DNA fiber autoradiography of tobacco mesophyll protoplasts

DNA fiber autoradiography was successfully applied to protoplasts isolated from tobacco leaf mesophyll. After an incubation period of 37 h, DNA began to be labeled with ³H-thymidine. These cells, with regenerated thin cell walls, were then effectively disrupted in a lytic solution containing sodium dodecyl sulfate. DNA fiber autoradiography confirmed that the mean replicon size of this plant is approximately 60 in good agreement with the values reported previously in animal cells.     

Energization of the sugar transport mechanism in the plasmalemma of isolated mesophyll protoplasts

The mechanism of 3-O-methyl-d-glucose transport through the plasmalemma has been investigated in protoplasts isolated from the mesophyll of Pisum sativum L. var. Dan.Analysis of the fluxes after 50 minutes of uptake showed that the gradual decrease in slope of the net uptake curve with time was not due to any decline in uptake capacity; it represented the approach to flux equilibrium of a small compartment of the protoplast, probably the cytoplasm.The energy of activation for initial flux into this compartment was 20 kilocalories per mole between 17 and 27 C. Very high discrimination was shown with regard to sugar isomers. Light strongly promoted flux (by a factor of 2.5 in the case of methyl glucose). Initial flux showed sharply contrasting inhibitor sensitivity in the light and the dark. Light uptake was sensitive to the proton conductor carbonyl cyanide m-chlorophenylhydrazone (CCCP), but stable for at least the first 10 minutes to the ATPase inhibitors quercetin, rutin, and diethylstilbestrol, as well as to arsenate. Dark uptake, on the other hand, was stable to CCCP but was immediately depressed by quercetin, rutin, diethylstilbestrol, and arsenate.Protoplasts which received a light pretreatment before incubation in the dark took up methyl glucose at the accelerated light rate for the first 7 minutes. Moreover, the light pretreatment sensitized subsequent initial dark uptake to CCCP, and conferred on it the stability to ATPase inhibitors and arsenate characteristic of light uptake. After about 7 minutes the characteristic inhibitor responses of dark uptake were resumed.It is proposed that more than one mode of energy-coupling for sugar transport may operate in these protoplasts.     

Perception of the auxin signal at the plasma membrane of tobacco mesophyll protoplasts

Auxin-induced variations of transmembrane potential difference have been shown to be a useful tool for analyzing hormone sensitivity in tobacco protoplasts. Using this technique, we demonstrated that protoplasts derived from wild-type, an auxin-resistant mutant and Agrobacterium-rhizogenes transformed plants differed widely in the sensitivity of their electrical response to naphthalene acetic acid. We have used different antibodies, raised to auxin binding proteins (ABP) from maize coleoptiles, or to the axr1 gene product (ABP1), to test whether changes in auxin sensitivity can be correlated with the presence of tobacco proteins immunologically related to this ABP. Titrations indicated that 0.4 nM anti-ABP IgG inhibited 50% of the auxin-specific response of wild-type protoplasts, whereas 0.04 nM or 4 nM anti-ABP IgG were necessary to inhibit the response of mutant and transformed protoplasts, respectively, to the same extent. On wild-type protoplasts, blocking part of the immunoreactive sites with anti-ABP antibodies resulted in a decrease in auxin sensitivity of the electrical response (0.4 nM anti-ABP IgG inducing a 10–fold decrease), whereas addition of maize ABP increased this auxin sensitivity (1 pM ABP1 raised the sensitivity more than 1000–fold). The results obtained suggest that the auxin sensitivity detected by our assay system correlates with the amount of tobacco proteins immunologically related to the axr¹ gene product from maize. A hypothesis accounting for the presence of these proteins at the external surface of tobacco protoplasts and for the effects of hetero-logous maize ABP on auxin sensitivity is proposed.     

Fine structure of isolated mesophyll protoplasts of tobacco

Summary Protoplasts of palisade cells isolated enzymatically from mature leaves of tobacco were studied with the electron microscope. A cell wall was completely absent, and the chloroplasts contained large inclusion bodies which were believed to be a crystalline form of fraction I protein. The fine structure of the protoplasts was otherwise that of healthy mesophyll cells, indicating that they are in a good physiological state. Some protoplasts were multinucleate as a result of fusion during the isolation process.     

A non-coat protein synthesized in tobacco mesophyll protoplasts infected by tobacco mosaic virus

Summary A high molecular weight protein unrelated to the viral coat was detected in tobacco mesophyll protoplasts infected by tobacco mosaic virus.     

Plating of isolated tobacco mesophyll protoplasts on agar medium

Summary A technique was developed to derive cell and plant clones from isolated mesophyll protoplasts of tobacco. The protoplasts, plated on a fully defined agar medium, divided and grew actively forming visible colonies after one month of culture. Efficiency of colony formation depended on cell density and light condition during incubation. Under standard conditions, 60% of plated protoplasts formed colonies. Upon transfer onto suitable media, these colonies differentiated shoots and roots, and eventually regenerated whole plants. Advantages of mesophyll protoplasts as the source of clones as well as implication of the plating technique for genetical studies are discussed.     

A novel method for regenerating plants from mesophyll protoplasts of Arabidopsis thaliana line WS

A procedure has been developed for the successful regeneration of plants from mesophyll protoplasts of Arabidopsis thaliana line WS. The protocol is an improved version of that of Damm and Willmitzer (1988). The main changes in original procedure are as follows:

1. A mixture of Cellulase Y-C (0.5%) and Pectolyase Y-23 (0.05%) is used for the isolation of protoplasts. Use of these enzymes reduces the incubation time to 50 min.
2. α-Naphthaleneacetic acid is used as the auxin throughout cultures of protoplasts and calli.
3. Protoplasts and calli are incubated under dim white light (0.8–8 μW/cm²) during culture.

With these modifications, we were able consistently to obtain regenerated shoots from about 70% of calli that had been transferred to shoot-forming medium even though the plating efficiency was rather low (about 0.5–1.5%).     

Characterization of the auxin-regulated par gene from tobacco mesophyll protoplasts.

The auxin-regulated par gene from tobacco mesophyll protoplasts was characterized in detail to deduce its possible function. An homology search of the par gene in the NBRF databases revealed that the par gene has homology to the stringent starvation protein (ssp) gene of Escherichia coli, which is induced under starved conditions and binds in an equimolar ratio to a holoenzyme of RNA polymerase. Hence, it is supposed that the par gene product could play a similar role to that of ssp. Although sequence homology of the par gene to the Gmhsp 26-A gene from soybean was observed, both genes were shown to respond differently to plant hormones and stresses. Gmhsp 26-A is induced by heat shock, 2,4-dichlorophenoxyacetic acid (2,4-D), cytokinin and abscisic acid (ABA), whereas the par gene was induced only by auxins. Furthermore, cycloheximide treatment prevents 2,4-D-mediated accumulation of Gmhsp 26-A mRNA, but not that of par mRNA. Both par and Gmhsp 26-A respond to CdCl2, but splicing of the par pre-mRNA proceeded in a normal way, whereas splicing off the Gmhsp 26-A pre-mRNA was inhibited. Hence, the par and Gmhsp 26-A genes should have a common ancestor, but have evolved in different directions. Detailed time-course experiments confirmed that the par gene was induced immediately after the addition of auxin and expressed upon the initiation of meristematic activity in tobacco mesophyll protoplasts. As the par gene was induced by the sole treatment of cycloheximide, it was proposed that the par gene belongs to a category of 'superinduction' genes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Integration of foreign DNA following microinjection of tobacco mesophyll protoplasts

Summary DNA from a bacterial plasmid containing the T-DNA border sequences of Agrobacterium tumefaciens was transferred into the nucleus or the cytoplasm of tobacco mesophyll protoplasts by microinjection. Following culture in hanging drops, some of these protoplasts produced calli containing the foreign DNA sequences. Evidence for the presence of the injected plasmid DNA in these calli was provided by Southern hybridization analysis. The results demonstrated that random portions of the bacterial plasmid were linked to plant DNA and that integration did not occur at the T-DNA borders present on the injected plasmid. The average number of integrated copies ranged from less than one to 1–2 per tobacco genome. The frequency of integration averaged 14% with intranuclear injections compared to 6% with cytoplasmic injections. With further refinement, the use of microinjection may allow the introduction of many different types of genetic elements into plants.     

Studies of the recovery of tobacco mesophyll protoplasts from an evacuolation treatment

Summary Mesophyll protoplasts ofNicotiana tabacum cv. Xanthi were subjected to centrifugation through a Percoll gradient. This resulted in the removal of the central vacuole from each protoplast, and improved mechanical and osmotic stability. Electron microscope studies showed that the remaining cell contents, including small vacuoles, were of normal morphology. Fixation of protoplasts at various times during subsequent culture showed that the central vacuole was restored after about 12 hours. Cell-wall formation was well advanced after 24 hours of culture. These results are discussed in terms of the potential use of evacuolate protoplasts and the mechanisms of vacuole formation.Abbreviations ER endoplasmic reticulum     

Effect of poly-l-ornithine on isolated tobacco mesophyll protoplasts: Evidence against stimulated pinocytosis

Summary The effects of poly-l-ornithine on the surface membrane of isolated tobacco protoplasts have been examined in the electron microscope using a colloidal metal oxide and a spherical virus as marker substances. No evidence was found to suggest that isolated protoplasts take up either of these markers by a pinocytotic process. Poly-l-ornithine increased the degree of damage observed in fixed preparations, and specifically caused lesions of the plasmalemma which were favoured sites for the binding of both external marker substances. It is suggested that the function of poly-l-ornithine and other treatments used to obtain virus infection of protoplasts is to stress the cell membrane to allow a non-physiological entry of high molecular weight materials. Pinocytosis appears not to occur nor to be necessary for uptake of these materials under conditions of membrane stress.     

Subcellular localization of glucose-6-phosphate dehydrogenase in tobacco mesophyll protoplasts

Subcellular localization of glucose-6-phosphate dehydrogenase (EC 1.1.1.49.) isoenzymes was determined in mesophyll protoplasts prepared from Nicotiana tabacum L. cv. Samsun. Intact chloroplasts and soluble cytosolic proteins were obtained by means of differential centrifugation. The 1000 g pellet contained 97 % of chloroplasts and 16.8 ± 2.1 % of the total activity of glucose-6-phosphate dehydrogenase. The rest of the enzyme was localized in the cytosol which also contained 91 % of the total activity of phosphoenolpyruvate carboxylase.     

A simple plant and tissue culture growth chamber for the regeneration of tobacco mesophyll protoplasts

The isolation and regeneration of tobacco mesophyll protoplasts from fully developed leaves, an important methodological step in plant genetic engineering as well as in plant cell biology and physiology, has been proven unreliable to the extent that it has become a significant setback to basic research. This unfortunate situation is primarily due to the suboptimal physiological state of greenhouse-grown protoplast donor plants. A technically simple and inexpensive method, based on the utilization of commercial Phototron units, is described for the production of suitable tobacco donor plants. Furthermore, a modified version of such a culture unit can be used to regenerate plants from protoplast-derived calli.