f3-glucan synthase (Callaghan, T., Ross, P., Weinber- ger-Ohana, P., and .... (8-10 Hz) coupling constants indicate that all five protons H-. I', -2', -3', -4', and -5' .... Blakeney, A. B., Harris, P. d., Henry, R. J., and Stone, B. A. (1983). New York. 9.
THEJOURNALOF BIOLOGLCAL CHEMISTRY c! 1991 by
Vol. 266, No. 21, Issue of July 25, pp. 13742-13745,1991 Printed in U.S. A.
The American Society for Biochemistry and Molecular Biology, Inc.
&Furfuryl-&glucoside ANENDOGENOUSACTIVATOROFHIGHERPLANTUDP-GLUCOSE:
(1 + 3)-@-GLUCANSYNTHASE* (Received for publication, January 11, 1991)
Patricia OhanaS5, DeborahP. DelmerllII, John C. Steffens**$$,David E. Matthews**$$, Raphael Mayer$, and MosheBenziman$#$6 From the Department of $Biological Chemistry, Department of llBotany, Institute of Life Sciences, The Hebrew Universityof Jerusalem, Jerusalem 91904, Israel and the **Departmentof Plant Breeding and Biometry, New York StateCollege of Agriculture and Life Science, Cornell Uniuersity, Ithaca, New York 14853
We have recently established the existence of endogenous activators of higher plant UDP-glucose: (1 + 3),f3-glucan synthase (Callaghan, T., Ross, P., Weinberger-Ohana, P., and Benziman, M. (1988) Plant Physiol. 86, 1099-1 103). Here we report the purification and chemical analysis of the most abundantand specific compound,termed Activator I, isolated from Vigna radiata. This compound was extensively purified by a multistep procedure which yielded 0.1 mg of purified activator/g of fresh tissue. Enzyme digestion, neutral sugar analysis, GC/MS ofpermethylated derivatives, and NMR analysis of native Activator I indicated that the compound contains a single &linkedglucosyl residue. High resolution FAB-MS indicated an elemental composition of Cl1HlaO7(Mr = 260), with a calculated M , of 98 for the aglycone. 13C, DEPT, and COSY NMR spectra showed that the aglycone molecule is an oxygen heterocycle of 5 carbons, consistent with a structure of &furfuryl alcohol. Comparison of IR and GC/EI-MS spectra of authentic @-furfurylalcohol with nativeaglycone confirmed the conclusion that Activator I is &furfuryl-& glucoside. Chemically synthesized /3-furfuryl-B-glucoside has identical chemical properties and biological activity when compared with the purified endogenous activator ( K . = 50 PM).
an absolute requirement for Ca2+, and it has been suggested that perturbation of the plasma membrane may lead to an increasein cytoplasmic[Ca”) andthereby trigger callose synthesis (2).Callose synthesis in uiuo can also be induced by many biochemically unrelated substances such as saponins, polyene antibiotics, acylated cyclic peptides, and certain detergents (2,3), the presumed mode of action of which includes an induction of net ea2+ uptake. However, Kohle et al. (3) recently showed that while increased ea2+ uptake is an event common to all elicitors of callose synthesis, it is not always sufficient for induction, and they suggested that additional regulatory parameters must beinvolved. It is well established that various sugars and, in particular, a variety of simple @glucosides stimulate callose synthasein uitro, andkinetic studies haveshown that the enzyme is virtually inactive unless both ea2+ anda @-glucosideare supplied as effectors (4,5 ) . These @-linkedglucosides are apparently not incorporated into the glucan product, suggesting that they do not serve as primers, but rather as allosteric effectors (4, 5 ) . I n uitro studies indicate that suchglucosides affect both the K,,, for UDP-glucose and the V,, of the synthase (4,5 ) . It was shown that@-linkedterminal glucosylated hydrophobic moiety is a necessary structural requirement for activation ( 5 ) .While theK,, of most of the @-linkedglucosides studied is in the millimolar range, that of the alkyl @-monoglucosides such asoctyl glucoside is in the micromolar range, suggesting the the latter may mimic some specific natural activator ( 5 ) . Thus, it would appear that one or more endogenous @-glucoAn intriguing problem surrounding the study of @-glucan sides provideadditional regulationof callose synthesis inuiuo, synthesisin higher plantsystemsisthat while cellulose but their precise structure has not beenelucidated. We have previouslyreported (6) the existence of heat-stable (Glcpl-4Glc glucan) is the most abundant natural polymer activators of membranous @-glucan synthase, isolated from produced in uiuo, callose (Glc@l-3Glcglucan)isoftenthe the soluble fraction of crudemungbean (Vigna radiata) predominantproductsynthesizedin uitro from thesame extracts, and one such activator was tentatively characterized substrate, UDP-glucose.Callose hasanimportant rolein as a @-linkedglucoside having a hydrophobic aglycone moiety. plant development; in addition to occurring in the walls of Following extensive purification and rigorous characterizasome specialized cells such as pollen tubes and cottonfibers, tion, we are now able to report the precise structure of this it is synthesized in massive amounts in most plant cells as a activator as@-furfuryl-@-glucoside. Chemically synthesized @wound polymer in response tocell damage or pathogen inva- furfuryl-@-glucosideexhibits biological activity identical with sion (1).This “wounding” seemingly induces a switch from that of the activatorpurified from mung bean tissue. cellulose to callose synthesis inhigher plants (1).The plasma membrane-localized UDP-glucose: 1,3-@-glucan synthase has MATERIALS AND METHODS’ * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Supported by Weyerhaeuser Go. 11 Supported by a contract from the United States Department of Energy. $$ Supported by Hatch Project 149417. To whom correspondence and reprint requests should be addressed.
RESULTS
Isolation-Extraction of tissue was performed withhot isopropyl alcohol followed by water in order to prevent any Portions of this paper (including “Materials and Methods” and Figs. 1 and 3-5) are presented in miniprint at the endof this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that isavailable from Waverly Press.
13742
Structure of Plant UDP-glucose: /3-Glucan Synthase Activator
13743
glucosyl moiety in the activator. Fast atom bombardment MS (thioglycerol matrix) of underivatized Activator I identified m/z 283 (M Na+) as a possiblemolecularion. GC/MS (ammonia) of theactivatoras a trimethylsilyl derivative yielded m/z 566 ([M NH,]'), consistent with the addition FIG. 2. Structure of Activator I. of four trimethylsilyl groups to a compound of M , = 260. The activator was acetylated (pyridine, acetic anhydride,1:l)and yield m/z 446 (M + H + NHJ, enzymic degradation of endogenous activators. In this original submitted to CI/MS (NHJ to extract, by TLC in solvent A, only two species of activators indicating tetraacetylation anddefining the massof the aglywere detected; one, called Activator I, migrated with anRf of cone as98. Acetylation moved the 'H NMR signals for H-2', -3', -4', 0.35, and the second (Activator 11) was immobile. Analysis of -6'a, and -6'b downfield, relative to underivatizedActivator I the fractions during the purification procedure described in -5' and (see Fig. 3), completelyresolving H-3',-4',and Fig. 1 did not reveal any additional activators, other than a allowing measurement of their coupling constants. The large neutral sugar fraction whichwas separated and discarded five protons Hduring chromatography onsilica gel. Furthermore, we did not (8-10 Hz) coupling constants indicate that all find any activators extractable from membrane fractions withI', -2', -3', -4', and -5' are axial. That H-1' is axial demonorganic solvents. Thus, we conclude that Activators I and I1 strates that the glycosidic bond is @ rather than a, and the axial conformation of the other four protons establishes the are the major activator compounds in this plant tissue, and, of these two, the extracts contained substantiallymore activ- relative configurations of all the secondary hydroxyls in the sugar residue of theactivator,therebydistinguishingthe ity for Activator I. Our purification procedure yields highly purified Activator I, resulting in a single peak on HPLC' at proposed structure containing l-@-D-glUCOSefrom all other R T 6.5 (inset, Fig. 1).A highly purified preparation of Acti- hexoses except 1-@-L-glucose. In addition to the glucosyl moiety, the 'H NMR spectrum vator I1 was also obtainedby this procedure, but its biological activity was found to be unstable upon storage-20 at "C, and of the underivatized Activator I (Fig. 3) indicated thepresence itsstructureisstillunderinvestigation.Thispurification of a weakly coupled aromatic spin system possessing three procedure easily provides sufficient amounts (15-20 mg) of protons at 6 7.43, 7.35, and 6.39 (all broad singlets). 'HCOSY highly purified Activator I for structural analysis andbiolog- spectra revealed a cross-peak from 6 7.43 to the proton at 6 ical assay. 7.35 and a weak correlation to the proton a t 6 6.39. More structure of Acti- intense cross-peakswere evident from6 7.35 and to the proton Structure of Activator I-The proposed vator I is shown in Fig. 2. The abilityof the purified activator at 6 6.39. In deeper contour plots, the protons at6 7.43 and 6 tostimulate @-glucan synthaseactivity was destroyed by 6.39 exhibited weak cross-peaks to two geminally coupled ( J strong acid (2 N HC1, 100 "C, 30 min) or by digestion with @= 11.9 Hz) protons at 6 4.62 and 4.48. Acquisition of the glucosidase, indicating the presenceof a @-glucosidiclinkage. spectrum at 40 "C shifted the DHO signal upfield and per(8) and mitted observation of the signal at6 4.62. The two methylene Followinghydrolysis, reduction,andacetylation analysis by GLC, the only peak eluted corresponded to that protons were also weakly (detectable only as COSY crossof the glucitol hexaacetate standard, indicating that glucose peaks) coupled to H-1' of glucose. is the only sugar in the native activator. GC/MS analysisof High resolution FAB/MS (3-nitrobenzylalcohol matrix) of the permethylated derivative of Activator I showed that the the tetraacetate provided m/z 429.1404 (M + H)+ (calculated solemonosaccharidederivative was 1,5-di-O-acetyl-2,3,4,6-m/z 429.1397 for[C19H24011]H+),indicatinganelemental tetra-0-methyl glucitol, which corresponds to a nonreducing composition for the aglycone of C5H60,. The 13C NMR specterminal glucose residue. The I3C NMR spectrumof Activator trum of Activator I did not indicate the presence of carbonyl I also provided evidence for glucose as the glycosidic portion groups in the aglycone. However, DEPT experiments showed of the activator: 6 102.77 (C-1', d), 78.05 (C-5', d), 77.99 (C- that threeof the sp2hybridized carbons ( 6 144.48,142.47, and 3', d), 75.03 (C-4', d),75.02 (C-2', d), and63.08 (C-6', t) (13). 111.68) possessed one proton each, accounting for the three In the 'H NMR spectrum (Fig. 3), the large (8-Hz) coupling aromatic protonsobserved in the 'H NMR spectrum. A fourth constant of the anomeric proton of glucose ( 6 4.33, d) conquaternary olefinic carbon (6 123.19) along with the2 carbons firmed the assignment of the glucosyl linkage configuration at approximately 6 140, a shift characteristicof carbons bound as @.The following additional glucose protons could be asto oxygen in 5-memberedoxygen heterocycles, suggested that signed by COSY experiments (Fig. 3): H-2' ( 6 3.09, bt, J = the aglycone was composed of a monosubstituted furan ring 8.5 Hz), H-6a' ( 6 3.74, dd, J = 12.4, 2.1 Hz), and H-6b' ( 6 system. A triplet at 6 62.79, taken together with the AB spin 3.54, dd, J = 12.3, 5.9 Hz). The resonances for H-3', H-4', system identified at 6 4.56 in 'H spectra, indicated hydroxyand H-5' (6 3.17-3.30) overlapped considerably. methylsubstitutionand suggested thattheactivator was The isobutane CI/MS of acetylated Activator I was comcomposed of a @-glucosyl-linked hydroxymethyl-substituted parable to thatof the acetate of methyl-@-glucoside (see Ref. furan.Thisassignment was supported by theFAB/MS 14). Itlacked a molecularion, and most of the major fragments (3NBA) fragmentation of the activator tetraacetate:m/z 429 arose from the sugarresidue alone: 331 (81) [MH+-aglycone], (29) [M + HI+, m/z 331 (52) [tetraacetylated glucosyl frag271 (20) [MH+-aglycone-HOAc], 211 (6)[MH+-aglycone-2 ment], m/z 169 (29), and m/z 81 (24) [furfuryl fragment]. 3HOAc], 169 (77) [MH+-aglycone-2HOAc-CH2CO]. However, Substitution was suggested by the upfield shift of the quatera major fragment a t m/z 81 (100) appeared to correspond to nary carbon ( 6 123.19) in the presence of the two deshielded [aglycone-OH] and thus to indicate a M , of 98 for the aglycarbons(C-2,C-5) a tothefuranring oxygen (6 144.48, cone. 142.47, respectively). Similarly, the presence of two downfieldAdditional mass spectrometryprovided evidence fora single shifted protonssuggested that theaglycone was @ rather than alcohol. ' The abbreviationsused are: HPLC,high performance liquid chro- a-furfuryl of the Aglycone-The aglycone of Activator I was Structure matography; EPPS, N-(2-hydroxyethyl)piperazine-N"3-propanesulfonic acidDTT,dithiothreitol; EI, electronimpact; CI, chemical prepared by treatment with @-glucosidaseand extraction into ionization; EGTA, [ethylenebis(oxythylenenitrilo)]tetraaceticacid. dichloromethane. Thepurified aglycone possessed a UV chro-
+
+
13744
Structure of Plant UDP-glucose: P-Glucan Synthase Activator
mophore similar to that obtained from the intact activator fury1 compoundscan be readily synthesized and might be (maximum 220 nm, t = 6160, witha minor peak at 260). biosynthetically derived, by dehydration of pentafuranoses, of @-furfuryl Electron impact GC/MSof the underivatizedaglycone yielded but for this route to function in the biosynthesis a spectrum identicalwith that of authentic P-furfuryl alcohol compounds, an additional rearrangement of the carbon back(Fig. 4). As expected,majordifferences were not apparent bone is required. between thefragmentations of @- anda-furfuryl alcohol. It is now clear that our earlier structural characterization However, these two compounds are readily differentiated by of the impure activatorwas incorrect since we had tentatively ‘ H NMR. In agreement with results obtained from the gly- concluded that the activator consisted of a molecule of dicoside (see Fig. 3), the isolated aglycone was shown to possess acylglycerol joined to glucose residues in @-linkage (6). The threearomaticprotons.Thefactthat two of these were incorrect identification of the aglycone is undoubtedlydue to was impure at that stage, and strongly deshielded (6 7.40 and 7.37) suggests that there are the fact that the activator two protons adjacent to the furan oxygen atom, and therefore analyses were based only on patterns of migrations in TLC that the hydroxymethyl substitution is a t position 3. H-4 following enzyme and chemical degradation; for example, the appeared at 6 6.40 and the two equivalent hydroxymethyl mobility of the native aglycone and that of a diacylglycerol protons (H-6a,b) as a singlet at 6 4.55. This spectrum was standard is similar insome solvent systems. It is well established that a variety of sugars or polyols at found to be identical with that of authentic @-furfuryl alcohol, whereas a-furfuryl alcoholshowedonly one proton in the millimolar concentrations show stimulatory activity on calregion near 6 7.4 and two near 6 6.3 as expected. lose production i n vitro (4). Of the total stimulatory activity The infrared spectrum of the aglycone corresponded very in crude extracts, 50% is attributable to Activator I, roughly closely with that of @- but not a-furfuryl alcohol (data not 10-20% to Activator 11, and the remainder to the high conshown). In addition, the aglycone co-chromatographed with centrations of neutral sugars (mainly glucose, some sucrose) authentic @-furfuryl alcohol and was separable from a-furfuryl present in these tissues. A regulatory role for neutral sugars alcohol in HPLC, TLC, andGLC. cannot be completely excluded. However, the findings that Synthesis of Glucan Synthase Activator I-The acetylated Activator I is the predominant activatingcompound and has derivative of the proposed structure was synthesized by con- high affinity for the enzymesuggest thatitplays a key densation of @-furfurylalcohol with acetobromo-a-D-glucose. function in theregulation of callose synthase i n uiuo. ’H NMR of the synthetic tetraacetylated activator (CDC1;J A general role for P-furfuryl-@-glucoside in the activation exhibited resonances for the @-furfurylmoiety at 6 7.39, 7.38 of plant glucan synthases would require its presence in a large (H-2 and H-5, bs), 6.33 (H-4, bs), and 4.70 and 4.52 (H-6a number of plant species. We have recently detected both @and H-6b,d, J = 11.5 Hz). TheP-glucosyl residue showed the furfuryl-@-glucosideand its aglycone in four other plant speexpected five axial protons H-1’(6 4.53, d, J = 8.1 Hz), H-2’ cies chosen for examination (pea, cotton, barley, and ( 6 5.00, dd, J = 8.0, 9.4 Hz), H-3’ and 4‘ (6 5.16 and 5.07, dd, sorghum). The data, as well as other studies concerning the J = 9.7 Hz), H-5’ ( 6 3.66, ddd, J = 2.4, 4.7, 9.8 Hz), and the biosynthesis and function of this activator, will be reported two nonequivalent hydroxymethyl protons H-6’a and 6’b (6 elsewhere.:’ 4.24, dd, J = 4.8, 12.3 Hz; 6 4.14, dd, J = 2.4 Hz, 12.2 Hz). Acknowledgments-We are grateful to Dr. Anthony Alexander and The four acetate signals appeared at 6 2.081, 1.998, 1.988, David J. Fuller for acquiring mass andNMR spectra, respectively. 1.976 (all 3H, s). Synthetic tetraacetyl 0-furfuryl-@-glucoside was identical REFERENCES with the acetateof natural Activator I with regard to its FAB 1. Delmer, D. P. (1987) Annu. Reu. Plant Physiol. 38,259-290 2. Kauss, H. (1987) Annu. Reu. Plant Physiol. 65,67-71 mass spectrum and TLC mobility. After deacetylation with 3. Kohle, H., Jehlick, W., Poten, F., Blascheck, W., and Kauss, H. (1985) sodium methoxide, it co-chromatographed with the isolated Plant Physiol. 77,544-551 4. Hayashi, T., Read, S. M., Bussell, J., Thelen, M., Lin,F. C., Brown, R. M., activator both in TLC and HPLC andproduced identical ‘H Jr.. and Delmer. D. P. (1987) Plant Phvsiol. 83, 1054-1062 NMR and COSY spectra. 5. Callaghan, T., Ross, P., Weinherger-Ohana, P., and Benziman, M. (1988) Plant Physiol. 86, 1104-1107 Biological Actiuity-The stimulatory effect of purified Ac6. Callaghan, T., Ross, P., Weinherger-Ohana, P., and Benziman, M. (1988) tivator I on @-glucan synthase activity was compared with Plant Physiol. 8 6 , 1099-1103 7. Genshirt, H., Waldi, D., and Stahl, E. (1965) in Thin Layer Chromatograthat of the synthetic activator (Fig. 5). Both the synthetic p h y : A Laboratory Handbook (Stahl, E., ed) pp. 344-371, Academic Press, and the isolated activators gave identical results. When calNew York 8. Blakeney, A. B., Harris, P. d . , Henry, R. J., andStone, B. A. (1983) culated from Lineweaver-Burk plots, the K, of Activator I Carbohydr. Res. 113,291-299 was calculated to be 50-55 KM. For comparison, stimulation 9. Harris,P. J., Henry, R. J., Blakeney, A. B.,andStone, B. A. (1984) Carbohyr. Res. 127,59-73 by sucrose results in the same V,,,,., but the K,, was found to 10. Benn, R., and Gunther, H. (1983) Angew. Chem. Znt. Ed. Engl. 2 2 , 350be 2 orders of magnitude higher (8 mM, inset to Fig. 5). 380 DISCUSSION
@-Furfurylalcohol has been reported from plants as free the alcohol in the essentialoils of Stellaria aquatica (15) and Aloe pyrrole-2-carboxylic esterin arborescens (16),andasthe Pseudostellariaheterophylla (17).We have found only one report of the occurrence of @-furfuryl-P-glucoside, which was isolated from extracts of azuki beans (Vigna angularis) (19). Thequantityextracted (0.14 mg/g freshtissue) wasvery similar to that found by us (0.1 mg/g fresh tissue) in mung beans. The work presented here represents the first identification of a physiological role for this compound in plants. The biosynthetic origin of this compound is unclear. a-Fur-
11. Schroeder, L. R., and Green, J. W. (1966) J. Chem. Soc. (C) 1 9 6 6 , 530531 12. Talley, E. A. (1963) Methods Carbohydr. Chem. 11, 337-340 13. Stothers, J. B. (1972) in Carbon-13 N M R Spectrocopy (Blomquist, A. T., and Wasserman, H., eds) p. 461, Academic Press, New York 14. Harrison. A. G. (1983) Chemical Zonizatmn Mass Smctrometry, . DD. .. 119120, CkC Press, Boca Raton 15. Kameoka, H., Wang, C. P., and Yamaguchi, T. (1978) Nippon Nogeikagaku Kaishi 5 2 , 335; (1979) Chem. Abstr: 9 0 , 690712 16. Kameoka, H., Maruyama, H., and Mlyazawa, M. (1981) Nippon Nogeikagaku Kaishi 55,997; (1982) Chem. Abstr. 9 6 , 114962 17. Reinecke, M. G., and Zhao, Y. (1988) J . Nat. Prod. (Lloydia) 51, 12361240 18. Synder, C. F., Frush, H. L., Ishell, H. S., Thompson, A., and Wolfrom, M. L. (1962) Methods Carbohydr. Chem. I, 524-534 19. Kitagawa, I., Wang, H. K., Saito, M., and Yoshikawa, M. (1983) Chem. Pharm. Bull. 31 , 664-673
P. Ohana and M. Benziman, manuscript in preparation.
Structure of Plant UDP-glucose: P-Glucan Synthase Actiuator
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