~mol) was added as internal standard. The supplemented eluate was subjected to HPLC analysis using a reversed-phase column (Whatman C18 PartiSphere 5 ~m particle size) and an acetonitrile in water gradient of 1.25-22.5% [11]. Separated metabolites were detected by UV absorption at 226 nm.

Isolation and identification of desulfated metabolites from 2-nitrobenzaldoxime feeding To obtain quantities of metabolites sufficient for preparative chromatography and structural analysis, the above cell cultivation, extraction and desulfation procedures were scaled up. Column eluates containing desulfated metabolites were reduced to dryness and acetylated by treatment with acetic anhydride/pyridine (1 : 2, v/v) for 17 h at ambient temperature. Concentration of the sample in vacuo followed by TLC of the residue on silica gel using diethyl ether as the mobile phase, led to the isolation of the two major UV-absorbing components. The less polar component (Rf ~0.8) was identified as (E)2-nitrobenzaldoxime-/3-D-glucopyranoside tetraacetate, 1HMR (C6D6): 8.47 (1H, s), 7.56 (1H, d, J = 81, 7.35(1H, d, J = 8), 6.70(1H, t, J = 8), 6.52 (1H, t, J = 8), 5.70 (1H, dd, J = 8.6 + 9.4), 5.47 (1H, t, J = 9.4), 5.34 (1H, t, J = 9.7), 5.26(1H, d , J = 8.6}, 4.27 (1H, dd, J = 4.2 + 12.4), 4.03 (1H, dd, J = 2.2 + 12.4), 3.32 (1H, ddd, J = 9.7 + 2.2 + 4.2), 1.73 (3H,s), 1.71

13 (3H,s), 1.65 (3H,s), 1.64 (3H,s). The more polar component (Rf ~0.4) was identified as the 2nitrophenyl desulfoglucosinolate pentaacetate, 1HMR (C6D6): 7.50 (1H, d, J = 8), 6.78 (1H, t, J = 8), 6.63 (1H, t, J = 8), 5.17 (1H, t, J = 9.5), 5.12 (1H, t, J = 9.5), 5.04 (1H, t, J = 9.5), 4.21 (1H, d, J = 9.5), 4.02 (1H, dd, J = 4.4 + 12.3), 3.78 (1H, dd, J = 2.5 + 12.3), 2.53 (1H, ddd, 2.5 + 9.7 + 12.3), 1.75 (3H,s), 1.67 (3H,s), 1.59 (3H,s), 1.55 (3H,s), 1.51 (3H,s). The solution of the pentaacetate in deuterobenzene was quite dilute and thus the amount of contaminating benzene was enough to mask the fourth (missing) aromatic resonance. The underivatized (E)~ and (Z)-2-nitrobenzaldoxime glucosides were isolated by subjecting the desulfated product mixture to HPLC (multiple runs), collecting and concentrating the appropriate fractions. The (E)-2-nitrobenzaldoxime-/~-glucopyranoside was the major component (~8.5 mg), ~HMR (D2O): 8.77 (1H,s), 8.12 (1H, d, J = 8), 7.76 (1H, t, J = 8), 7.70 (1H, d, J = 8), 7.64 (1H, t, J = 8), 5.09 (1H, d, J = 7.9), 3.84 (1H, dd, J = 2.2 + 12.4), 3.66 (1H, dd, J = 5.7 + 12.4), 3.3 - 3.6 (4H, overlapping peaks). The (Z)-2-nitrobenzaldoxime-/3-glucopyranoside was a minor component (~1.5 mg), ~HMR (D20): 8.09 (1H, s), 8.14 (1H, d, J = 8), 7.72 (1H, t, J = 8), 7.63 (1H, d, J = 8), 7.61 (1H, t, J = 8), 4.97 (1H, d, J = 8.4), 3.82 (1H, dd, J = 2.2 + 12.4), 3.64 (1H, dd, J = 5.6 + 12.4), 3.45 (1H, ddd, J = 2.2 + 5.6 + 9.4), 3.44 (1H, t, J = 9), 3.30 (1H, t, J = 9.4), 3.22 (1H, t, J = 9).

Isolation and identification of desulfated me tabolite from Z-nitrobenzaldehyde feeding The desulfated product mixture was analyzed directly by NMR and found to consist predominantly of 2-nitrobenzyl alcohol-f3glucopyranoside, 1HMR (D20): 8.04 (1H, d, J = 8), 7.72 (1H, d, J = 8), 7.68 (d, 1H, J = 8), 7.51 (1H, d, J = 8), 5.18 (1H, d, J = 13.8), 5,15 (1H, d, J = 4), 5.07 (1H, d, J = 13.8), 4-2 (numerous overlapping peaks due to sugar impurities). Isolation and identification of (E)-~-nitrobenzaldoxime-~f6-sulfato)glucopyranoside Cell extracts were applied to DEAE-

Sephadex A-25 columns and unadsorbed material washed off with water. Anionic compounds, including sulfatoglucosides were desorbed with 0.3 M NaC1. The eluate was concentrated to dryness under vacuum, redissolved in water and subjected to preparative TLC on silica gel or cellulose. Chromatograms on silica gel were developed with n-butanol/ ethanol/water (52: 32 : 16, by vol.) and on cellulose with ethanol/ water (80/20, v/v). The dominant UV-absorbing band (Rf = 0.41 on silica gel and Rz = 0.53 on cellulose) was recovered, eluted with water, and concentrated to dryness under vacuum (~4 mg). ~HMR (D2O): 8.81 (1H, s), 8.18 (1H, d, J = 8), 7.79 (1H, t, J = 8), 7.70 (1H, t, J = 8), 5.18 (1H, d, J = 7.9), 4.45 (1H, dd, J = 2.1 + 12.4), 4.36 (1H, dd, J = 4.6 + 12.4), 3.8 - 3.4 (4H, overlapping peaks).

Miscellaneous Precursor administration was carried out by direct addition (1 mM final concentration) of an ethanol solution of specified aldoximes, 2-nitrobenzaldehyde or 2-nitrobenzyl alcohol (200 raM; Aldrich) to sterile nutrient media of in-vitro cultures or to water for organ or whole plant feeding. Enzymatic deglycosylation of metabolites was carried out by overnight incubation at 37°C with purified almond /~-glucosidase (EC 3.2.1.21; Sigma), purified Escherichia coli f3-glucuronidase (EC 3.2.1.31; type X, Sigma) or purified white mustard thioglucosidase (myrosinase, EC 3.2.3.1; Bio-Chem-Color Co., Goettingen, F.R.G.) dissolved in water. Enzymatic reactions were stopped with heat treatment for 3 rain in a boiling water bath. A sample containing a mixture of (Z)-2-nitrobenzaldoxime and (E)-2-nitrobenzaldoxime was prepared by photoisomerization of the latter in benzene [12]. The structures were confirmed by 1HMR spectrometry. The diastereoisomers were separated by the above outlined HPLC procedure. Results

Detection of aldoxime derivatives 2-Nitrobenzaldoxime (90--95% E-isomer) was

14

2 ( I S)

fed to 7-day-old shake flask cultures ofB. juncea cv. Cutlass, and accumulation of sulfated metabolites determined by HPLC analysis after desulfation with unfractionated arylsulfatase. Control cultures (Fig. 1A) varied in content from undetectable to minor amounts of 1-methoxy-, 4-methoxyand unsubstituted indolylmethylglucosinolate, while test cultures (Fig. 1B) accumulated compound 1 (2-nitrophenylglucosinolate, see Methods for tHMR) and unknown compounds 3, 4, 5 and 6. When cell extracts were treated with thioglucosidase

4

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RETENTION Fig. 1.

15

20

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T I M E (rain)

HPLC analyses of desulfated metabolites of a B. juncea cell culture fed 2-nitrobenzaldoxlme. A: unfed control culture; B: desulfation with unfractionated arylsulfatase; C: desulfation with fractionated arylsulfatase; D: desulfation with fractionated arylsulfatase followed by deglycosylation with/3-glucosidase. Peak identification, 1, 2nitrophenyl desulfoglucosinolate; 2, internal standard (I.S.), ONPG, 3, {Z)-2-nitrobenzaldoxime-f~-v-glucopyranoside; 4, (E)-2-nitrobenzaldoxime-~-D-glycopyranoside; 5, 2-nitrobenzyl alcohol; 6, 2-nitrobenzaldoxime.

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RETENTION

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HPLC analyses of desulfated metabolites of a B.

juncea cell culture fed 2-nitrobenzaldehyde. A: desulfation with unfractionated arylsulfatase; B: desulfation with fractionated arylsulfatase; C: desulfation with fractionated arylsulfatase followed by deglycosylatlon with fl-glucosidase. Peak identification, 2, internal standard; 4, 2-nitrobenzyl alcohol-fl-D-glucopyranoside; 5, 2-nitrophenyl alcohol.

15

prior to desulfation, natural and artificial glucosinolates were degraded while the unknown compounds remained intact. Screening of several possible conversion products by co-chromatography identified compounds 5 and 6 as 2-nitrobenzyl alcohol and 2-nitrobenzaldoxime, respectively. Control experiments with authentic samples showed that the two compounds had not passed through the DEAE-Sephadex column unmodified but were clearly generated by the sulfatase. This initially but incorrectly suggested that the 2-nitrobenzaldoxime was converted by the cells to its sulfate ester as well as to the sulfated alcohol. Subsequently it was determined that the unfractionated sulfatase contained /3glucosidase which was responsible for their formation. The alcohol presumably arose via conversion of the aldoxime to the aldehyde followed by reduction. Separate feeding of 2nitrobenzaldehyde and 2-nitrobenzyl alcohol gave identical HPLC profiles (Fig. 2A) that included the alcohol (compound 5).

and 2A). Structural elucidation was achieved by NMR analysis of compounds isolated from desulfated cell extracts (Fig. 3). The structure of (E~2-nitrobenzaldoxime-/3-D-glucopyranoside was clearly established from its 1HMR spectrum as well as the spectrum of its tetraacetate derivative. Thus, the anomeric proton exhibited a large coupling constant in accord with the /]-assignment, while the chemical shift of the aldoxime hydrogen was appropriate for the (E)-configuration [13,14]. Similarly, the structure of (Z)-2-nitrobenzaldoxime-/3-D-glucopyranoside (compound 3, Fig. 1B) was established from its 1NMR spectrum via the chemical shift of the aldoxime hydrogen which was at 0.68 ppm higher field than that of the (E)-isomer [13,14]. The structure of 2-nitrobenzyl alcohol-/3-Dglucopyranoside (compound 4, Fig. 2A) was suggested by the ~HMR spectrum of the desulfated metabolite mixture obtained from 2nitrobenzaldehyde feeding.

Identification of glucosides Separate feedings of both the aldoxime and aldehyde yielded other unkown compounds of identical retention times (compound 4, Figs. 1B

Effect of fl-glucosidase on the new compounds Treatment of cell extracts from the aldoxime- and aldehyde-fed cultures with /3-glucosidase or/3-glucuronidase prior to desulfation did not alter the HPLC profiles. However,

HO~OH 8H~77 N/ H~509

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~B336684

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~ ~ H

~I M~497 N 0 92N O 8

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HO~oH O~/f/~ ~

8:1 /N H~518

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OH H O ~ O H

0.../~--o---~

B506 H-,/ H I B518 H " ~ ~515

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....

02N~_~ x C

Fig. 3. Structures of some glucosides and selected chemical shifts. A: (E)-2-nitrobenzaldoxime-fJ-D-glucopyranoslde; B: (Z)-2-nitrobenzaldoxime-/3-D-glucopyranoside; C: (E)-2-nitrobenzaldoxime-fYD-(6-sulfato)glucopyranoside; D: 2-nitrobenzyl alcoholq3-~glucopyranoside.

16

treatment with/3-glucosidase after desulfation caused a marked reduction in the amount of compounds 3 and 4, the glucosyl conjugates. Subsequently, it was established that the commercial sulfatase being used was contaminated with fJ-glucosidase, thus generating the 2-nitrobenzyl alcohol and 2-nitrobenzaldoxime (compounds 5 and 6) as artifacts of the desulation process. Removal of the/3-glucosidase from the sulfatase yielded an enzyme preparation that no longer produced the free alcohol and aldoxime (Figs. 1C and 2B). Sequential treatment with the purified sulfatase (on DEAESephadex column) and fJ-glucosidase (in solution} showed the virtual complete conversion of the (E)-aldoxime and alcohol glucosides (compounds 4, Figs. 1D and 2C) to the free aldoxime and alcohol. The (Z)-aldoxime glucoside (compound 3) was hydrolyzed only partially, yielding the (Z)-2-nitrobenzaldoxime, identified by co-elution at 20.5 min with a authentic sample.

Position of sulfate substitution The only possible position of the sulfato substituent was on the glycone moiety, rendering the glucosides resistant to/3-glucosidase, as was observed. In order to corroborate this, ~HMR analysis of the intact 2-nitrobenzaldoxime sulfatoglucoside was carried out and suggested the presence of the sulfate group at the 6 position of the sugar. This conclusion was based on comparison of the chemical shifts of the C-6 hydrogens of the sulfatoglucoside with those of the glucoside. In the former case the resonances were at lower field by at least 0.6 ppm, and were the most significant difference between the spectra of the two compounds.

Mass spectrome try Fast atom bombardment/mass spectrometry was attempted on the aldoxime glucoside and the corresponding sulfated derivative. The glucoside gave small peaks at M + 1 and M + 23, however, the spectra did not appear reliable. The sulfated compound did not afford any interpretable results. Similarly, thermospray/ mass spectrometry on both compounds was unsuccessful.

5

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4-0

80

120

160

200

Incubation time (rain) Fig. 4. Time course of release of 2-nitrobenzaldoxime glucoside from 2-mtrobenzaldoxime sulfatoglucoside by arylsulfatase.

Desulfation of sulfatoglucoside To establish a hydrolysis pattern for sulfatoglucoside by arylsulfatase in free solution, product release was monitoried by HPLC (Fig. 4). The relatively high level of product present at the beginning of the reaction reflects the instability of the substrate, in particular during enzyme inactivation in a boiling water bath. The combination of a low concentration of the sulfatoglucoside (~0.5 mM) and a low affinity of the enzyme for the substrate resulted in extremely low reaction rates; the initial rate was only ~ 1 % of that measured with a saturating concentration (7.5 raM) of the assay substrate 4-nitrophenylsulfate. Thus, relatively large amounts of arylsulfatase were required for the quantitative recovery of desulfated metabolites in on-column reactions, used routinely in this study for analytical purposes. The

17

wide range of aromatic and aliphatic sulfate esters hydrolyzed by Helix pomatia arylsulfatase suggests either a great lack of specificity or a mixture of different enzyme species. Hydrolysis of sulfatoglucosides might be more specifically accomplished by glycosulfatases (sugar-sulfate sulfohydrolases, EC 3.1.6.3) [15]. Desulfation of 2-nitrobenzaldoxime sulfatoglucoside increased the mobility in cellulose TLC (ethanol/water, 8 : 2, v/v) from Rf 0.53 to 0.82, consistent with a decrease in polarity. The final identity of the sulfate substituent was confirmed by ion chromatography of an acid {2 N HCI, 5 min, 90°C) hydrolysate of a sulfatoglucoside sample prepared by cellulose TLC.

tion system showed typical saturation kinetics with half maximum rate of sulfatoglucoside formation at ~0.18 mM 2-nitrobenzaldoxime (Fig. 5B).

Sulfatoglucosides of different aldoximes Using the criterion that sulfatase treatment was a prerequisite of susceptibility to f3-glucosidase, B. juncea cell cultures were shown to also form sulfatoglucosides from benzaldoxime, 3nitrobenzaldoxime, 2-chlorobenzaldoxime, 2,6dichlorobenzaldoxime, 2-fluorobenzaldoxime, 3fluorobenzaldoxime, hexanaldoxime and 2-hexenaldoxime.

Sulfatoglucoside formation in different species and tissues In addition to B. juncea cell cultures, tissues of several other Brassica species as well as a

Kinetics of sulfatoglucoside formation The rate of total sulfatoglucosides formed from 1 mM 2-nitrobenzaldoxime in B. juncea cell culture was constant for at least 24 h (Fig. 5A). Thus, the enzymes and metabolic precursor flow required for the transformations were not adversely affected by the high aldoxime concentration which completely arrested cell multiplication [9]. The transforma-

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number of unrelated species were able to form sulfatoglucosides from 2-nitrobenzaldoxime. The conjugation reactions occurred in tissues from plant organs and in-vitro cultures, regardless of the explant origin {Table I). 2-Nitrobenzaldoxime was taken up by the roots of whole plants and accumulated as conjugates mainly in the roots, with very low levels in the leaves and seed pods. All glucosinolate producing species (Brassica, Arabidopsis, nasturtium, stinkweed) and cyanogenic glucoside producing species (flax, sorghum) produced sulfatoglucosides. However, this relation with known aldoximederived products was not obvious in carrot, bromegrass, soybean, sunflower and tobacco. Discussion

(raM)

Fig. 5. Kinetics of sulfatoglucoside formation from exogenous 2-nitrobenzaldoxime in B. juncea cell culture. A: time course, feeding I mM 2-nitrobenzaldoxime. B: effect of precursor concentration after 24 h incubation. FCW, fresh cell weight.

Although in plants, low molecular weight secondary metabolites are very often glycosylated and/or, less frequently, sulfated [16], only a limited number has been described in which the sulfate ester is a substituent of the glycone moiety. All of the known low molecular weight sulfatoglycosides are conjugates of phenolic compounds, predominantly flavonoids [16], but also include hydroxycinnamic acid [17], anthraquinone [18] and betanidin [19]. The present study revealed that the enzy-

18 Table I.

Detection of 2-nitrobenzaldoxlme sulfatoglucoside in tissues of several plant specms fed 2-nitrobenzaldoxime.

Species Brassica juncea cv. Cutlass (brown mustard)

B. yancea cv. Domo B. napus cv. Westar B. napus cv. J e t Neuf B. napus (F1 cross) B. oleracea (kohlrabi) B. nigra (black mustard) Sinapis alba (B. hirtaJ (white mustard) A rabidopsis thaliana Tropaeolum mayus (nasturtium} Thlaspi arvense (stinkweed) Linum usitat~ss~mum (flax) Sorghum bicolor (sorghum) Catharanthus roseus (periwinkle) Daucus carota (carrot) Bromus merinos (bromegrass) Glycine max (soybean) Medicago sativa (alfalfa) Axyris amaranthoides (pigweed} Hehanthus annuus (sunflower} Capswum annuum (pepper) Nwotiana tabacum (tobacco)

Tissue

Origin

Sulfatoglucoslde (relative amount) ~

Cell suspension Callus

Hypocotyl hypocotyl, root, petiole, leaf, epidermis-free leaf, 4-day~)ld cotyledon

+++

Leaf Pod Immature seed from pod feeding Root, leaf, pod from plant feeding Cell suspension Callus Cell suspension Embryo Callus Callus Callus Leaf Leaf Leaf Callus Leaf Cell suspension

+++ + + b

+ Hypocotyl Hypocotyl Hypocotyl Microspore Hypocotyl Hypocotyl

+ + + + + +

Hypocotyl

+ + ++ ++

Hypocotyl

+ + + + + +

+ + + +

+ + + +

Anther

Cell suspension Cell suspension Leaf Leaf Leaf

Hypocotyl

+ + + +++ ++

Cell suspension Leaf Cell suspension

Hypocotyl

+ + +

Hypocotyl

+ +

•Comparisons based on i g fresh tissue weight. bNo incorporation.

matic capability of forming sulfatoglucosides occurs widely. However, the finding was based on biotransformation of exogenous synthetic aldoximes, an aldehyde and an alcohol, posing the question as to the physiological function of the enzymes involved. The possibility exists that the observed conjugations were non-specific side reactions, invoked during experimental exposure of plant cells to unphysiologically high concentrations of metabolic intermediates.

Identification of naturally occurring sulfatoglucosides in the commonly grown crop plants tested in this study, such as Brassica spp, flax, sorghum, carrot, bromegrass, sunflower or tobacco, should provide information as to their function and significance. In Brassica spp, the relation of aldoxime sulfatoglucosides to the sulfated S-glucosyl thiohydroximates, i.e., glucosinolates, is of particular interest. Diversion of endogenous aldoxime

19

precursors of glucosinolates to their O-glucosides or sulfatoglucosides has not been reported. The question arises whether this side pathway normally competes for substrate or whether the experiments described here with exogenous aldoximes created an artificialsituation. Furthermore, would a desired genetic block of glucosinolate biosynthesis downstream from the aldoxime intermediate promote operation of the side pathway in order to prevent aldoxime accumulation? Exogenous aldoximes are toxic to plant cell cultures [9] and plant organs, necessitating their neutralization, perhaps through conjugation and cellular isolation. The glucose and sulfate moieties m a y play an important role in intracellular localization and inter-tissue transportation. It is worth noting here that partially purified desulfoglucosinolate sulfotransferase of B. napus [20] showed no sulfation activity towards 2-nitrobenzaldoxime glucoside, suggesting involvement of enzymes with distinct specificity. Previous failure to detect endogenous sulfatoglucosides in glucosinolate producing plants might have been partially due to their instability. The O-sulfate ester bond with the glucose moiety is quite labile. W e found methanol unsuitable as a solvent since it invariably led to the loss of the sulfate ion, concurring with earlier experience with anthraquinone sulfatoglucosides [18]. Therefore, it is likely that in the f e e d i n g e x p e r i m e n t s of K i n d l a n d S c h i e f e r [6] w i t h w h i t e m u s t a r d (a B r a s s i c a s p e c i e s ) , t i s s u e extraction with hot methanol had precluded d e t e c t i o n of a r y l a l c o h o l s u l f a t o g l u c o s i d e s derived from aryl aldoximes. In contrast to extensive biochemical knowle d g e of s u l f a t i o n r e a c t i o n s in a n i m a l s y s t e m s , s t u d i e s of s u l f a t e e s t e r b i o g e n e s i s a n d e n z y m o l o g y in p l a n t s a r e f e w , f o c u s i n g p r i m a r i l y on flav o n o i d s f r o m n o n - f o o d p l a n t s [16]. T h e p r e s e n t work may provide the basis for a convenient e x p e r i m e n t a l s u l f a t i o n s y s t e m p r e s e n t in c r o p p l a n t s . T h e r o l e in t h e d e t o x i f i c a t i o n of e n v i r o n mental and agricultural chemicals with passage of s u l f a t e e s t e r c o n j u g a t e s t h r o u g h t h e f o o d c h a i n m i g h t b e of p a r t i c u l a r i m p o r t a n c e [21].

Acknowledgement

We would like to thank Dr. Lloyd Nelson from this laboratory for his generous advice and Mr. Michael Chester for the fractionation of t h e a r y l s u l f a t a s e . References

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