Purification and characterization of 2-enoyl-CoA reductase from bovine liver.

Mitochondrial 2-enoyl-CoA reductase from bovine liver was purified and characterized. A simple three-step purification was developed, involving ion-exchange chromatography to separate the bulk of the NADPH-dependent 2,4-dienoyl-CoA reductase, followed by chromatography on Blue Sepharose and adenosine 2',5'-bisphosphate-Sepharose. Homogeneous enzyme with a subunit Mr of 35 500 is obtained in 35% yield. The Mr of the native enzyme, determined by three different methods, yielded values that suggest that the enzyme is dimeric. NADPH is required as cofactor, and cannot be replaced by NADH. The activity of the purified enzyme towards 2-trans-double bonds in 2-monoene and 2,4-diene structures was investigated. 2-Enoyl-CoA reductase reduced the double bonds in a series of 2-trans-monoenoyl-CoA esters with different chain lengths, but did not exhibit significant activity towards 2-trans-double bonds of 2,4-dienoyl-CoA esters. This result is discussed in the light of analogous observations with enoyl-CoA hydratase.     

Diurnal Rhythmicity in the Pattern of mRNAs in the Leaves of Sinapis alba

Previous studies have shown that certain specific leaf mRNAs exhibit a diurnal rhythmicity in their quantity in higher plants. To determine whether this situation is restricted to a few mRNAs, or affects a large number, we have used in vitro translation and two-dimensional polyacrylamide gel electrophoresis to analyze the mRNA complement in leaves of Sinapis alba at different times during an 8-hour/16-hour day/night cycle. A method for the visual analysis of two-dimensional polyacrylamide gel electrophoresis was also developed. This method selected, at each sampling time, spots that were significant. It then selected, between two sampling times, intensity changes that were significant at the 0.02 confidence level. During a day/night cycle, complex rhythmic changes affected about 10% of the mRNAs. Nineteen different rhythm patterns were found. These 19 patterns fell into four main classes: mRNAs that increase during the light period and decrease during the dark, mRNAs that increase and then decrease during the light period, mRNAs that decrease during the light period and increase during the dark period, and mRNAs that increase and then decrease during the dark period.     

Tuber formation and development of <Emphasis Type="Italic">Dioscorea cayenensis–Dioscorea rotundata</Emphasis> complex <Emphasis Type="Italic">in vitro</Emphasis> effect of polyamines

Tuberisation was obtained in vitro on yam (Dioscorea cayenensis–Dioscorea rotundata complex). The effect of exogenous polyamines on tuber formation and development (length and weight of microtubers) was investigated and discussed in relation with changes in endogenous polyamines. Application of exogenous polyamines, inhibitors of their metabolism, and polyamines precursors in various concentrations positively affected microtuber formation by yam nodal cuttings and their further development. In control conditions, 3 wk are needed to obtain 100% of tuberisation. With low concentrations of putrescine (10⁻⁵ or 10⁻⁶ M), tuber formation occurred earlier. Polyamine endogenous level and metabolism can be significantly affected by exogenous polyamines, but modifications of endogenous free polyamines could not be directly correlated to the tuber formation process. Increases in endogenous putrescine and auxins were observed in tubers showing a better development in the presence of putrescine. These results can be used for optimising in vitro conditions for mass production of larger microtubers of the D. cayenensis–D. rotundata complex.     

Axillary proliferation and tuberisation of Dioscorea cayenensis–D. rotundata complex

Yams (Dioscorea spp) are tuber crops used as staple food in Africa because of their nutritional value. However agronomic constraints, phytosanitary problems and the lack of good healthy planting material restrict their production. In contrast to the inefficiency of traditional method of planting, tissue culture techniques allow to increase the multiplication and the rapid production of pathogen- free plant material. This work was undertaken to provide farmers in African countries with healthy microplants and microtubers as seeds. In vitro nodal segments of two varieties of local yams D. cayenensis–D. rotundata complex (cv. ‘Singo’, cv. ‘Singou’ and cv. ‘Gnidou’) were micropropagated on the modified medium of Murashige and Skoog. The morphogenesis, the growth of microplants and microtuber formation have been found to be controlled by external factors that act individually and synergistically. Addition of kinetin (2 mg l⁻¹) to the culture media could reduce multiplication rate (node number) of some clones. An increase of the sucrose concentration from 3% to 5% induced no change in the multiplication and tuberisation parameters. An important reduction of the multiplication (shoot number, height and node number) and the tuberisation (tuber number and length) was observed with 8% sucrose. Multiplication (shoot and node number) was increased in the presence of jasmonic acid (10 μM). JA also induced an increase of tuber number in the absence of Kin. Multiplication of yam by in vitro growth of nodal segments is a way for rapid clonal multiplication and could allow solving the problem of lack of seed material faced by farmers. This method could also be used for multiplication of elite cultivars, independently of the growing season.     

Hyperhydricity of Micropropagated Shoots: A Typically Stress-induced Change of Physiological State

Hyperhydricity of micropropagated shoots, formerly called vitrification, undoubtedly results from growth and culture conditions, subjectively reputated as stressing factors: wounding, infiltration of soft culture medium, generally of a high ionic strength, rich in nitrogen and in growth regulators in a special balance, in a humid and gaseous confined atmosphere. Stress is (objectively) defined as a disruption of homeostasis resulting from a constraint escaping the usual flexibility of metabolism. It induces another temporary (reversible) or definitive (irreversible) thermodynamic physiological state. The state-change concept developed by Strasser (1988) and Strasser and Tsimilli-Michael (2001) is applicable to the phenomenon of hyperhydricity. An appraisal of the redox capacities of hyperhydrated shoots together with a study of some enzymic activities that catalyse pentose phosphate and glycolytic pathways has indeed shown that such shoots have evolved towards a temporary state of lower differentiation or a juvenile state with a sufficient activity to survive and to defend themselves.     

Systemic resistance and lipoxygenase-related defence response induced in tomato by <Emphasis Type="Italic">Pseudomonas putida</Emphasis>strain BTP1

Background  

Previous studies showed the ability of Pseudomonas putida strain BTP1 to promote induced systemic resistance (ISR) in different host plants. Since ISR is long-lasting and not conducive for development of resistance of the targeted pathogen, this phenomenon can take part of disease control strategies. However, in spite of the numerous examples of ISR induced by PGPR in plants, only a few biochemical studies have associated the protective effect with specific host metabolic changes.     

Hyperhydricity of Prunus avium shoots cultured on gelrite: a controlled stress response.

Hyperhydricity is a physiological disorder frequently affecting shoots vegetatively propagated in vitro. Hyperhydric shoots are characterised by a translucent aspect due to a chlorophyll deficiency, a not very developed cell wall and a high water content. Hyperhydricity of Prunus avium shoots was expressed in vitro in one multiplication cycle by replacing the gelling agent agar (normal shoots: NS) by gelrite (hyperhydric shoots: HS). P. avium shoots evolving towards the hyperhydric state produced higher amounts of ethylene, polyamines (PAs) and proline, which are substances considered as stress markers. A higher activity of glutathione peroxidase (GPX; EC 1.11.1.9), involved in organic hydroperoxide elimination, suggested an increased production of these compounds in HS. The unchanged free fatty acid composition indicated no HS membrane damages compared to NS. The ploidy level of HS nuclei was not affected, but the bigger size and the lower percentage of nuclei during the S phase suggested a slowing down of the cell cycle. The results argued for a stress response of the HS, but no signs of oxidative damages of lipid membrane and nucleus were observed. The discussion points out paradoxical results in a classical analysis of stress and suggests an alternative way of defense mechanisms in HS, involving homeostatic regulation and controlled degradation processes to maintain integrity and vital functions of the cell.     

Effects of storage conditions on sprouting of microtubers of yam (Dioscorea cayenensis–D. rotundata complex)

The control of field tuber dormancy in the yam (Dioscorea cayenensis–D. rotundata complex) is poorly understood. Although studies have examined single environmental factors and chemical treatments that might prolong tuber dormancy and storage, only a few were focused on further tuber sprouting. The present study concerns microtubers obtained by in vitro culture. When microtubers were harvested (after 9 months of culture) and directly transferred on a new medium without hormones, the tubers rapidly sprouted in in vitro conditions. No dormancy was observed in this case. Harvested microtubers were also stored dry in jars in sterile conditions during 2 to 18 weeks before in vitro sprouting. In this case, microtubers stored during 18 weeks sprouted more rapidly than those stored 8 weeks. A constant “dormancy-like period” (storage duration + sprouting delay) was observed, between 20 and 28 weeks respectively for the more rapid and the slower microtubers. The size of the tubers used for the storage had great influence on further sprouting. The larger they were, the better they sprouted. Light during storage had no effect on the sprouting delay while a temperature of 25 °C permit a quicker sprouting than 18 °C. The medium used to obtain microtubers could also have an effect on sprouting rate.     

Polysome formation and incorporation of new ribosomes into polysomes during germination of the embryonic axis of maize

Isolated axes of Zea mays L. cvs CiV₂ and CUZCO were imbibed for different periods of time, and free polysomes were extracted and analysed by centrifugation in a sucrose gradient. The amount of rRNA per axis was determined at different moments of germination. Polysome reassembly was practically completed by 8 h and 54% of the preformed ribosomes were found in the polysome fraction. An increase in the proportion of large polysomes was also observed during this period of germination. During the following period, the polysome content and the distribution of the various classes of polysomes remained unchanged.
The time of appearance of newly synthesized ribosomes into the polysomes was investigated using axes germinated in the presence of [³H]-uridine. Centrifugal analysis of EDTA-dissociated polysomes and gel electrophoretic analysis of polysomal RNA showed that new ribosomes appeared into polysomes a few hours after completion of the initial polysome assembly. When released into the cytoplasm, the new ribosomes were preferentially incorporated into polysomes rather than stored as free ribosomes.