Suberin Production by Isolated Tomato Fruit Protoplasts

Abstract

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...

Purification and characterization of hydroxycinnamoyl-coenzyme A: ?-hydroxypalmitic acid O-hydroxycinnamoyltransferase from tobacco (Nicotiana tabacum L.) cell-suspension cultures

“…The physiological significance of the occurrence of HHT activity in tobacco cells grown in suspension is, however, presently difficult to assess, whereas the occurrence of HHT in wound-healing potato tuber discs was clearly correlated with suberization (Lofty et al 1994). Very little is known about suberin formation in tobacco (Kolattukudy and Espelie 1989) and, to our knowledge, suberin, or suberin-associated alkyl ferulates, have never been characterized in cell suspensions, although suberin synthesis has been observed in ABAtreated potato callus cultures (Cottle and Kolattukudy 1982) and in isolated tomato fruit protoplasts (Rao et al 1984). We have checked that HHT activity is not enhanced when tobacco cell cultures are treated with 10 -4 M ABA (see Materials and methods).…”

Suberization of tomato (Lycopersicon esculentum) locule tissue

“…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.…”

“…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.…”

Isolation and culture of cotton ovule epidermal protoplasts (prefiber cells) and analysis of the regenerated wall

“…In an intact plant, a process that is analogus to cell wall regeneration by protoplasts is the formation of the phragmoplast which forms the cell plate. A similar process in which a wall is formed de novo is also encountered in cellular response to injury [9] and many protoplast systems regenerate cell walls containing increased levels of/3.1,3-1inked glucans similar to callose [2,7,20,28,34], or in the case of tomato fruit protoplasts, the wound response is the production of subrin [31 ].…”