Journal o f CI en i "al Ecology. Vol. 2 I. No. I I. 1995

PHLOEM TRANSPORT OF ANTIRRHINOSIDE, AN IRIDOID GLYCOSIDE, IN Asarina scandens (SCROPHULARIACEAE)

E S T H E R G O W A N , t B E T T Y A. LEWIS,-" and R O B E R T T U R G E O N I * ~Sec'tion ~1 Phmt Biology :Division o f Nutritional Sciem'es Cornelf UnivetMtv Ithaca, New t)~rk 14853

(Received March 23. 1995: accepted July 8. 19951 Abstract--lridoid glycosides, tcrpene-derived compt~unds found in many plant thmiiies, protect the plant against generalisf and nonadapled specialist insect herbivores, fungi, and bacteria. Antirrhinoside. a common iridoid glycoside in lhe tribe Antirrhineae (Scrophulariaceae), was nlpidly labeled when mature leaves of ,4sarina scamh'ns were exposed to ~4CO,. Antirrhinoside was finnslocated in the phloem along with sucrose. Radiolabeled amirrhinoside appeared in the petiole of the labeled leaf within 20 rain of the beginning of the labeling period. Amirrhinoside was also flmnd in phloem sap obtained by the EDTA method. Key Words--Antin-hinoside. ,4sarim~ .~camtens, iridoid glycoside, phloem, Scrophulariaceae. mmslocation.

INTRODUCTION Plants synthesize a wide array of c o m p o u n d s to protect them against herbivores and pathogens. If any o f the c o m p o n e n t s o f the chemical defense system are translocated, the plant gains a potential defense against p h l o e m - f e e d i n g organisms and also gains the ability to coordinate the synthesis and use o f protective resources in various organs, Note, for example, the systemic nature of signals in acquired resistance to virus, bacteria, and fungi (Ryals et al., 1994) and to insects ( N a r v a e z - V a s q u e z et al., 1994).

*To whom correspondence should be addressed. 1781 00t18-033119511100-178 I~J7.50/0 ' ' [995 Plenum Puhli~hila[l Corp~.witlion

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An ideal candidate class of phloem-mobile defensive chemicals is the group of terpene-derived compounds known as iridoid glycosides (Bowers, 1991). There are almost 600 known iridoid glycosides in at least 57 families of plants (Bowers, 1991). Some of these compounds, which can comprise almost one quarter of the dry weight of certain leaves (Bowers, 1991; Boros et al., 1991), are among the most bitter substances known and are effective in protecting many plant species against generalist and nonadapted specialist insect herbivores. They may also inhibit seed germination and seedling growth (Adam et al., 1979) and act as toxins to both fungi (van der Sluis et al., 1983) and microbes (Kubo et al., 1985). Many insects sequester iridoid glycosides that they obtain from plants in order to defend themselves against potential predators (Bowers, 1991: Bowers and Farley, 1990). Iridoid glycosides are synthesized via the mevalonic acid pathway (Inouye and Uesato, 1986). Many are potentially phloem-mobile because they are of low molecular weight, water soluble, and nonreducing, as are other phloemmobile substances. We report here that antirrhinoside, a common iridoid glycoside found throughout the tribe Antirrhineae in the family Scrophulariaceae (Kooiman, 1970) and sequestered by aposematic moth larvae (Boros et al., 1991 ) is translocated in Asarina scandens. METHODS AND MATERIALS Plant Material. Seeds of Asarina scamlens (Cav.) Penn. were obtained from Thompson and Morgan (Jackson, New Jersey). Plants were grown in a greenhouse in artificial soil (Metro-Mix 350: E.C. Geiger, Harleyville, Pennsylvania) and used for experinaentation when approximately 3 months old (Turgeon et al,, t993). Daylight in the greenhouse was extended to I6 h with fluorescent lamps. Analytical Methods. Mature leaf blade tissue was frozen and ground in liquid N~ and extracted at 50°C in methanol-chlorofoma-water ( 1 2 : 5 : 3 , v/v/v) (Haissig and Dickson, 1979). Water was then added to obtain biphasic partitioning of the extract (3 parts water per 5 parts extract). Following centrifugation (2,000g, 10 rain), the separated aqueous phase was passed through ionexchange membranes [Bio-Rex AG50W cation membrane (H ~ form) and AG1 anion membrane (carbonate form)] (Bio-Rad, Richmond, Cali[bmia) (Turgeon and Gowan, 1992), filtered through a 0.22-#m nylon spin filter, and separated by HPLC using a Waters (Milford, Massachusetts) model 510 pump, a Waters SugarPak 1 column held at 90°C, and a Waters model 410 differential refractometer, Water was used as the mobile phase. A large peak migrating at 10.5 rain was isolated by pooling three 0.5-rain fractions and dried down at 70°C under a stream of N 2 gas. When rechromatographed by the same methods, 96% of the pooled material ran as a single peak.

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Two-dimensional TLC on plant extracts and purified compounds was performed using silica gel GHL plates (Analtech, Newark, Delaware), pretreated by dipping in a solution of 0.03 M boric acid in 60% ethanol (v/v), and activated at 95-110°C for 1 h. The first development was in chloroform-acetic acidwater (6 : 7 : 1, v/v/v) and the second in isopropanol-water (5 : 1, v/v). NMR spectroscopy of the purified material was performed in a 5-mm tube in D~O on a Varian VXR-400S with acetone as internal reference. The ~3C spectrum was obtained in the proton-decoupled mode. Carbon-13 chemical shifts were corrected to 61.55 ppm for C-6 of the glucosyl residue. Radiolabeling Methods. Plants were brought into a laboratory fumehood illuminated by a water-filtered incandescent 1000-W metal-halide lamp providing approximately 400 /~mol photons/m2/sec (photosynthetically active radiation) at plant ]evel. Approximately 1 hr later a mature leaf blade was enclosed in a plastic bag and I4CO2 (1.5 MBq), generated in the barrel of a syringe by addition of excess 80% lactic acid to [~4C]Na~CO~ (6.6 x 10-s MBq/mmol), was iniected into the bag without delay. The petiole of the labeled leaf was covered with aluminum toil to prevent accidental photosynthetic incorporation of leaked gas, After a 5-rain exposure to HCO~, the bag was removed and, following a chase period in room atmosphere, the leaf blade and petiole were quickly removed and separately plunged into liquid N~. Tissue was extracted and analyzed by TLC as described above. Radiolabeled spots were detected with X-ray film, scraped from the plate, and quantified by scintillation counting. Phloem Exu~kue. Ten leaves were cut at the base of the petiole, in the middle of the day, and immediately recut under water. The leaves were then placed in a single group in a small tube so that the petiole bases were immersed in 0.2 ml of a solution of 20 mM EDTA in 20 mM PIPES buffer (pH 6.8). The leaves were then placed~ in their tube, in a humid chamber in complete darkness at room temperature. The EDTA solution was entirely removed for analysis and replaced every hour for 4 hr. The solutions were purified by ion-exchange chromatography and the neutral fraction analyzed by HPLC as described above.

RESULTS

ldentificatirm c~/'Antirrhinoside. In extracts of A. scandens leaf blade tissue we detected an HPLC peak that eluted at 10.5 rain from the SugarPak column, while sucrose eluted at 7.8 rain. There was considerably more of this material (9.5 mg/g fresh weight) than sucrose (1.4 mg/g fresh weight) or any other component of the neutral fraction. This substance was present in all five species of the Antirrhineae that we previously studied (Turgeon et a[., 1993). It cochromatographed with several monosaccharide standards on the SugarPak column but tested negatively as a reducing compound with Benedict's solution or 2,2'-bicinchoninate (Sturgeon, 1990). On TLC plates it produced an intense

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GOWAN, LEWIS~ANDTURGEON

OH

CH3

H O- Glu

FIG. 1. Structure of amirrhinoside.

dark brown to black color with Seliwanoff's reagent (Zweig and Shemm, 1972) or vanillin-H2SO4 reagent (Touchstone and Dobbins, 1983) and a blue fluorescence with p-anisidine phosphate reagent (Kooiman, 1970). Glucose was produced upon hydrolysis with 2 N tfifluoroacetic acid (room temperature tor 2 hr). The compound was identified as antirrhinoside (Figure 1) by IH and t3C NMR. Chemical shifts and coupling constrants tbr protons of the aglycone and the anomeric carbon (HI') of the glucosyl unit agreed closely with reported values tot antirrhinoside (Scarpati et al.. 1968). Carbon-13 NMR data confirmed the identity of antirrhinoside. The chemical shifts lbr all carbons in the aglycone and glucosyl unit were in close agreement with published values (Bianco et al., 1981). Translocation Eweriments. Antirrhinoside in the lamina became labeled. although not as quickly as sucrose, tbllowing exposure of the leaf to ~4CO, (Figure 2). With increasing time the anaount of labeled antirrhinoside increased in both absolute terms and in proportion to sucrose so that within an hour the label in antin-hinoside was approximately one quarter that of sucrose. To determine if antirrhinoside is translocated, the petioles of the same leaves were extracted. Radiolabeled sucrose and antirrhinoside were first found in the petioles in measurable quantities 20 rain after exposure of the leaves to HCO~. and the amount of these radiolabeled compounds increased rapidly over the next 40 rain (Figure 2). The proportion of labeled antirrhinoside in the petiole increased in the same way that it did in the lamina. It is important to note that radioactive antirrhinoside was already present in the translocate when label was first detected in the petiole. This rules out the possibility that labeled antirrhinoside is synthesized in the petiole from transported sucrose, rather than being translocated to the petiole from the leaf blade. At any given time point, the ratio of labeled antirrhinoside to labeled sucrose was lower in the petiole than in the leaf blade. This lag in the labeling pattern

PHLOEM TRANSPORT OF ANTIRRHINOSIDE

[ 785

30 • O ~

25-

ffl 211-

.c_ .D

~ 10~ u

o

Petiole o

o

i 30

i 40

5 0

i 10

i 20

Time

i 50

i 60

(rain)

Fic. 2. Labeling of antirrhinoside in the lamina and petiole of Asarina scandens leaves following exposure of the leaf blade to t4CO~. The amount of label in antirrhinoside is expressed in a percentage of that in sucrose.

of the compound in the petiole in comparison to the blade is to be expected, since the composition of translocate entering the petiole is a reflection of the labeling pattern in the mesophyll cells at an earlier point in time. While labeling experiments demonstrated that antirrhinoside is transported, the experiments did not reveal the concentration of this compound in the phloem sap. Samples of sap, obtained by the EDTA method (King and Zeevart, 1974), were composed mainly of sucrose with smaller amounts of raft]nose and stachyose, as previously shown (Turgeon et al., 1993), and also contained antirrhinoside (Figure 3), as well as monosaccharide. The amount of antirrhinoside was approximately 7% that of sucrose. If it is assumed that the concentration of sucrose in the phloem is in the range of 1 M (Winter et al., 1992), the concentration of antirrhinoside would be approximately 70 raM.

DISCUSSION

The data reported here indicate that antirrhinoside is translocated from source leaves to other parts of the plant in the phloem of Asarina scandens. Very little work appears to have been done on the transport of iridoid glycosides. Aucubin is transferred from Penstemon teucrioides to the parasitic plant Castilleja integra (Stermitz et al., 1993) but the route of transport has not been determined. An aphid, Acyrthosiphon nipponicus, sequesters paederoside, an

1786

GOWAN, LEWIS, AND TURGEON

2O

~,_ 10

l

0

Flcl. 3. Sugars in the phloem sap obtained from leaves ofAsarina scandens by the EDTA method and measured by HPLC. The amount of each compound is expressed as a percentage of the amount of sucrose in the sap. Fructose and myoinositol coeluted. Bars = SE (N = 4).

iridoid glycoside that protects it from ladybird beetles (Nishida and Fukami, 1989); since aphids are known to feed in sieve elements, it is reasonable to conclude that this iridoid is also phloem mobile. Considered together the results indicate that phloem transport of iridoid glycosides may be a common occurrence.

The labeling of antirrhinoside after exposure of the leaf to ~4CO2 follows a complex pattern, increasing in the leaf blade and lamina over time in proportion to sucrose. While there is more antirrhinoside than sucrose in the leaf blade, there appears to be a higher proportion of sucrose in the transport system, as determined by the EDTA exudation method. These data are consistent with the occurrence of two pools of antirrhinoside in the leaf blade, a large nonmobile pool, presumably in the vacuoles, and a high-specific-activity transport pool that is the source of antirrhinoside in the phloem. It should be noted, however, that the EDTA procedure on which these conclusions are partially based provides only a rough estimate of phloem sap composition (Girousse et al., 1991). While it is clear from our labeling experiments that antirrhinoside is transported, the data we report here on the composition of the phloem sap should be considered tentative until they are confirmed by other methods, such as collection of sap from aphid stylets. On the basis of the intbrmation at hand it is difficult to know whether the

PHLOEM TRANSPORT 01- ANTIRRHINOSIDE

1787

a m o u n t o f a n t i r r h i n o s i d e in the p h l o e m w o u l d be high e n o u g h to d e t e r p h l o e m f e e d i n g o r g a n i s m s such as a p h i d s . First, we have b e e n unable to d e t e r m i n e the c o n c e n t r a t i o n o f t r a n s p o r t e d a n t i r r h i n o s i d e with c o m p l e t e c o n f i d e n c e , as disc u s s e d a b o v e . S e c o n d , d e t e r r e n c e tests are usually c o n d u c t e d with dry diets, so it is not p o s s i b l e to c o m p a r e t h o s e results with o u r o w n . H o w e v e r , the fact that a n t i r r h i n o s i d e is p r e s e n t in the p h l o e m in easily d e t e c t a b l e quantities indicates that such d e t e r r e n c e m a y be p o s s i b l e . F u r t h e m l o r e , if i m p o r t e d a n t i r r h i n o s i d e is stored in sink tissues, it will a c c u m u l a t e to high levels o v e r time; thus the t r a n s p o r t e d c o m p o u n d c o u l d be an i m p o r t a n t factor in p r o t e c t i n g g r o w i n g r e g i o n s o f the plant from h e r b i v o r e s . Acknowledgments--Support Ior this work was derived I'mm NSF grant DCB-91(MI59 and Department of Agriculture Competitive Grant 94-37306-0351.

REFERENCES ADAM. O., KHOt, N,H., BERGNER, C.. and LI~N, N.T. 1979. Phmt growth inhibiting properties of plumieride l?om Plumeria otmfs(folio. Phymchemistry 18:1399-14(~. BI..x~:c~. A.. C,~cioI.A.P.. GuIso. M.. LAv.\Ro~4F.. C.. and TI~(~(;~L(). C. 198l. lridoids. XXX1 Carbon-13 nuclear magnetic resonance specm~scopy of free iridoid glucosides in D:,O solution. Gazz. Chim. hal. 111:201-206,

Bolos, C.A.. S'rv~Mlrz, FR.. MCFARLANI),N. t991, Processing of iridoid glycoside anlimnoside from Maurandya antirrhiniflora (Scrophulariaceae) by Meris paradoxa (Geometridae) and Lepipolys species t Noctuidae). J. Chem. Ecol. 17:tl23-1t33. B()\~,I~Rs, M.D. 1991, Iridoid glycosides, pp. 297-325, in G.A. Roscnthal and M.R. Bcmnbaum (eds.). Herbivores, Their Interactions with Secondary Plant Metabolites, Vo[. I, Academic Press. San Diego. BOWERS, M,D., and F,~.RLEY~S. 1990. The behavior of grey jays, Perisoreus canadensis, towards palatable and unpalatable Lepidoptera. Anita. Behav. 39:699-705. GIROUSSE, C.. BONNI:MAIN,J .L, DELROT. S., and BOURNOVILLE. R, 1991. Sugar and amino acid composition of the phloem sap of Medi('ado safiva. A comparative study of two collecting

methods. Phmt Physiol. Biochem. 29:41-48. HAISSlG, B.E., and DICKSON, R.E. 1979. Starch measurement in plant tissue using enzymatic hydrolysis, PhysioL Plant, 47:151 - 157. INOUVE, H., and U~:SATO,S. 1986. Biosynthesis of iridoids and secoiridoids. Prwg, Chem. Org. Nat, Prod. 50:169-236. KING, R.W., and ZEEVAR'r,L A D . 1974. Enhancement of phloem exudation from leaf petioles by chelating agents. Plant PhysioL 53:96-103. KOOIMAN, P. I970. The occurrence of iridoid glycosides in the Scrophutariaceae. Acta Bot. NeerL 19:329-340. KUBO, I,, MATSUMOTO.A., and TAKaSE. 1. 1985. A multichemical defense mechanism of bitter olive Olea europaea (Oleaceae). Is oleuropein a phytoalexin precursor? J. Chem. Ecol. 11:251263. NARVAEZ-VASQUEZ,J., OROZCO-CARDENAS,M . L , and RYAN. C.A. 1994. A sulfhydryt reagent modulates systemic signaling for wound-induced and systemin-induced proteinase inhibitor synthesis. Plant Physiol. 105:725-730.

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NISHIDA, R.. and FUKAMh H. 1989. Host plant iridoid-based chemical defense of an aphid, Ao'rthosiphon nipponicus, against ladybird beetles. J. Chem. EcoL 15:1837-1845. R'T'ALS, J., UKNES, S., and WARD, E. 1994. Systemic acquired resistance. Phmt PhysioL 104:11091112. ScarPazl, M.L , Gulso, M.. and Es~SlTO, P. 1968. Iridoidi IVL Struttura e configurazione dell" Antirrinoside. Gazz. Chim. ltal, 98:177-190. STErMtTZ. F,R., FODERARO,T.A.. and Lh Y-X. I993. Iridoid glycoside uptake by Castill~(ja int¢gra via root parasitism on Penstemon teucrioides. Phytochemis,3'. 32:1151-t 153. STURGEON, R.J. 1990. Monosacchaddes, pp. 1-37. i, P.M. Dey (ed.l. Methods in Plant Biochemistry, Vol. 2. Carbohydrates. Academic Press, New York. TOUCHSTONF, J.C., and DOBBINS, M.F. 1983. Practice of Thin Layer Chromatography, 2nd ed. Wiley, New York. p, 174, TUrGEON, R., and GOW.aN, E, 1992. Sugar synthesis and phloem loading in Coleus blumei leaves, Pluntu 187:388-394. TURC3FON, R.. BEEBF., D.U,, and Gowan, E. 1993. The intemaedi~,ry cell: Minor~vein anatomy and rattinose oligosaccharide synthesis in the Scrophulariaceae. Pla,ta 191:446-456. VAn DERSLU~S, W,G,, VaN I~F~RNAr, J.M.. and LaBADIE. R.P. 1983. Thin-layer chromatographic bioassay of iridoid and secoiridoid glucosides with a fungitoxic agtycone moiety using ~-glucosidase and the fungus Pe*m'iflium e.vpans,m as a test organism, J, Chroma,~gr. 259:522526. WINTER, H., LOHAUS, G., and HELDT, H.W. 1992. Phloem transport of amino acids in relation to their cytosolic levels in barley leaves. Plant Ph3wiol. 99:996-1004. ZWEIC;, G., and SHErMA, J. (eds). 1972. CRC Handbook of Chromatography. Vol. 2. CRC Press, Ohio. p. 129.