Occurrence of unstable PEP carboxylase in C4 grasses in the genus Spartina Schreb.

Abstract The aims of this work were to discover the distribution within the C₄ grass Spartina anglica of a PEP carboxylase which is very unstable during and after extraction, and to determine whether this unstable form occurs in other members of the genus. In S. anglica, only the leaf contains an unstable PEP carboxylase. Within the leaf only the major one of two isoenzymes is unstable, and this is located in the mesophyll cells. The unstable isoenzyme is inactivated during extraction and storage unless protected by bovine serum albumin or Triton X-100, and is inactivated in assay mixtures at optimum pH in the absence of PEP. Evidence is presented that inactivation is not due to degradation or inhibition during extraction and storage. The enzyme from leaves of Spartina species taxonomically closely related to S. anglica is also very unstable during and after extraction, but that from less closely related species is much more stable.     

Progress in cellular engineering of plants: biochemical and genetic assessment of selectable markers from cultured cells

Summary Recent availability of stable and well characterized selectable markers and ability to combine alien genomes parasexually have contributed to the development of molecular biology in higher plants, including gene expression and genetic manipulation.Several types of biochemical mutants (resistant to inhibitory concentrations of aminoacid(s) or aminoacid analogs as well as deficient for enzyme activity) have recently been isolated and characterized biochemically and genetically. Among them, mutants with alterations in the nitrogen and aminoacid metabolism, or in the activity of alcohol dehydrogenases are being used in the development of more efficient techniques of gene transfer.The manipulation of whole genomes by sexual or somatic cell fusion offers new potential in this field, but refinement of transfer techniques is desirable. The new set of selectable markers obtained through advanced cellular technology, as well as our ability to regenerate plants from manipulated cell lines are expected to play a major role in cellular engineering.     

Properties of phosphoenolpyruvate carboxylase in desalted extracts from isolated guard-cell protoplasts

Properties of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) obtained from isolated guard-cell protoplasts of Vicia faba L. were determined following rapidly desalting of the extract on a Sephadex G 25 column. The activity of PEP carboxylase was measured as a function of PEP and malate concentration, pH and K⁺ concentration within 2–3 min after homogenization of the guard-cell protoplasts. The activity of this enzyme was stimulated by PEP concentrations of 0.1 to 0.75 mM and by K⁺ ions (12 mM), but inhibited by PEP concentrations above 1 mM and by malate. Changes in the K_m(PEP) and V_max values with increasing malate concentrations (2.5 and 5 mM) indicate that the malate level, varying in relation to the physiological state of guard cells, plays an important role in regulating the properties of phosphoenolpyruvate carboxylase.Abbreviations CAM Crassulacean acid metabolism - GCP guard-cell protoplast - PEP phosphoenolpyruvate Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday     

Regulation of the cytosolic adenylate ratio as determined by rapid fractionation of mesophyll protoplasts of oat

Adenylate levels in chloroplasts, mitochondria and the cytosol of oat mesophyll protoplasts were determined under light and dark conditions, in the absence and presence of plasmalemma-permeable inhibitors of electron transfer and uncouplers of phosphorylation. This was achieved using a microgradient technique which allowed an integrated homogenization and fractionation of protoplasts within 60 s (Hampp et al. 1982, Plant Physiol. 69, 448–455), under conditions which quench bulk activities of metabolic interconversion in less than 2 s. In illuminated controls, ATP/ADP ratios were found to be 2.1 in chloroplasts, about unity in mitochondria, and 11 in the cytosol; whereas, in the dark, this ratio only showed a large drop in chloroplasts (0.4). None of the compounds used [carbonylcyanide m-chlorophenylhydrazone (CCCP), carbonylcyanide p-trifluoromethoxy-phenylhydrazone (FCCP), antimycin A, dibromothymoquinone (DBMIB), dichlorophenyldi-methylurea (DCMU), or salicylhydroxamic acid (SHAM)] affected the stroma adenylate ratio in the dark. Under illumination, however, the ATP/ADP ratios were partly reduced in the presence of antimycin (inhibitor of cyclic photophosphorylation) and of DCMU (inhibitor of linear electron flow), while in the presence of DBMIB, DCMU+ antimycin (inhibition of both cyclic and linear electron flow), and CCCP (uncoupling) the ratio obtained was the same as that occurring in the dark. In contrast, mitochondrial adenylate levels did not exhibit large variations under the various treatments. The cytosolic ATP/ADP ratio, however, showed dramatic changes: in darkened protoplasts, cytosolic values dropped to 0.2 and 0.1 in the presence of uncouplers and antimycin, respectively, while SHAM did not induce any significant alteration. In the light, a similar pronounced decrease in ATP levels was observed only after the application of uncouplers or inhibitors of both mitochondrial and photosynthetic electron transport, whereas selective inhibition of the latter was largely ineffective in reducing the cytosolic ATP/ADP ratio. Thus, the results show that the antimycin-sensitive electron transport is, potentially, equally active in light and darkness. In addition, they indicate that antimycin-insensitive electron transport in mitochondria (alternative pathway) does not significantly contribute to the cytosolic energy state.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DBMIB dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropy-p-benzoquinone) - DCMU dichlorophenyldimethylurea - FCCP carbonylcyanide-p-trifluoromethoxy-phenylhydrazone - SHAM sancylhydroxamic acid     

Transference of the high molecular weight carbohydrate sequences of fetuin to the surface of carrot embryo protoplasts

A method is described for the removal of the carbohydrate sequences of glycoproteins, and their covalent attachment to hydrocarbon chains. These synthetic membrane components may then be incorporated into liposome and cell membranes. Pronase-liberated glycopeptides derived from fetuin were linked by a reduced Schiff's base linkage to tetradecyl aldehyde. The resulting glycolipid was incorporated by external addition, into phosphatidylcholine liposomes. Glycolipid transfer to these liposomes rendered them suseptible to agglutination by wheat germ lectin, which binds N-acetylneuraminic acid, the terminal carbohydrate of the high molecular weight fetuin sugar sequence. Sequential removal of the terminal sugars, and subsequent agglutination behaviour towards various lectins, suggests that the carbohydrate sequence had been transfered intact. The glycolipid was incorporated into plant protoplast membranes by incubation with glycolipid-containing liposomes for 2 h at 37°C. These synthetic glycolipids may find a use in the study of carbohydrate-based recognition systems in animal and plant membranes. In addition they may prove useful in the development of cell and membrane tagging and handling techniques, by the insertion of sugar groups not normally present in these membranes.     

Gametophyte Regeneration and Apospory from Archegoniate Protoplasts Under Conditions Devised for Higher Plants*

Developmental pathways from isolated protoplasts were investigated with the moss Funaria hygrometrica, the liverwort Anthoceros crispulus, and the fern Anogramma leptophylla. Gametophytic protoplasts regenerated gametophytes. Apospory was obtained with sporophytic protoplasts of the moss and the fern. The archegoniates also grew in interdivisional co-cultures with solanaceous species.     

Herbicide-resistant Indica rice plants from IRRI breeding line IR72 after PEG-mediated transformation of protoplasts

The commercially important Indica rice cultivar Oryza sativa cv. IR72 has been transformed using direct gene transfer to protoplasts. PEG-mediated transformation was done with two plasmid constructs containing either a CaMV 35S promoter/HPH chimaeric gene conferring resistance to hygromycin (Hg) or a CaMV 35S promoter/BAR chimaeric gene conferring resistance to a commercial herbicide (Basta) containing phosphinothricin (PPT). We have obtained so far 92 Hg^r and 170 PPT^r IR72 plants from protoplasts through selection. 31 Hg^r and 70 PPT^r plants are being grown in the greenhouse to maturity. Data from Southern analysis and enzyme assays proved that the transgene was stably integrated into the host genome and expressed. Transgenic plants showed complete resistance to high doses of the commercial formulations of PPT.     

Survival and growth of pea protoplasts after transformation by electroporation

Protoplasts from two different pea cultivars, Belman and Filby, were stably transformed by direct gene transfer using electroporation. Transgenic calli could be obtained after selection, when hygromycin resistance was used as the selective trait introduced into the protoplasts, while no transformants were obtained when kanamycin resistance was used as selective marker in either of the two pea cultivars tested. The effect of the field strength on survival and division rates of the protoplasts was studied. Two different culture systems and osmotica were compared for induction of sustained divisions in and regeneration of transgenic callus from the protoplasts. The choice of the culture system had a considerable effect on the initial division frequency of the treated protoplasts, as well as on the later growth of the colonies. Transformation efficiency was monitored by histochemical GUS assay, and the transgenic nature of the calli selected for resistance against antibiotics was confirmed by DNA analysis.     

Protoclonal variation of plant regeneration in rice

Protoplasts were isolated from callus derived from a single homozygous seed of Oryza sativa L. var. Norin 8. Thirty protoclones were randomly selected and these showed variation in regeneration frequency ranging from 0–87% with an average of 52%. The potential for regeneration of each protoclone as reflected in the regeneration frequency was analyzed five times over a period of 250 days and showed that the protoclones can be classified into three types, namely: protoclones with high regeneration frequency; protoclones with low regeneration frequency, both of which maintained their respective levels of regeneration potential; and protoclones with gradually decreasing regeneration frequency. Secondary protoclones established from protoplasts isolated from some of these protoclones and regenerated 2–3 times for a period of 120 days also showed further reduction in regeneration frequency. The polypeptide composition analyzed by two dimensional gel electrophoresis suggests the presence of specific polypeptides related to regeneration potential. Analysis of ploidy level based on plant morphology and pollen size suggests the predominance of tetraploids among the regenerated plants.