Induction of “Pore” Formation in Plant Cell Membranes by Toluene

Abstract

Treatment with aqueous toluene-ethanol has been shown to induce "pore" formation in plant cell membranes. The evidence i as folows: Althoug the phenomenon of pore induction is qultatively simlar to that in microorganisms, the pores induced appear to be smaler. It is proposed that induced leakage could be the bas for the development of simple and rapid methods for plant biochemial studies.Biochemical studies of cells provided with hard outer walls are hindered by the technical difficulties involved in homogenization. In the case of microorganisms a way of bypassing these difficulties has been developed. The formation of small holes is induced in the cell membrane by suitable techniques, the simplest being treatment with toluene (2, 5-7, 10, 12, 14-16, 19, 20 Preloading of the cytoplasm with fluorescein was achieved by incubating the roots for 10 min at room temperature in a 10 mm solution of KH2PO4 containing 1 mg of fluorescein/10 ml.Loading with ANS2 was achieved by incubating the roots for 10 min at room temperature in a 50 gM solution of ANS brought to pH 6 by addition of dilute NaOH containing 1% toluene and 4% ethanol.Cytoplasmic fluorescence induced by either fluorescein or ANS was observed with a Leitz Ortholux fluorescence microscope using transmission filters BG38 (4 mm) plus BG12 (3 mm) and suppression filter K530. Preloading of the vacuoles with neutral red was achieved by incubating the roots in an aqueous solution of the dye (1 mg/10 ml). Potassium phosphate buffer (pH 7.5) was then added to a final concentration of 10 mm and incubation continued for 1 hr. Although neutral red precipitates at this pH, the non-ionized species which is formed is lipid-soluble and penetrates the cellular membranes. After incubation the roots were rinsed several times in distilled H20.The procedure for compartment analysis was essentially similar to that described by Glinka (3). Course of influx of '4C-thiourea into Atriplex root at room temperature was found to be considerably faster than into carrot root. After 1 hr the labeled thiourea had reached equilibrium with the tissue. Atriplex roots were therefore loaded by incubation in 50 mm 14C-thiourea for 1 hr at room temperature. Efflux was measured by transferring the roots, in a holder, sequentially through a series of vials containing 6 ml of 50 mm nonradioactive thiourea. The vials were shaken constantly and were maintained at 4 C to slow down the process. During the first 2 min the roots were transferred every 15 sec to wash off the radioactivity adhering to the holder and to the surface of the roots. Subsequent efflux periods were progressively longer and continued for 3 hr.

Fractal morphology of Beta vulgaris L. cell suspension culture permeabilized with Triton X-100®

“…This can be analogous to the change in shape and size caused by the shrinkage and therefore, the increase in FD that certain biological materials undergo when dried (Chanona et al, 2003;Alamilla-Beltrán et al, 2005). It has been reported that one of the mechanisms of the permeabilization process is the formation of micropores or microchannels through the cell wall that allow the flow of metabolites (Lerner et al, 1978;Brodelius, 1988). The quantity of micropores and their temporality are functions of the type, concentration and contact time of the permeabilizing agent.…”

Kinetics of Toluene-Induced Leakage of Low Molecular Weight Solutes from Excised Sorghum Tissues

“…Lerner et al (7), showed that toluene treatment induced pore formation in A triplex roots and allowed the leakage of low mol wt substances. Only compounds of mol wt less than that of NAD were able to leak out of roots.…”

“…Not only did low mol wt solutes diffuse out of the cells, but other compounds could be introduced into the cells to serve as substrates for in situ enzymic reactions. Lerner et al (7) have applied the toluene treatment method to plant roots. From their experiments they concluded that the size of the pores formed by toluene in Atriplex root cell membranes are smaller than those formed in membranes of microbial or mammalian cells.…”

Exogenous DNA Uptake via Cellular Permeabilization and Expression of Foreign Gene in Wheat Zygotic Embryos.

“…(Parihar et al, 1994). Interestingly, the embryos treated with toluene, a widely known inducer of transient `pore' formation of specific sizes (Lerner et al, 1978), exhibited significantly higher frequency of GUS expression (78%) at 1:20 v/v dilution with permeabilization buffer (Fig. 1).…”