Suberization of tomato (Lycopersicon esculentum) locule tissue

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It was demonstrated by electron microscopy that wounded and cultured tomato fruit outer placental (locule) tissue generated lamellated (secondary) walls.
Suberization of tomato (Lycopersicon esculentum) locule tissue GONELLAS. R. L. RAOAND J. H. MARTINWILLISON' Biology Department, Dalhousie University, Halifax, N.S., Canada B3H 4JI AND

W. M. NIMALRATNAYAKE Canadian Institute of Fisheries Technology, Technical University of Nova Scotia, P.O. Box 1000, Halifax, N.S., Canada B3J 2x4

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Received March 12, 1985 and W. M. N. RATNAYAKE. 1985. Suberization of tomato (Lycopersicon esculentum) RAO,G. S. R. L., J. H. M. WILLISON, locule tissue. Can. J. Bot. 63: 2177-2180. It was demonstrated by electron microscopy that wounded and cultured tomato fruit outer placental (locule) tissue generated lamellated (secondary) walls. BF-,- methanol depolymerization of an extract from these walls and its chromatographic analysis showed the presence of the polymer suberin in the tissue adjacent to the wound after 7 days in culture. Quantitative studies using Iatroscan thin-layer chromatography coupled with flame ionization detection showed close similarities between the aliphatics of this wound suberin (which constituted 70% of the total monomers recovered) and that generated by protoplasts isolated from the same tissue, particularly among the monofunctional products, but some striking differences among the difunctional products. It is proposed that the results support the concept that protoplast isolation elicits a wound response similar to that elicited by mechanical wounding of the mother tissue, but that the physiological conditions obtained during protoplast isolation and culture result in a modification of this response. et W. M. N. RATNAYAKE. 1985. Suberization of tomato (Lycopersicon esculentum) RAO,G. S. R. L., J. H. M. WILLISON locule tissue. Can. J. Bot. 63: 2177-2180. L'on a dCmontrC par la microscopic Clectronique chez des fruits de tomate blessCs, en culture in vitro, que le placenta externe (locule) produit des parois secondaires. La dCpolymCrisation par le BS-mCthanol d'un extrait de ces parois et l'analyse chromatographique de cet extrait rCvklent la presence de subkrine dans le tissu adjacent B la blessure aprks 7 jours de culture. Des Ctudes quantitatives utilisant la chromatographie sur couche mince (Iatroscan) couplCe B la detection par l'ionisation de flamme ont montri de fortes ressemblances entre les composCs aliphatiques de cette subCrine (elle constitue 70% des monomkres totaux recouvrCs) et ceux produits par des protoplastes isolCs des mCmes tissus, en particulier au niveau des produits non fonctionnels, mais certaines diffkrences frappantes entre les produits disfonctionnels. On propose que les rCsultats appuient le concept selon lequel l'isolation des protoplastes provoque une rCponse aux blessures similaire B celle des blessures micaniques du tissu maternel. Cependant, les conditions physiologiques au cours de l'isolation et de la culture des protoplastes modifient cette rCponse. [Traduit par le journal]

Introduction Protoplasts obtained from the placental tissue lining the locules of green tomato fruits synthesize a multilamellar wall (MLW) that has been shown to contain suberin (Rao et al. 1984). Many plant tissues respond to experimental wounding by suberization of the cell walls near the wound both in vivo, as in the case of jade leaves, tomato fruit pericarp, and bean pods (Dean and Kolattukudy 1976), and in vitro, as in the case of potato tubers (Kolattukudy and Dean 1974). Based on these observations, we proposed (Rao et al. 1984) that the suberization of the isolated tomato fruit protoplasts might represent a wound response. If this is so, one might expect that the wound tissue itself would respond comparably, but there has been no previous study of the wound response of this particular tissue. This paper reports the results of a study in which superficial layers of the placenta of tomato fruit (sometimes referred to as "locule tissue") have been mechanically wounded and examined for evidence of a response to wounding using electron microscopy and chromatographic analysis. Materials and methods Tomato plants (Lycopersicon esculentum L. cv. Scotia) were grown in a roof-top greenhouse at Dalhousie University. Immature green 'Author to whom all correspondence should be addressed.

fruits of about 50 mm diameter were harvested and were surface sterilized by immersing them in 80% ethanol for 2-5 min. Later, they were washed in sterilized water. Part of the pericarp was removed with a razor blade. Areas of the placental tissue lining the locule that were firm and free of seed and seed pouches were selected, cut into about 2- to 4-mm' portions, and transferred to sterile Petri dishes containing a sterile nutrient medium previously described (Rao et al. 1984). The dishes were sealed with Parafilm and stored in the dark at room temperature. Cultures remained sterile throughout the incubation period. At various times, samples of the tissue were observed using a Zeiss (West Germany) large fluorescence microscope to check for yellow green fluorescence. After 7 days of culturing, small portions of the tissue samples were prepared for electron microscopy and the remainder were analyzed chemically. Methods for electron microscopy and chemical analysis for the detection of suberin were as described previously (Rao et al. 1984).

Results and discussion The outer placental (locule) tissue of tomato fruit is composed of large, vacuolate, thin-walled parenchymatous cells (Pearce 1972) having similarities with those of the outer pericarp tissue of the fruit (see Mohr and Stein 1969). There has been no report of cell wall associated lamellae of the sort found around cultured locule tissue protoplasts (Willison 1973; Rao et al. 1984) in locule tissue from whole uninjured fruit. Seven days after wounding, however, a MLW very similar in appearance to that generated by locule tissue protoplasts in isolation

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FIGS.1-3. Thin-section electron micrographs of cell peripheries from the outer placental tissue of tomato fruit, wounded and cultured, for 7 days. Fig. 1. A lamellated envelope (MLW) lies between the primary wall (PW) and the plasma membrane (PM). The cell appears dead, presumably as a result of suberization. A secondary pectocellulosic envelope (arrowheads) appears to have been generated after suberization. Fig. 2. Part of the suberized regenerated wall showing sublamellation within a compacted region of the MLW. Fig. 3. A portion of the wall generated by a tomato fruit protoplast exhibiting loose arrangement of the MLW. Two types of lamellae are evident: a dark-stained thick type (large arrows) and a light thin type (small arrows).

arose beneath the original walls of cells associated with the wound (Figs. 1-3). The precise form of this wound-induced wall material varied from cell to cell within the tissue blocks examined (Figs. 1-3). It is possible that this variation was related to the distance from the wound surface, but the large cell size meant that insufficiently large blocks could be examined to allow any such relationship to be determined unequivocally. It was clear, however, that the range of variation was comparable to that found in populations of cultured isolated tomato locule tissue protoplasts (Willison and Cocking 1972; Willison 1973). This variation included the presence (Fig. 1) and absence (Fig. 3) of a microfibrillar wall, which we presume to be pectocellulosic, beneath the MLW (i.e., deposited after the deposition of the MLW). The form of the MLW also varied (Figs. 1-3) notably with respect to the relative quantities of the thinner and thicker types of lamellae (for description of these lamellae in isolated protoplasts see Willison, 1973). There was also variation in the degree to which the lamellae were compacted, with open arrangements predominating over close compaction (the latter being visible in Fig. 2). Correspondingly, using ultraviolet microscopy, yellow green autofluorescence

was evident in the walls of cells surrounding the wound 7 days after the wounding, but was completely absent when samples of locule tissue were first excised. Yellow green autofluorescence is characteristic of phenolic compounds associated with suberized layers in green cotton fibres (Ryser et al. 1983). Iatroscan thin-layer chromatography coupled with flame ionization detection (TLC-F1D) of depolymerized extracts obtained from the wounded tissue (Fig. 4) showed the presence of fatty acid methyl esters (FAME), diacid dimethyl esters (DME), fatty alcohols (FAI), o-hydroxy acid methyl esters (o-OH), and two unidentified components with R Fvalues 0 and 0.5. These compounds are diagnostic of suberin (see Dean and Kolattukudy 1976; Kolattukudy 1978). The aliphatics comprised 70.2% of the total monomers, the remainder being aromatic compounds, presumably phenolics of the types usually associated with suberins. The proportion of aliphatics contrasts with that for the MLW suberin secreted by protoplasts isolated from the locule tissue (Rao et al. 1984), in which only 32% of the monomers were aliphatic. The relative proportions of the classes of aliphatic compounds (FAME, DME, FAl, o-OH) in locule tissue suberin is given in Table 1 together with those

RAO ET

AL.

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TABLE1. Aliphatic monomer compositions (weight (percent)) of suberins Lipid class

Locule tissue

FAME FA1 DME o-OH Unknown

47 12 20 21 -

Protoplasts"

Carrot"

Sweet potato"

51 8 21 5 15

14 3 24 31 28

9 4 21 36 30

Rutabaga"

Potato skinc

Ribes"

4 5

15

-

11

22

44

36 33

-

1 36 50 13

30

NOTE:Each value indicates the percentage of the toral aliphatic monomers given in that column, values having been adjusted to the nearest whole numbers. " Rao er a / . 1984. Kolattukudy 1978. 'Kolattukudy and Agrawal 1974. "Holloway 1972.

TABLE2. Monofunctional products (weight (percent)) from the depolymerized wall extract of tomato locule tissue as determined by GLC Chain length of monofunctional product"

FAME

FA1

"The general formula for monofunctional molecules is n : x w y , where n is the number of carbon atoms in the chain, x is the number of double bonds, and w y is the position of the double bond closest to terminal CH, group.

FIG.4. Iatroscan TLC-FID of the depolymerized wall extract obtained from tomato locule tissue cultured for 7 days. One microlitre of the sample was spotted on Chromarod S-I1 and developed in a solvent system consisting of hexane - diethyl ether - formic acid (95 :5 :0.1, by volume) for 30 min and scanned on the Iatroscan FID. D, diacid dimethyl esters (DME); E, end of scan; F, fatty alcohols (FAI); M, fatty acid methyl esters (FAME); 0 , origin; S, start of scan; U, unidentified; W, o-hydroxy acid methyl esters (o-OH).

reported from isolated protoplasts (Rao et al. 1984), carrot, sweet potato, rutabaga (Kolattukudy 1978), Ribes (Holloway 1972), and potato skin (Kolattukudy and Agrawal 1974) for the purpose of comparison. Both the locule tissue suberin and that

of the isolated protoplasts show comparatively high levels of FAME, a result which corresponds with the findings of Dean and Kolattukudy (1976) on wounded tomato pericarp. Details of the gas-liquid chromatographic (GLC) analysis of the monofunctional products (FAME and FAl) are given in Table 2. The major fatty acids were CI6,CI8,CI8:,,(unsaturated), CZ0,and CZ2,as was the case with the FAME of tomato fruit protoplast suberin (Rao et al. 1984). Even within the predominant C 18;,, group, there were close parallels between the suberins generated by protoplasts in isolation and by the mother , and Czz locule tissue. Among the fatty alcohols, C16, C I S CZ0, were the major components. Although a somewhat similar distribution was reported for tomato fruit protoplast suberin (Rao et al. 1984), locule tissue suberin differs from the former by containing relatively large amounts of C14and C16fatty alcohols. GLC analysis of the difunctional products (DME and o-OH) is given in Table 3. Notable differences between the difunctional products of the locule tissue suberin and those of the isolated locule protoplast suberin are (i) the absence of C14 DME from the tissue suberin (while these constitute 24% of the

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TABLE 3. Difunctional products (weight (percent)) from the depolymerized extract of tomato locule tissue as determined by GLC.

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Chain length of difunctional product

DME

w-OH

products. An equally interesting possibility, which we plan to investigate, is that in addition to physical wounding, the presence of fungal enzymes contributes to the precise suberization response elicited from isolated protoplasts. Acknowledgements We are grateful to Dr. R. G. Ackman for providing access to lipid analytical facilities. This work was supported by Natural Sciences and Engineering Research Council of Canada grant A0507 to J. H. M. Willison. ASAMIZU, T., and A. NISHI.1980. Regenerated cell walls of carrot

"Unsaturated.

,,

protoplast DME), (ii) the relative abundance of C in the tissue DME (43% vs. only 2% in the protoplast suberin), and (iii) the absence of C 1 3 , C C IX:,, , CIO,and C12 W-OH from the tissue suberin, despite their presence in that of the protoplasts. COUpled with the difference in the relative proportions of w-OH in the suberins generated by the protoplasts and by the locule tissue (Table I), the difference between the detailed responses of the two systems is striking. While there are differences between the suberins generated by the locule tissue and its isolated protoplasts, it is important to see these differences in the context of the range of variability that has been reported for suberins. This is not an appropriate place to attempt a detailed analysis, but Table 1 permits an approximate judgement of the extent to which the act of protoplast isolation has affected the suberization response. While the locule tissue suberin and the corresponding protoplast suberin are related (more so than, for example, locule tissue suberin and that of potato skin), the closeness of this relationship appears to be less than that between, for example, the suberins generated by the wounded tubers of rutabaga and sweet potato, despite the taxonomic gap. Tentatively, we suggest not only that tomato locule tissue protoplasts exhibit a wound response as they generate cell walls (which the present work has established to be a response that is characteristic of the mechanically wounded mother tissue), but also that this wound response is aberrant. In this respect, there are perhaps parallels between suberization of isolated protoplasts and the regeneration of polysaccharidic walls (for reviews, see Willison and Klein 1982; Burgess 1983). Several recent reports (Asamizu and Nishi 1980; Blaschek et al. 1981; Takeuchi and Komamine 1981; Franz et al. 1983) have established that the polysaccharide compositions of the walls regenerated by isolated protoplasts are distinctly different from those of the walls surrounding the cells from which the protoplasts were isolated. It has previously been suggested (Hanke and Northcote 1974) that an important contributing factor in the determination of the composition of the polysaccharidic regenerated wall is the loss of certain fractions to the incubation medium of isolated protoplasts in culture. Perhaps the same might apply to suberization, there again being no preexisting wall to confine the secreted

protoplasts isolated from suspension-culture cells. Plant Physiol. 48: 207-212. BLASCHEK, W., D. HAASS,H. KOEHLER, and G. FRANZ. 1981. Cell wall regeneration by Nicotiarza trrbac~rrnprotoplasts: chemical and biochemical aspects. Plant Sci. Lett. 22: 47-57. BURGESS, J. 1983. Wall regeneration around isolated protoplasts. Int. Rev. Cytol. Suppl. 16: 55-77. DEAN,B. B., and P. E. K O L A ~ U K U 1976. D Y . Synthesis of suberin during wound-healing in jade leaves, tomato fruit and bean pods. Plant Physiol. 58: 41 1-416. FRANZ, G., W. BLASCHEK, D. HAASS,and H. KOEHLER. 1983. Biosynthesis of cellulose: studies with tobacco protoplasts and cultured cells. J. Appl. Polymer Sci. Appl. Polymer Symp. 37: 145-155. HANKE, D. E., and D. H. NORTHCOTE. 1974. Cell wall formation by soybean callus protoplasts. J. Cell Sci. 14: 29-50. HOLLOWAY, P. J. 1972. The suberin composition of the cork layers from some Ribes species. Lipids, 9: 158- 170. K O L A ~ U K U DP.Y ,E. 1978. Chemistry and biochemistry of the aliphatic components of suberin. In Biochemistry of wounded plant tissues. Edited by G. Kahl. Walter de Gruyter, Berlin. pp. 43-48. KOLAT~UKUDY, P. E., and V. P. AGRAWAL. 1974. Structure and composition of aliphatic constituents of potato tuber skin (suberin). Lipids, 9: 682-69 1. KOLAT~UKUDY, P. E., and B. B. DEAN.1974. Structure, gas chromatographic measurement, and function of suberin synthesized by potato tuber tissue slices. Plant Physiol. 54: 116- 121. MOHR,W. P., and M. STEIN.1969. Fine structure of fruit development in tomato. Can. J. Plant Sci. 49: 549-553. PEARCE, R. S. 1972. The culture of isolated higher plant protoplasts. Ph.D. thesis, University of Nottingham, Nottingham, U.K. RAO, G. S. R. L., J . H. M. WILLISON,and W. M. N. RATNAYAKE. 1984. Suberin production by isolated tomato fruit protoplasts. Plant Physiol. 75: 716-7 19. RYSER,U., H. MEIER, and P. J. HOLLOWAY. 1983. Identification and localization of suberin in the cell walls of green cotton fibres (Gossypiurn hirsuturn L., var. green lint). Protoplasma, 117: 196-205. TAKEUCHI, Y., and A. KOMAMINE. 1981. Glucans in the cell walls regenerated from Virzca rosea protoplasts. Plant Cell Physiol. 22: 1585- 1594. WILLISON,J. H. M. 1973. Fine structural changes occurring during the culture of tomato fruit protoplasts. Colloq. Int. C.N.R.S. No. 212. pp. 215-241. WILLISON, J. H. M., and E. C. COCKING. 1972. 'The production of microfibrils at the surface of isolated tomato fruit protoplasts. Protoplasma, 75: 397 -403. WILLISON, J. H. M., and A. S. KLEIN.1982. Cell-wall regeneration by protoplasts isolated from higher plants. In Cellulose and other natural polymer systems. Edited by R. M. Brown, Jr. Plenum Publishing Corp., New York. pp. 61 -85.