Anna H. Nagy

Restoration of morphogenic potential in Nicotiana by somatic hybridisation

SummaryKR103 is a kanamycin resistant cell line of Nicotiana sylvestris, deficient in inducible shoot redifferentiation. The existence of a genetically unaltered inducibility factor in KR103 was tested by fusing protoplasts of this line with those of Nicotiana knightiana, which can not be induced to form shoots in callus culture.Selection of somatic hybrids was based on screening for kanamycin resistance and green pigmentation, factors originally separated in the parental lines. The hybrid nature of two lines (H1 and H2) was shown by studies on esterase, alcohol dehydrogenase and glucose-6-phosphate dehydrogenase isoenzymes.In the somatic hybrids shoot formation on inductive medium was restored, indicating that the inducibility factor had not been altered in KR103. Genetic complementation and induced regulatory changes due to genome interaction are discussed as possible explanations for the restoration of morphogenic potential.

Genetic instability in somatic hybrids of Nicotiana tabacum and Nicotiana knightiana

SummarySomatic hybrids of Nicotiana knightiana (2n=2X=24) and an albino mutant of Nicotiana tabacum (2n=4X=48) were selected after polyethylene glycol induced protoplast fusion. Three lines were selected on the basis of the simultaneous expression of shoot inducibility and green pigmentation, traits originally separated in the parental species.The hybrid nature of the lines was confirmed by their characteristic isoenzyme patterns, the morphology of the regenerated plants, and by the appearance of heterochromatic blocks in the interphase nuclei.Chromosome numbers in the somatic hybrids varied greatly within individual plants. Variegation in leaf and flower colour and segregation for morphological traits in vegetatively multiplied plants are attributed to segregation of chromosomes in the somatic cells, a consequence of the numerical instability. Hybridity, caryotypic changes induced by tissue culture, and high chromosome numbers, are discussed as possible reasons for the observed genetic instability.

Acid phosphatase isoenzymes of Chlamydomonas reinhardii

SummaryAcid phosphatase isoenzymes of Chlamydomonas reinhardii were investigated by isoelectric focusing in polyacrylamide gel systems. In this paper we describe in detail an original method for isoelectric focusing of acid phosphatases extracted from wildtype and acid phosphatase-lacking mutant algae, obtained from Laboratoire de Génetique of University of Liège. Three isoenzymes can be separated from the buffer-soluble components of these cells. An additional isoenzyme type can be visualized using the nonionic detergent NP40 as solubilizer. We conclude that these four isoenzymes are releated to the structural gene of the soluble constitutive acid phosphatase, which was shown by their appearance in P2 and their total absence in mutant Pa. The pl values of soluble constitutive acid phosphatase isoenzymes range between pH 5.2 and 6.2. As a result of treatment with NP40 the extracts from both wild-type and mutant lines contain two additional active phosphatase forms which can be characterized by their high heat resistance and low pI values. These enzymes are fully active using either α-naphthyl phosphate or different acetate esters as substrates.

CHARACTERISTICS OF PIGMENT-PROTEIN COMPLEXES IN NORMAL AND CHLOROPLAST MUTANT LEAVES*

Abstract— The structural stability of chloroplasts was characterized by the proportion of leaf pigments present in the lipoprotein complex.

Analyses of thylakoid membrane polypeptides from photosystem I and II deficient mutants of Chlamydomonas reinhardii by two-dimensional electrophoresis

Summary Thylakoid membrane polypeptide composition in 3 light-sensitive and photosystem deficient mutants of Chlamydomonas reinhardii was studied using a modified 2-D technique of O’Farrel. The modifications were: LiDS solubilization instead of SDS, isofocusing with reverse polarity in first dimension and a very sensitive silver staining procedure. This high resolution technique allowed to separation of about 320–350 of polypeptides in the wild-type strain. The mutants lts-30 and lts-135 which are completely deficient in PSII activity, have 300 spots in their 2-D polypeptide map. The alterations — which are mostly missing — are localized mostly in the 20–35 kD region. These polypeptides are constituents of LHCP. The membrane polypeptide composition in lts-7 mutant with 70% decrease of P 700 activity, is rather similar to the wild-type.

Ribosome-deficient Mutants of Zea mays

Summary Ribosomes, ribosomal RNAs and soluble proteins of leaves were studied in wild-type and two chloroplast mutants of maize. The mutants are inherited in Mendelian manner and are characterized by accumulating ζ -carotene ( ζ -mutant) and lycopin ( ly -mutant) instead of β -carotene. Sedimentation pattern of the ribosomes shows a reduced amount of 70S ribosomes in the dark-grown mutants. There are even fewer 70S ribosomes present in the mutant leaves grown in the light. In parallel a low amount of chloroplast rRNA is present in the mutant leaves. Gel electrophoresis of leaf extracts shows a low content of Fraction I protein in the mutant. It is suggested that several abnormalities found in the mutants are due to the low level of 70S ribosomes.

Method for Simultaneous Determination of Glutamine Synthetase and Adenosine Triphosphatase Activities on Polyacrylamide Gel

Summary A method for the simultaneous localization of adenosine triphosphatase and glutamine synthetase after non-denaturing polyacrylamide gel electrophoresis is described. The determination of enzyme aetivities is based on the staining for the inorganic phosphate produced by aetivities of both glutamine synthetase and adenosine triphosphatase enzymes. The speeificity of the glutamine synthetase reaction was demonstrated by methionine sulphoximine (MSO) inhibition of the enzyme reaction. Mg 2+ -ATPase, Ca 2+ -ATPase and glutamine synthetase enzymes were sneeesfully separated on a continuous linear (3–10 %) polyacrylamide gradient. Glutamine synthetase and ATPase enzymes isolated from Azotobacter bejerinckii and from the green alga Chlamydomonas reinhardii showed different isoenzyme pattern after electrophoretic annlysis.

A rapid method for separation of thylakoid membranes by density gradient centrifugation in percoll

Summary Thylakoid membranes were purified from isolated spinach chloroplasts and from cell-free homogenates of Chlamydomonas reinhardii in a discontinuous Percoll gradient (7.5, 15, 30%). Purity of thylakoid membrane fraction were demonstrated by LiDS-gradient PAGE and electron microscopic examination.

Red and far-red effect on the isoenzyme composition of young fern gametophytes

Summary Changes in isoenzyme patterns, induced by active phytochrome were studied in germinating fern spores of Pteridium aquilinum and Dryopteris filixmas. The functional analysis of soluble proteins by a sensitive electrophoretic method supplemented with enzyme specific staining showed characteristic phytochrome effect in NADP-IDH of D. filix-mas and G6PD of both species. This effect was virtually prevented by inhibitors of protein synthesis.

Digitonin Fragmentation of Spinach and Maize Chloroplasts Distribution of Chlorophylls Between Particle Fractions

Although the available data are not yet definitive it seems likely that two kinds of particles, corresponding to the two photosystems of photosynthesis in green plants, exist as more or less separate entities and may be isolated by appropriate fractionation methods (BOARDMAN & ANDERSON 1964, KLOFAT & HANNIG 1967, MICHEL & MICHEL-WOLWERTZ 1968). An important point of these procedures is the specific fragmentation of chloroplast lamellae. Fragmentation has been performed by various detergents, ultrasonic treatment and hydrostatic pressure (VERNON et al 1965, BRIANTAIS 1966, JACOBI & LEHMAN 1968, BRIL et al. 1969). One of the most suitable detergent proved to be digitonin, because particles obtained by digitonin fragmentation preserve photochemical activities (WESSELS 1963, ANDERSON & BOARDMAN 1966). Despite the extensive use of this method, kinetics and conditions of its application have not been stud ied on details. Kinetics of fragmentation by digitonin cannot be studied on a time scale. The treatment cannot be stopped at a definite moment, because the long-lasting separateon of particles is also performed in the presence of digitonin. In order to overcome this difficulty, variants in duration of the treatment were substituted by different relative amounts of digitonin. Both the variants of time and concentration produce a similar scale of probability of collissions between detergent molecules and chloroplast material, a reaction leading to fragmentation of the lamellae.