D. Lavergne

Immunological Detection of Potential Signal-Transduction Proteins Expressed during Wheat Somatic Tissue Culture

An immunochemical approach was used to detect the expression of putative guanine nucleotide-binding proteins (G-proteins), arrestin, and nucleoside diphosphate kinases during wheat (Triticum aestivum) tissue culture initiated from immature embryos. Both the soluble and membrane extracts from the immature embryos revealed bands of 58, 40, and 16 kD with antibodies to G-protein ([alpha] subunit), arrestin, and nucleoside diphosphate kinase, respectively. These proteins were overexpressed in vitro in both nonembryogenic callus and embryogenic cultures. An additional soluble protein (32 kD) was detected by anti-G[alpha] antibodies in cultured tissues but not in immature embryos, suggesting a possible function in cell multiplication. Moreover, somatic embryogenesis was associated with the appearance of a 29-kD protein reactive with anti-arrestin antibodies, both in soluble and membrane fractions. Tissue-cultured genetic stocks of Chinese Spring wheat, including the disomic, 36 ditelosomic, and 6 nullisomic-tetrasomic wheat lines, were used to ascertain the chromosomal location of the genes encoding the 29-kD arrestin-like protein. The lack of a signal with the nonembryogenic ditelosomic 3 D short chromosome arm line suggests that the 3 D long chromosome arm possesses at least one gene involved in the expression of the 29-kD protein. The putative role of the 29-kD protein in signal-transduction regulating embryogenesis is discussed.

Further studies of phosphoglycerate kinase and ribulose-5-phosphate kinase of the photosynthetic carbon reduction cycle: Regulation of the enzymes by the adenine nucleotides

Abstract Phosphoglycerate (PGA) kinase was purified from spinach chloroplasts. Its activity was determined in a single enzymatic reaction without any coupling with the glyceraldehyde-3-phosphate dehydrogenase. With respect to ATP it exhibits non-Michaelis kinetics and behaves like an allosteric enzyme. The Hill coefficient is 1.35 and the calculated Km(ATP) is 2 mM. Ribulose-5-phosphate (Ru5P) kinase was purified from spinach leaves. It exhibits Michaelis kinetics with respect to ATP. The Km(ATP) is 0.42 mM. Both kinases from spinach chloroplasts are controlled by the adenylate energy charge.

Photosynthetic ability of in vitro grown coconut (Cocos nucifera L.) plantlets derived from zygotic embryos

Photosynthetic parameters have been investigated using complementary approaches throughout the in vitro development of coconut zygotic embryos into plantlets. Patterns of chlorophyll fluorescence were comparable in in vitro grown coconut plantlets (OFAx = 0.72 and O, = 0.45) and in autotrophic adult palms (OpAX = 0.76 and Bp=0.5O). Chlorophyll content was lower in in vitro-cultured plantlets (0.92 mg/g fresh weight (FW)) than in autotrophic plants (2.43 mg/g FW). The photosynthetic rate (1.14 pmol CO,/m’ per s) of autotrophic palms was half that of in vitro grown plantlets, while transpiration rates were similar in both. Changes in the PEPC:RubisCO ratio during the development of in vitro grown plantlets (from 89.17 to 0.04 pmol COJh per mg total soluble protein (TSP)) reflected a transition from a heterotrophic towards a RubisCO-mediated mode of CO, fixation. The RubisCO enzyme capacity (2.83 pmol CO,/h per mg TSP) and content (172.8 mg/g TSP) measured in in vitro-cultured plantlets were lower than those measured in autotrophic palms (6.60 pmol CO,/h per mg TSP and 217.6 mg/g TSP respectively). Transmission electronic microscopy (TEM) observations showed a complete ultrastructural organisation of chloroplasts in plantlets at the end of the in vitro culture process (6 weeks under light). All the studied parametersj Abbreoiatiotis: Chl a, Chlorophyll a; Chl b, Chlorophyll b; FW, Fresh weight; PAR, Photosynthesis active radiations; PEPC, Phosphoenolpyruvate carboxylase; PVP, Polyvinyl pyrrolidone; RH, Relative humidity; RubisCO, Ribulose 1,5-bisphosphate carboxylase oxygenase; To, Temperature; TCA cycle, Tricarboxylic acid or Krebs cycle; TEM, Transmission electronic microscopy; TSP, Total soluble proteins.

Simultaneous purification of two kinases from spinach leaves: Ribulose-5-phosphate kinase and phosphoglycerate kinase

Abstract Ribulose-5-phosphate kinase (PRK) and phosphoglycerate kinase (PGK) are isolated from a single spinach leaf homogenate. PRK is purified 5300-fold to an homogeneous state, free of phosphoribo-isomerase and ribulose diphosphate carboxylase. The specific activity is about 320. The PGK has a purification factor of 230, with a specific activity of 78; the pure enzyme is free of glyceraldehyde-phosphate-dehydrogenase. The molecular weights estimated by sodium dodecyl sulfate polyacrylamide electrophoresis experiments are about 46 000 for PRK and 48 000 for PGK. Both kinases are stable for a period of at least three months.

Nitrate reductase activity changes during a culture cycle of tobacco cells: the participation of a membrane-bound form enzyme

Abstract The in vivo and vitro nitrate reductase (NR; EC 1.6.6.1) activities of suspension cell cultures were investigated in two strains of Nicotiana tabacum : a photomixotrophic green wild type (WT) and non-photosynthetic green type RubisCo − (SP25). During a batch culture cycle of 21 days, two peaks of in vivo NR activity (NRA) were observed: an early peak at 7 h and a second peak which reached a maximum at 6–8 days. At the beginning of the culture, in vitro activity was detected early for the WT cells compared to SP25 cells for which the activity was very low or absent. During the second peak, in vitro activity was present in the two strains and correlated very well with in vivo activity. Immunoblot experiment suggested that NR 120 kDa polypeptide did not accumulate in the cytosol at the beginning of the first phase of activity and the NR protein seemed to be labile. In contrast, the NR protein was stable during the second peak. On the other hand, it was observed that the dividing cells at 5–8 days seemed to accumulate nitrate, while the non-dividing cells, at the beginning of the culture, are unable to do that. This feature might be related to a change of cell permeability for nitrate because NR activity causing nitrate reduction is better in the second peak than in the first one. In correlation with this likely permeability change, external nitrate concentration modulated the first peak NRA and not the second one. The differential sensitivity of NRA of the two peaks to nitrate, led us to rediscuss the localization site of NR and to focus our experiments on a membrane-bound form of this enzyme.

Expression of auxin and light-regulated arrestin-like proteins, G proteins and nucleoside diphosphate kinase during induction and development of wheat somatic embryos.

Abstract The modulation of three signal transduction elements: arrestin-like proteins, G proteins and NDPK was assessed during the induction of wheat (Triticum aestivum L.) somatic embryogenesis under different auxin (2,4-D) and light conditions. Immunological approaches using specific antibodies, kinase activity measurement and [α-32P]-GTP-binding assay were performed. The induction of embryogenic capacity by 2,4-D was characterised both by the increased expression of the classical 40-kDa arrestin-like form and by the appearance of an additional arrestin-like protein of 29xa0kDa. The 40-kDa arrestin-like soluble form was unaffected by light stimuli. On the other hand, the 29-kDa arrestin-like form, specific of the embryogenic tissue culture, was found to be light regulated. From embryogenic cultures grown under light or dark, different soluble G proteins from 22 to 48xa0kDa were detected by probing polyvinylidene fluoride (PVDF) blots with [α-32P]-GTP. In addition, in the microsomal fraction from light-grown cultures, a polypeptide of 20xa0kDa was heavily labelled. Under light conditions, cell proliferation induced by 2,4-D stimulated the appearance of a 32-kDa nucleoside diphosphate kinase (NDPK) form in addition to the classical 16–18-kDa protein, without a significant change in the NDPK activity. The modulated expression of plant arrestin-like proteins, G proteins and NDPK molecules in response to auxin and light support the view that they play key roles in signalling cascades participating in plant development.

Physiological Studies on two Cultivars of Pennisetum: P. americanum 23 DB, a Cultivated Species and P. mollissimum, a Wild Species I. Photosynthetic Carbon Metabolism

Summary The photosynthetic carbon metabolism and the anatomical features of the leaves of twO series of plants of Pennisetum have been studied: plants from 23 DB line from the cultivated Pearl millet P. americanum (thyphoides), plants from an ecotype of P. mollissimum , one of its related wild species originating from Mali. The activities and intracellular location of the enzymes of carbon metabolism are very similar in the two plants. These characteristics together with the leaf anatomy indicate that both Pennisetum have the C4 pathway of CO 2 assimilation and belong to the NADP-ME-type. In mesophyll cells two enzymes function to reduce oxaloacetate: the light-activated chloroplast NADP-malate dehydrogenase and the cytoplasmic NAD-malate dehydrogenase. Like malic acid, aspartic acid participates to the transport of CO 2 from the mesophyll to the bundle sheath cells. The labelling pattern of aspartic acid and malic acid during a 14 C0 2 pulse and chase experiment indicate that the turn-over rates of the carbon chains of malate and aspartate are very different and that there are two ways of decarboxylation. While malate synthesized in the mesophyll cells is rapidly decarboxylated in the presence of the bundle sheath chloroplast NADP-malic enzyme, malate originating in the bundle sheath mitochondria from the carbon chain of aspartic acid is more slowly decarboxylated in the presence of the mitochondrial NAD-malic enzyme. The two C4 acids are described as representing two different compartments and having distinct roles in the photosynthetic carbon metabolism. Malic acid is the single CO 2 donor while aspartic acid plays the role of a reservoir for C4 acids. It is assumed that the major and the minor ways of decarboxylation could at least partly be controlled by the nucleotide supply via photosynthesis (NADP) and (or) mitochondrial respiration (NAD). Except for the fact that P. mollissimum fixes more actively CO2 and accumulates more starch than P. americanum 23 DB, it is noteworthy that the two plants have developped similar metabolic adaptation.

Some regulatory properties of glutamine synthetase isoforms in a C4 plant: Pennisetum americanum (L.) Leeke

Abstract Some properties of cytosolic (GS 1) and chloroplastic (GS 2) glutamine synthetases were studied and compared in a C 4 ( Pennisetum americanum ) and a C 3 plant ( Oryza sativa L.). Although similar kinetic and regulatory properties were found for the enzymes of each plant type the relative proportions of GS 1 and GS 2 were markedly different. The possible physiological role of each isoform is discussed in relation to their regulatory properties and their levels of activity in the cytosol and chloroplast during the light/dark transition-period.

Physiological Studies on two Cultivars of Pennisetum: P. americanum 23 DB, a Cultivated Species and P. mollissimum, a Wild Species: II. Effects of Leaf Age on Biochemical Characteristics and Activities of the Enzymes Associated with the Photosynthetic Carbon Metabolism

Summary Two genotypes of Pennisetum were studied for biochemical characteristics and enzyme activities associated with photosynthetic carbon metabolism with regard to the effect of leaf age. Research was conducted with fully just expanded 4th leaves from 12 to 35 day old plants. Growth analysis, leaf area, protein and starch content, chlorophyll content, and dry weight were investigated. All these parameters were higher for P. mollissimum compared to P. americanum . Starch content of leaves was always higher in P. mollissimum . The PEP carboxylase activity was highest in young leaves and decreased rapidly with subsequent full leaf expansion. For RuBP carboxylase, the activity increased quickly until full leaf expansion. The ratio PEP carboxylase/RuBP carboxylase activity was high in young tissue as expected for C 4 type plants. Mature leaf tissue had a ratio of 1, a value nearer to the C 3 type plant. These data indicate that there are physiological changes which occur during development of Pennisetum leaves.

Thermal sensitivity of NAD-malate dehydrogenase isoforms from various Pennisetum ecotypes

Thermal sensitivity of NAD-malate dehydrogenase (NAD-MDH) was studied in several Pennisetum ecotypes. The effect of temperature was investigated using either crude extracts from shoots or separated isoforms. In all the ecotypes tested, in vitro temperature optima were found to be near 45°C. A high heat stability of NAD-MDH was observed for all the ecotypes studied. Three isoenzymes were isolated by DEAE-cellulose chromatography and analyzed by starch gel electrophoresis. Three anodic isoenzymes were separated on the electrophoretic pattern, they are known to be associated with peroxisomes, mitochondria and soluble leaf compartments, respectively. The soluble NAD-MDH isoenzyme was found to be more affected at high temperature than the peroxisomal and mitochondrial forms.