Dane R. Roberts

Exogenous polyamines alter membrane fluidity in bean leaves — a basis for potential misinterpretation of their true physiological role

Changes in the rotational motion of paramagnetic and fluorescent lipid-soluble probes were used to assess the effects of putrescine, spermidine and spermine on the fluidity of microsomal membranes from primary leaves of bean (Phaseolus vulgaris L.). Surface probes were more strongly immobilized by physiological concentrations of the polyamines than probes that partitioned deep into the bilayer interior. Spermidine and spermine were more effective than putrescine at reducing membrane fluidity, and at equimolar concentrations, the polyamines and calcium had similar effects on the mobility of the membrane probes. Spermine had essentially equivalent effects on the fluidity of native membranes, heat-denatured membranes and liposomes prepared from the total lipid extract of the membranes, indicating that polyamines associate with membrane lipid. These results raise the possibility that some of the physiological effects previously attributed to exogenously added polyamines could reflect membrane rigidification rather than a true physiological response.

Differential changes in the synthesis and steady-state levels of thylakoid proteins during bean leaf senescence

During senescence of primary bean leaves (Phaseolus vulgaris), there are differential changes in the rates at which thylakoid proteins are synthesized. In particular, synthesis of the 32 kD herbicide-binding protein continues throughout senescence, whereas formation of the α and β subunits of ATPase, the 68 kD photosystem I reaction center polypeptide, cytochrome f, cytochrome b6 and the structural apoprotein of the lightharvesting chlorophyll protein complex (LHCP) declines. Pulse-chase experiments with intact leaves indicated rapid degradation of the 32 kD protein, which is consistent with its known rapid rate of turnover. This degradation was light-dependent and inhibited by DCMU, and the kinetics of degradation were similar for young and senescent membranes. In Coomassie-stained gels, the 68 kD reaction center polypeptide of photosystem I, the α and β subunits of ATPase and the LHCP were the dominant proteins for all ages of membranes. Western blot analysis indicated that cytochrome f and cytochrome b6 are selectively depleted during senescence. The data have been interpreted as indicating that translational disruptions in both the cytoplasmic and chloroplastic compartments may contribute to the decline in photosynthetic electron transport in the senescing leaf.

Evaluation of somaclonal variation during somatic embryogenesis of interior spruce (Picea glauca engelmannii complex) using culture morphology and isozyme analysis

Somaclonal variation during interior spruce (Picea glauca engelmannii complex) somatic embryogenesis was evaluated using culture morphology and isozyme analysis. Genotype-specific abscisic acid-dependent developmental profiles and isozyme patterns were similar for subclone and parent line embryogenic cultures and cotyledonary somatic embryos. Extensive analysis of fifteen hundred subclone embryos of one genotype revealed no isozyme pattern variation. Initiation of embryogenic cultures was dependent on the developmental stage of the explant although cultures derived from different stages were morphologically similar. The embryogenic cultures initiated from interior spruce embryos show a high degree of genetic stability in that the morphological behavior and isozyme phenotype were always consistent with that of the explant genotype. These results support the conclusion that this culture system is appropriate for clonal propagation of interior spruce.

A delivery system for naked somatic embryos of interior spruce

Interior spruce, a mixture of P. glauca and P. engelmannii, is an important forestry species in British Columbia. In recent years significant advances have been made in the protocol for interior spruce somatic embryogenesis. Maturation in the presence of abscisic acid resulted in greater production of late cotyledonary embryos, increased storage protein accumulation, and improved overall quality by inhibiting precocious germination (Roberts et al. 1990a). Subsequently, a high-relative-humidity (HRH) treatment which causes partial drying of the embryos, effects a more synchronized and vigorous germination (Roberts et al. 1990b). Following germination in vitro, emblings (plants produced through somatic embryogenesis) can be planted in soil and grown in the nursery. Assessments of emblings versus control seedlings grown under nursery and field conditions indicate comparable morphological and physiological development (Grossnickle et al. 1992). Silvagen Inc., a Canadian Canadian company, formed an alliance with the Forest Biotechnology Centre (FBC) and Nova Scotia Research Foundation (NSRF) in 1990 to commercialize spruce somatic embryogenesis (Cervelli and Senaratna 1994, this volume). It was clear that in order to produce a large number of emblings, in a commercial setting, there was a need to decrease production costs.

Mass spectral determination of benzamide derivatives of polyamines separated by HPLC

Abstract The identities of benzamide derivatives of putrescine, spermidine and spermine separated from plant tissue extracts by HPLC are confirmed by mass spectral analysis and recoveries are reported.

In Vivo Low Temperature-Induced Decrease in 3-Transhexadecenoic Acid Influences Oligomerization of LHCII

Winter rye (Secale cereale L cv Puma) is dependent on low growth temperature in order to develop a photosynthetic apparatus capable of efficient processing of light energy and reduction of CO2 to carbohydrates (1). Functionally, Puma rye leaves developed at 5°C exhibit a 70% greater capacity to utilize CO2 at low temperature than rye leaves developed at 20°C (2). This was correlated with light-saturated rates of whole chain electron transport (H2O ----> MV) which were 40% higher in thylakoids isolated from 5°C leaves than those isolated from 20°C leaves (3). In addition, 77°K fluorescence emission spectra and room temperature fluorescence induction measurements in the presence of DCMU indicated that the energy distribution between LHCII and PSII reaction centres and between PSI and PSII reaction centres had been altered upon growth and developement at low temperature (4). Griffith et al (5) concluded that development of rye leaves at low temperature results in an alteration in protein-protein interactions associated with LHCII. This is consistent with an earlier report which indicated that 5°C thlakoids exhibited a decrease in particle size on the EF fracture face (6). However, Huner and co-workers (6, 7) have reported that no significant differences between 5°C and 20°C thylakoids exist with respect to pigment or polypeptide composition. In this report we present preliminary evidence which indicates that low temperature developemnt results in a specific alteration in the fatty composition of thylakoid phosphatidylgycerol which, in turn, affects the structural organization of LHCII.