Danielle L. Laval-Martin

Tween-20 Activates and Solubilizes the Mitochondrial Membrane-bound, Calmodulin Dependent NAD+ Kinase of Avena sativa L.

Abstract. Among different treatments assayed, a mix of a nonionic detergent (5% Tween-20) with 0.5 m NaCl was found to solubilize a large part of the calmodulin-dependent NAD+ kinase bound to the inner mitochondrial membrane. It also stimulated its activity by increasing 7 times the maximal velocity. Activity stimulation was also observed with phosphatidylcholine, phosphatidylethanolamine and with reductants (HSO3 and DTT). This solubilized NAD+ kinase and the calmodulin-dependent cytosoluble isoform displayed distinct molecular masses, as well as different kinetic parameters. We propose that solubilization of membrane-bound NAD+ kinase could occur in vivo in Avena sativa and could generate a soluble isoform.

Pyridine nucleotides and redox charges during germination of non-dormant and dormant caryopses of Avena sativa L.

Summary In order to investigate the possible biochemical mechanisms involved in triggering the early steps of germination, reduced and oxidized pyridine nucleotides (PN) were analysed and compared in embryos separated from endosperms during the imbibition of non-dormant and dormant caryopses of Avena sativa L. (van Noire de Moyencourt). Experiments were conducted at the restrictive temperature of 30°C, and PN contents were also determined after imbibition for 6 h in the presence of either sodium azide (NaN 3 ), gib-berellin (GA 3 ), or ethanol, all compounds known to stimulate the germination of dormant seeds. The main differences detected were: a quick rise in the catabolic redox charge (CRC = NADH/ [NADH+NAD + ]) in the embryos of non-dormant caryopses during the sensu stricto germination phase, followed by an increase in all contents of cofactors in the growing embryos. Since comparable observations could be done in embryos of dormant caryopses after addition of either NaN 3 or ethanol, this increase of the CRC, probably due to an activation of glycolysis, could initiate germination. The effect of GA 3 , found to be very different, is discussed. In addition, stimulation of the pentose phosphate pathway through the synthesis of NADP + by NAD + kinase (E.C. 2.7.1.23) appears to be essential for the growth of the seedlings. In the embryos of dormant caryopses, the lack of rise of CRC and the subsequent weak synthesis of PN could be one of the limiting factors for germination.

Changes in soluble and membrane-bound isoforms of calcium-calmodulin-dependent and -independent NAD+ kinase, during the culture of after-ripened and dormant seeds of Avena sativa

Activities of the soluble and membrane-boundisoforms of Ca 2+ calmodulin (CaCam)-dependent and-independent NAD + kinases, were followed in theembryos during the culture of dormant (D) and after-ripened (AR) seeds ofAvena sativa L. Embryos of D and AR seeds differ mainly in the evolution ofmembrane-bound activities, the majority of which are CaCam-dependent andlinked to mitochondria. The in vivo application ofgibberellic acid, CaCl2 andH2O2, which enhanced germination,induced an enhancement of all CaCam-dependent isoforms. Trifluoperazine (TFP),a calmodulin antagonist, greatly enhanced all CaCam-dependent isoforms andabolished the differences between the NAD + kinaseactivities of the two kinds of embryo. In addition, TFP rendered embryosunable to resume axis growth, probably due to pleiotropic effects. In contrastto H2O2, the reducing agentdithiothreitol diminished the soluble CaCam-dependent enzyme and blocked thegermination of both types of seed, whereas it increased the dependentmembrane-bound activities. The results demonstrate (1) that theCaCam-dependent NAD + kinase isoforms —amongst which is the isoform bound to mitochondrial membranes — play animportant role at the end of sensu stricto germinationand during the following growth of Avena sativa; and (2)that an excess of activity of these isoforms could be markers of stress orlethal conditions.

NAD+ Kinase Activities in Euglena Gracilis and Phaseolus Vulgaris

After an electrophoretic separation of proteins from Euglena gracilis and dry seeds of Phaseolus vulgaris in native conditions in polyacrylamide gels, gels were incubated in mixtures containing NAD+, Mg-ATP2-, glucose 6-phosphate, G6P dehydrogenase, and either phenazine ethosulfate and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (PES/MTT) or phenazine methosulfate and nitro blue tetrazolium (PMS/NBT) as coupled redox system for NAD+ kinase activity detection. In the presence of PES/MTT, 4 bands were revealed for E. gracilis, among which two corresponded to NAD+ kinase activity, the other corresponding to a NAD+ reductase activity due to alcohol dehydrogenase (ADH). In the presence of PMS/NBT, only the bands of NAD+ kinase activity were revealed. With Phaseolus vulgaris, 3 bands of ADH were always revealed in both mixtures, and only the use of PMS/NBT allowed the detection of NAD+ kinase as a fourth band. With both materials, NAD+ reductase staining in gels was intensifed in the presence of GTP or ATP and even further with ADP or GDP. The results demonstrate that: 1) the NAD+ kinase and NAD+ reductase are two distinct enzymes; 2) the NAD+ reductase corresponds to ADH.

Evolution of PS IIα and PS IIβ centers during the greening of Euglena gracilis Z: Correlations with changes in lipid content.

The building up of the two types of reaction centers, PS IIα and PS IIβ, was investigated during the greening of Euglena gracilis Z cells in resting medium. The maximal values in the proportion of PS IIα centers (55%) and in the oxygen evolved per chlorophyll were reached at the outbreak of greening, when accumulation of galactolipids (MGDG and DGDG) rich in unsaturated fatty acids occurred, and when anionic lipids (SQDG and PG) emerged. As the greening progressed, the chlorophyll accumulation corresponded to a secondary enrichment in PS IIβ centers, which built up more rapidly than PS IIα centers; correlatively, a general saturation of the fatty acids constitutive of all lipid classes took place.

Changes in NAD+ kinase activity during germination of Phaseolus vulgaris and P. acutifolius, and effects of drought stress

Summary In embryonic bean axes during germination, it can be seen that there is a decrease of ADH activity, an increase of NAD + kinase activity, and then an emergence of G6PDH activity. Ethanol and hydrogen peroxide, which are shown to enhance the efficiency of P. vulgaris germination, accumulate to a significant degree during germination, hydrogen peroxide being much more concentrated than ethanol at the radicleprotrusion stage. Five forms of NAD + kinase can be distinguished: a soluble Ca 2+ sensitive form; two Ca-CaM independent forms; one soluble, one membranal; and two Ca-CaM dependent forms, one soluble and one membranal. In dry seeds, the membranal Ca-CaM dependent form is represented more in P. vulgaris (100% of the total pelletable activity) than in P. acutifolius (30%). This membranal Ca-CaM dependent NAD + kinase, prevalent after radicle protrusion, might be important for early growth metabolism, since an ethanol treatment applied until radicle protrusion reduces membranal NAD + kinase and G6PDH activities as well as growth, while an H 2 O 2 treatment slightly reinforces G6PDH and membranal NAD + kinase activities. Except for drought-stressed P. vulgaris at the stage of radicle protrusion in which NAD + kinase activities are not perturbed, in both P. vulgaris and P. acutifolius NAD + kinase activities temporarily decrease in response to drought stress, these being restored after subsequent rehydration.

EFFECT OF ALUMINIUM ON THE NAD+ KINASE ACTIVITY OF EUGLENA GRACILIS GROWN HETEROTROPHICALLY

The effect of 2 mM AlCl3 on NAD+ kinase (E.C. 2.7.1.23) activity was studied using Euglena gracilis strain Z grown heterotrophically in darkness at pH 3.5 in the presence of lactate as sole carbon source. The Al-treatment slowed down the culture growth and suppressed the peak of NAD+ kinase activity, which characterizes the beginning of the exponential phase of growth of the control cell cultures. There are two possible explanations of the Al effect: it 1) either prevents the enzyme activation by the Ca-calmodulin (CaM) complex; or 2) suppresses the CaM-dependent NAD+ kinase form. In Euglena cells, a part of the NAD+ kinase activity is enhanced by EGTA and lowered by Ca2+: this peculiar NAD+ kinase activity is unaffected by the Al treatment.

Long-Term Effect of Diuron on Chlorophyllous Callus of Bromus erectus: Lipid Composition

Abstract The selection of plants resistant to photosynthetic herbicides is limited by the low rate of success in obtaining a photosynthetic callus. An attempt was made to induce diuron tolerance with chlorophyllous tissue strains of Bromus erectus: growth, chlorophyll contents and total fatty acids were studied during four months of photoheterotrophic growth in the presence or absence of 6 μM diuron. The presence of the herbicide, which reduced the fresh weight by 40%, provoked increases in chlorophyll levels by 2-or 3-fold, and in galactolipids (especially DGDG) which were enriched in linolenic acid content. These results, closely related to already described characteristics of herbicide-resistant material, suggest that callus of Bromus erectus could constitute an interesting photosynthetic in vitro material.

NaCl and wounding induced changes in NAD reductase in hypocotyls and root tips ofPhaseolus vulgaris L.

The effect of 300 mM NaCl and wounding on the nicotinamide adenine dinucleotide (NAD) kinase, revealed by the guanosine triphosphate-dependent NAD reductase activity, was studied in two differently resistant bean cultivars using densitometric analysis of electrophoretic gels. In the presence of NaCl the total activity of NAD reductase was increased, in hypocotyls and root tips of resistant cultivar. The contribution of each of NAD reductase isoforms to the total activity was not significantly different between cultivars. Conversely, after wounding the hypocotyl, an increase could be observed in both cultivars and differences were demonstrated in the contribution of the different isoforms.