The multilamellar wall secreted by protoplasts isolated from locule tissue of tomato (Lycopersico scuklntum L.) fruit was purified, nd an extract was obtained after depolymerization with BF3-methanol. Analysis of this extract using thin layer chromatography demonstrated the presence of fatty acid methyl esters, fatty alcohols, dicarboxylic add dimethyl esters, and w-hydroxy acid methyl esters. These components were quantified using an Iatroscan thin layer chromatography-flame ionization detection system. The different chain lengths in each group were identified and quantified using gas chromatography. The results clearly indicated the presence of suberin.Plant protoplasts are valuable experimental systems for the study of cell-wall secretion (see reviews: 14, 17): they permit the examination ofthe process from a unique wall-less starting point; they eliminate cellular interactions which complicate tissue experimental systems; and the lack of wall around the initial isolated protoplast makes it feasible to attempt to distinguish the interrelated processes of the biosynthesis, the secretion and the assembly of individual cell wall components.Fruit locule tissue protoplasts of tomato synthesize two types of envelope within 24 h of culture; some of these protoplasts make a wall having a pectocellulosic appearance, others make a wall which appears multilamellated in thin sections examined electron microscopically (13). After making the mlw2 some of the protoplasts secrete a second envelope of the pectocellulosic type (13,16 The protoplast band which formed at the top ofthe gradient was collected in glass Petri dishes and suspended in Murashige and Skoog culture medium (12) with 20 ml/l coconut milk. The dishes were sealed with Parafilm and stored in dark at room temperature (20-25°C). In general, the protoplast isolation and the culture procedures followed those of Gamborg and Wetter (4).For electron microscopy, 7-d-old protoplasts were harvested and fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer and similarly buffered 2% OSO4. They were embedded in TAAB embedding resin (TAAB Laboratories, Reading, U.K.), and sections were poststained with uranyl acetate and lead citrate.Preparations of the mlw material were obtained from protoplasts after 7 d culture, using a well-established procedure for the isolation of suberized lamellae (3). In this procedure, protoplasts were rinsed in distilled H20, ground with pestle and mortar, treated with pectinase and cellulase, then rinsed in water again. Soluble lipids wee extracted from the mlw isolate, and the polymer was depolymerized with BF3-methanol, again using procedures of Dean and Kolattukudy (3). Extracts were prepared from tomato periderm wound callus by the same methods.TLC was performed on Adsorbosil-5 Prekotes (Applied Science Laboratories Inc.) of dimensions 20 x 20 cm, activated at 1 1OC for 30 min. Separation of the lipid fractions was effected in the solvent system: hexane/diethyl ether/acetic acid (85/15/ 1, v/v/v). After spraying with 2', 7'-dich...
1985. Suberization of tomato (Lycopersicon esculentum) 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. RAO, G. S. R. L., J. H. M. WILLISON et W. M. N. RATNAYAKE. 1985. Suberization of tomato (Lycopersicon esculentum)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]
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