Aimé Nato

Development of photosynthetic characteristics in oil palm during in vitro micropropagation

Summary Various photosynthetic parameters (photochemical activities, CO 2 exchange and carboxylase enzymatic activities) were studied during four characteristic stages of oil palm ( Elaeis guineensis Jacq.) in vitro micropropagation through somatic embryogenesis: proliferating somatic embryos, shootlets (1st and 2nd caulogenesis cycles) and rooted plantlets. In vivo chlorophyll fluorescence measurements indicated that the maximum photochemical activity of photosystem II (PSII) was very low in proliferating embryos and strongly increased in later development stages, finally reaching an activity very close to that measured in acclimatized plants. The quantum yield of photosynthetic electron transport followed the same trend except that a marked depression of electron transport activity was observed in the rooted plandets. CO 2 exchange measurements showed that absolute levels of in vitro photosynthesis were low, but measurable. Photosynthetic activity was also investigated by focusing on certain aspects of carbon metabolism. The activities of two of the primary enzymes of CO 2 fixation, namely PEPC (EC 4.1.1.31) and RuBisCO (EC 4.1.1.39) were measured throughout the micropropagation process. The PEPC:RuBisCO ratio progressively decreased, due to a substantial depletion of PEPC activity. Specific RuBisCO activity did not show any significant alteration, except a transient increase during the first caulogenesis stage on gelified medium. Quantitation of RuBisCO was carried out via rocket immunoelectrophoresis, using a polyclonal antibody raised against RuBisCO from green tobacco leaves. The relative amount of RuBisCO increased during somatic embryo development (from 3.2 % in proliferating embryos to 38.8 % in 2nd-cycle-shoot-lets), then it decreased during the rooting treatment (26.4%). The impact of sucrose enriched media on photosynthetic activity at this stage is discussed.

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.

Changes in phosphoenolpyruvate carboxylase and ribulose-biphosphate carboxylase activities during the photoheterotrophic growth of Nicotiana tabacum (CV xanthi) cell suspensions

Abstract The capacities (in vitro ∗ measurements) of phosphoenolpyruvate carboxylase (PEPCase) and ribulose-biphosphate carboxylase (RuBPCase) have been studied in photoheterotrophically growing cell suspensions of Nicotiana tabacum L. (cv Xanthi). The most significant observation was the different behaviour of the two carboxylases. The PEPCase capacity expressed on a g dry wt. basis rises from an initial day O value of 200–300 μmol of CO 2 fixed to 600–800 μmol during the exponential phase and then returns to the initial value during the post-exponential and stationary phases. The RuBPCase capacity was nearly constant (90–140 μmol of CO 2 fixed) throughout the growth cycle. The peak of respiration and the high PEPCase capacity during the exponential growth phase were concomitant with an active process of soluble protein synthesis. After electrophoresis of cell-free extracts, two bands of PEPCase activity (forms I and II with R m values of 0.23 and 0.38 respectively)_and one band of RuBPCase activity ( R m of 0.27) were localized on polyacrylamide gels. The densitometric profiles of soluble proteins showed a marked enrichment of PEPCase form II band during the active phase of cell division as compared to that of cells in the stationary phase of growth. Moreover, the 2-fold increase of the specific activity (μmol CO 2 fixed/h and/mg of soluble proteins) of the PEPCase during the exponential phase suggested that the enhancement of the catalytic power of this enzyme could be due to de novo protein synthesis. The biosynthesis pattern of PEPCase was essentially that of peak enzyme in contrast with RuBPCase which showed only slight variations over the growth cycle.

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.

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.

Chloroplastic glutamine synthetase from tobacco leaves: a glycosylated protein

The amino acid and carbohydrate content of chloroplastic glutamine synthetase from tobacco leaves has been analysed. The enzyme subunit contanins 5% carbohydrate, mainly represented by glucosamine, galactosamine, glucose, galactose and mannose residues. The enzyme subunit displayed a single band of molecular mass 44000 Da after sodium dodecyl sulphate (SDS) electrophoresis. However, when isoelectrofocussing electrophoresis was performed, four subunits were evident differing by their charge. Furthermore, the four different subunits stained positively when tested with periodic acid Shiff reagent, showing that sugars and amino sugars were present within all the subunits.

Potential use of Cu2+, K+ and Na+ for the destruction of Caulerpa taxifolia: differential effects on photosynthetic parameters.

Chemical techniques were investigated in order to eradicate Caulerpa taxifolia, a green alga spreading at a remarkable rate in the Mediterranean Sea. The action of copper, potassium and sodium ions on survival rates and photosynthetic parameters was compared, in order to optimise the conditions of further in situ treatments. The lethal doses were determined and the impact of the studied cations on photosynthesis and respiration rates and PSII photochemistry was analysed from measurements of net oxygen exchanges and chlorophyll fluorescence. The Cu2+ concentrations required to obtain 100% mortality were 15 × 102 to 104 times lower than those of K+ and Na+. Respiration was slightly affected whatever the salt concentration,while photosynthesis could be totally inhibited depending on the applied treatment. Changes in the structure of the Ribulose-1,5-biphosphate carboxylase/oxygenase (RubisCO, EC: 4.1.1.39) were also detected when C. taxifolia under went cation treatments (10 mg L-1 Cu2+, 1h; 20 gL-1 K+, 3 h; 20 g L-1 Na+, 1 h). Given the high concentration and long incubation periods required with K+ and Na+ ions, these cations are not suitable to be used in situ. Our results make possible the utilisation of copper cations following technical approaches such asion-exchange textile covers, which allows a controlled release of cupric ions without dissemination in the marine environment.

Glutamate synthase isoforms in tobacco cultured cells

Glutamate synthase in tobacco cultured cells (Nicotiana tabacum L. cv. Xanthi) was assayed with different electron donors upon greening of etiolated cells. Ferredoxin-dependent glutamate synthase (EC 1.4.7.1) activity increased 4-fold, whereas NADH-dependent glutamate synthase (EC 1.4.1.14) and NADPH-dependent glutamate synthase (EC 1.4.1.13) activities remained constant during greening of the cells. Ferredoxin-dependent glutamate synthase and NAD(P)H-dependent glutamate synthases were distinguished by their chromatographic behaviour on ferredoxin-Sepharose affinity column. The immunoglobulin G (IgG)-anti-ferredoxin-dependent glutamate synthase from rice green leaves cross-reacted with ferrodoxin-dependent glutamate synthase, on the other hand, NAD(P)H-dependent glutamate synthases were not immunoprecipitated with the IgG, indicating also that ferredoxin-dependent glutamate synthase and NAD(P)H-dependent glutamate synthase activities are carried out by the distinct protein molecules in tobacco cultured cells, and some physiological functions of the enzymes are discussed.

Effect of medium sucrose on the photosynthetic capacity of coconut vitroplants formed from zygotic embryos

The coconut palm is an important source of oil and other products in tropical countries. Coconut populations in the Caribbean area, including Florida (USA), Mexico, Belize and Honduras have being lowered by a severe disease called lethal yellowing (LY) that has killed millions of plants (Oropeza and Zizumbo, 1997). In order to control this disease it is necessary to implement plant-breeding programs to increase both LY resistance and yields. For a successful breeding program it is important to have safe and efficient ways to exchange plant material from country to country. It is clear that the in vitro culture of zygotic embryos meets the need for phytosanitary requirements for plant material transport from country to country. However, the acclimatisation of plants derived from tissue cultured zygotic embryos is not effective enough and shows poor survival rates (Ashburner et al., 1994). It is important to study the physiology of these vitroplants to determine if the problem is related to problems in controlling water loss when vitroplants are transferred from the in vitro container to the field or to problems with the development of photosynthetic ability.

Genetic characterization of two traditional leafy vegetables (Sesamum radiatum Thonn. ex Hornem and Ceratotheca sesamoides Endl.) of Benin, using flow cytometry and amplified fragment length polymorphism (AFLP) markers

Amplified fragment length polymorphism (AFLP) markers and flow cytometry were applied for the genetic characterization of wild and cultivated accessions of Sesamum radiatum and Cerathoteca sesamoides ; two neglected and underutilized species of traditional leafy vegetable consumed in Benin. The average 2C nuclear DNA content per nucleus was found to be 1.99 ± 0.06 and 1.05 ± 0.06 pg for S. radiatum and C. sesamoides , respectively which correspond to estimated genome size of 1946,22 Mpb for S. radiatum and 1026,9 Mpb for C. sesamoides. No variation in DNA content could be detected within accessions from each analysed species. Also, no relation was found between nuclear DNA content, ecogeographical origin and the status (cultivated or wild) of the analyzed accessions. AFLP markers revealed low diversity within the accessions analyzed. Results from the study contributed to a better characterization of S. radiatum and C. sesamoides accessions and will help in defining both genetic resources conservation and breeding strategies. Key words : Amplified fragment length polymorphism (AFLP) markers, Ceratotheca sesamoides, flow cytometry, genetic diversity, ploidy, Sesamum radiatum, leafy vegetables.