Paul A. Castelfranco (1921-2021): a scientist par excellence, a man of lasting faith, and ever a humanist.

We present here the life and the work of Paul A. Castelfranco (1921-2021), a very special person who was not only a top chemist of chlorophyll biosynthesis, but also made major contributions on fatty acid oxidation, acetate metabolism and cellular organization. He led an extraordinary and exemplary life as a human being. We present here both his personal life as well as his scientific life, which is followed by reminiscences by William Breidenbach, Kevin Smith, Alan Stemler, Ann Castelfranco, and John Castelfranco. As the subtitle of this Tribute implies, till the end Paul was a scientist par excellence, an intellectual with unlimited curiosity, a humanist, and a man of enduring religious faith. We all miss him dearly.

In vivo modification of plant membrane phospholipid composition.

Tomato seedlings treated with ethanolamine showed altered phospholipid composition. The changes included altered acyl chain composition as well as changes in the relative amounts of the phospholipid classes. Specifically, there was an increase in phosphatidylethanolamine and phosphatidylserine with a concomitant decrease in phosphatidylcholine and no overall increase in phospholipids. Treatment with ethanolamine increased the relative amount of C18 acyl chains (especially 18 : 2) in phosphatidylethanolamine and phosphatidylcholine at the expense of 16 : 0 and 16 : 1. Acyl composition of other phospholipid classes were unchanged. Labeled ethanolamine was incorporated mostly into phosphatidylethanolamine and phosphatidylcholine. Ethanolamine-stimulated incorporation of labeled oleate was entirely into acyl chains and appeared only as 18 : 1 and 18 : 2. There was greater incorporation, but less conversion of 18 : 1 to 18 : 2 with choline. Stearate was incorporated but desaturated.

Pressure effects on metabolism in tissues from mice (Mus muscalis) and freshwater mussel (Elliptio complanata).

Metabolic rates of tissue sections from freshwater mussel gills and mouse brain and lung tissue were measured by calorimetry in ampules pressurized with gas mixtures. Increasing partial pressure of oxygen or total pressure with constant partial pressure of O2 does not affect the respiratory quotient but increases rates of tissue metabolism. Changes in metabolic activity occur over pressure and Po2 ranges commonly encountered by humans engaged in SCUBA diving.

Proteins of soybean seeds: I. Isolation and characterization of the major components.

Soybean (Glycine max) storage proteins were characterized by sedimentation and by polyacrylamide gel electrophoresis under dissociating (8 m urea) and nondissociating conditions. Three sedimenting classes of proteins were found, with sedimentation coefficients of 2.2S, 7.5S, and 11.8S. The coefficients were related to the bands obtained by electrophoretic separation. The results support the idea that relatively few proteins make up the bulk of the seed protein.

Proteins of Soybean Seeds: II. Accumulation of the Major Protein Components during Seed Development and Maturation.

Fresh weight and dry weight as well as quantitative and qualitative protein changes in the developing soybean (Glycine max) seed were described from 12 days after flowering until maturity. The seed proteins were separated on sucrose density gradients into three major fractions, having average sedimentation coefficients of 2.2S, 7.5S, and 11.8S. The 2.2S sedimenting proteins predominated at very early stages of development (12 days after flowering) and decreased proportionately throughout maturation. The 7.5S and 11.8S components appeared to be synthesized later in maturity and in larger amounts than the 2.2S proteins. Electrophoretic studies on extracts from whole seeds and on isolated protein fractions confirmed the early abundance of proteins in the 2.2S fraction and revealed temporal differences in the accumulation of three components of the 7.5S fraction. The 11.8S sedimenting fraction appeared throughout seed development as a homogeneous protein which accumulated in the seed with a time course similar to that of the total 7.5S protein fraction.

Response to chilling of tomato seedlings and cells in suspension cultures.

Tomato cell suspensions and seedlings (Lycopersicon esculentum) responded comparably when exposed to chilling temperatures (10 C or below). Seedling growth and cellular activities related to cell viability and culture growth (triphenyltetrazolium chloride reduction, fluoroscein diacetate uptake, and hydrolysis) were sharply diminished below 10 C. Arrhenius plots of the respiratory O(2) consumption by both seedlings and cell suspensions had a break at 10 C, as is characteristic for chilling-sensitive species. The acyl chains that were found in the phospholipids of both cell cultures and seedlings were similar. These results indicate the potential usefulness of plant suspension cultures for studies of chilling injury.

Receptor-Mediated Endocytosis in Plants is Energetically Possible.

A detailed examination of the arguments of Cram (1980 New Phytol 84: 1-17) against the uptake of major nutrients in plants by endocytosis shows that the arguments do not exclude the possibility of receptor-mediated endocytosis.

Plant plasma membrane proteins : immunological characterization of a major 75 kilodalton protein group.

A major 75 kD protein group from the tomato plasma membrane was semipurified on polyacrylamide gels and used to raise a rabbit antiserum. The resulting antiserum recognized a single 75 kilodalton band from phase partitioned tomato plasma membrane (from both suspension cells and mature, green fruit) after resolution on one-dimensional polyacrylamide gels. Two-dimensional polyacrylamide gel analysis of proteins from tomato plasma membrane showed that the 75 kilodalton antiserum recognized a group of proteins ranging from 63.1 to 88.2 kilodaltons (mean = 75.6 kilodaltons) and with isoelectric point values ranging from 5.7 to 6.3. No other spots were visible on the two-dimensional blots. This antiserum was shown to bind protoplast surface epitopes by indirect immunofluorescence. The presence of this protein group in both monocotyledonous and dicotyledonous plants was established by immunoblotting the tomato 75 kilodalton antiserum against proteins obtained from plasma membrane-enriched fractions from corn roots and soybean roots. The data suggest that this 75 kilodalton protein group is a major proteinaceous component of the plant plasma membrane.

Physical Properties of Mitochondrial Lipids from Lycopersicon hirsutum.

Mitochondrial lipids from Lycopersicon hirsutum undergo a broad thermal transition beginning well below 0 degrees C and ending at approximately 25 degrees C. Differential thermal analysis of mitochondrial lipids isolated from ecotypes of L. hirsutum that differ in chilling sensitivity indicates that these lipid preparations have physically similar properties. This was confirmed by electron-spin-resonance experiments, although this technique failed to detect the broad transition detected by differential thermal analysis. No quantitative differences were observed between the percentages of individual lipid classes (based on polar head group) or between the fatty acid compositions of mitochondrial lipids from the two ecotypes investigated. These results suggest that the observed differences between the responses of these ecotypes to prolonged exposure to 5 degrees C may not be related to differences between the physical properties of their mitochondrial lipids.

Characterization of deoxyribonucleic Acid species from castor bean endosperm: inability to detect a unique deoxyribonucleic Acid species associated with glyoxysomes.

Three DNA buoyant density species (nuclear, 1.692 g cm(-3); mitochondria 1.705 g cm(-3); and proplastid, 1.713 g cm(-3)) can be detected in extracts from castor bean endosperm. No other buoyant density species can be identified. DNA extracts from sucrose density gradient purified glyoxysomes exhibit varying amounts of each of the three identified DNAs but no other distinguishable DNA species. RNA synthesized in vitro by Escherichia coli RNA polymerase using purified castor bean nuclear DNA as a template, hybridizes equally well with its template and with the 1.692 g cm(-3) species from glyoxysome fractions. These results are discussed in terms of their relevance to microbody biogenesis.

Temperature-induced Phase Changes in the Membranes of Glyoxysomes, Mitochondria, and Proplastids from Germinating Castor Bean Endosperm.

Sucrose-gradient purified mitochondria, glyoxysomes, and proplastids from germinating castor bean (Ricinus communis L.) endosperm were examined by electron-spin resonance spectroscopy. A temperature-induced phase change was demonstrated in all of these organelles, their derivative membranes, and in micelles formed from the membrane phospholipids. The apparent transition temperature of the membrane lipids varied slightly between the samples, but in all cases, fell within the temperature range around 10 C where physiological and biochemical changes in the response to temperature for most chilling-sensitive plants occur.

Effect of chilling temperatures on the activities of glyoxysomal and mitochondrial enzymes from castor bean seedlings.

Castor bean (Ricinus communis L.) is included among the group of plants sensitive to chilling temperatures. Seedlings of this species were shown to exhibit visible symptoms of this injury, as well as impaired radicle growth and storage product mobilization. Respiration of intact seedlings and oxidation of succinate by isolated mitochondria displayed discontinuities in Arrhenius plots of their reaction velocities, characteristic of chilling species. However, gluconeogenic glyoxysomal enzymes do not display such discontinuities, indicating that there is probably no functional relationship between these enzymes and the glyoxysomal membrane.

Biogenesis of Mitochondria in Germinating Peanut Cotyledons II. Changes in Cytochromes and Mitochondrial DNA.

Biogenesis of mitochondria occurs in the germinating cotyledons of peanuts. This process was demonstrated by measuring both constitutive and enzymatic properties of mitochondria as a function of germination time. Direct counting by phase contrast microscopy of sucrose density gradient preparations showed that the number of mitochondria increased markedly during germination. DNA with a buoyant density distinct from the major cellular DNA was associated with these mitochondrial preparations. During germination the amount of this DNA in mitochondrial pellets increased. This increase closely paralleled the increase in number of mitochondria.Succinoxidase and succinic dehydrogenase increased during germination. Both activities were confined to the mitochondrial fraction. The rate of increase of succinoxidase activity was significantly greater than the rate of increase of succinic dehydrogenase and both increased at least initially at a greater rate than the amount of mitochondrial DNA or numbers of mitochondria.The amounts of cytochromes present in mitochondrial preparations were measured spectrophotometrically. All of the cytochromes increased in amount during germination. The rate of increase of cytochrome a - a(3) very close to the rate of increase in succinoxidase activity.

Biogenesis of mitochondria in germinating peanut cotyledons.

The increase in respiratory activity in germinating peanut cotyledons has been correlated with an actual increase in mitochondria. Using sucrose gradient centrifugation, it was shown that succinoxidase activity is associated with a well-defined band of cell particulates of uniform density, size and ultrastructure. During germination the population of succinoxidase-containing particles increases, as shown by enzymatic assay, protein assay and direct particle counts with the phase contrast microscope.

Characterization of glyoxysomes from castor bean endosperm.

Electron micrographs are presented which establish the identity of the components of the 3 major bands observed after sucrose density centrifugation of the crude particulate fraction from the endosperm of germinating castor bean seedlings. These are: mitochondria (density 1.19 g/cc), proplastids (density 1.23 g/cc) and glyoxysomes (density 1.25 g/cc). Further evidence is provided on the enzymatic composition of the glyoxysomes. Essentially all of the particulate malate synthetase, isocitrate lyase, catalase, and glycolic oxidase is present in these organelles. The distribution of glyoxysomal enzymes on sucrose density gradients is contrasted with that of the strictly mitochondrial enzymes fumarase, NADH oxidase, and succinoxidase. Malate dehydrogenase and citrate synthetase are present in both organelles. The functional role of glyoxysomes and their relationship to cytosomes from other tissues is discussed.

Heat generation and dissipation in plants: can the alternative oxidative phosphorylation pathway serve a thermoregulatory role in plant tissues other than specialized organs?

A number of hypothetical physiological roles have been proposed for the cyanide-insensitive alternative pathway in plants (Palmer, 1976; Laties, 1982; Meeuse, 1984; Purvis and Shewfelt, 1994; Wagner and Krab, 1995). The calorimetric observations of Raskin and co-workers (Ordentlich et al., 1991; Nevo et al., 1992; Moynihan et al., 1995) are significant contributions showing an interesting metabolic, chilling-induced response of the alternative pathway activity and differences in the low-temperature response among species adapted to different climates. Since different oxidative pathways do not have large differences in enthalpy, and observed heat rate increases are insufficient to cause significant temperature increases of physiological importance in nonthermogenic plants, other explanations must be developed for the relationship between the partitioning of electron flow and physiological conditions such as low temperature. The induction and engagement of the alternative respiratory pathway is involved in metabolic stasis, maintaining proper balance between carbon flow, ATP-ADP ratio, and electron flow during fluctuating or extreme temperature conditions. The alternative oxidase is engaged when ATP requirements are adequately met, as discussed by Palmer (1976), Meeuse (1983), Lambers (1985), and Wagner and Krab (1995). The expression and kinetic activity of the alternative oxidase are regulated by concentrations of key metabolites (Day and Wiskich, 1995; Siedow and Umbach, 1995; Wagner and Krab, 1995; Day et al., 1996). Dynamic partitioning of electron flow between Cyt oxidase and the alternative oxidase depends on the kinetic behavior of the two oxidases and the substrate dehydrogenases (Day and Wiskich, 1995; Siedow and Umbach, 1995; Wagner and Krab, 1995; Day et al., 1996). Furthermore, Moynihan et al. (1995) found that Episces cupreata Hook, adapted to the tropics, has very little alternative oxidase activity compared with wheat (Nevo et al., 1992), adapted to a large range of temperature climates. This results is consistent with the general relation between the apparent alternative oxidase activity and the climate of origin of the species.

Characterization of a differential low-temperature growth response in two species of Lycopersicon: the plastochron as a tool.

The plastochron model was used to evaluate the differences in the growth response of two Lycopersicon spp. grown under two temperature regimes (25/18 and 12/5°C). Two altitudinal accessions of L. hirsutum Homb. et Bnpl., from low and high altitude, a breeding line of L. esculentum (L.) Mill. and the hybrid between the latter and the high-altitude L. hirsutum were studied. The plastochron (P) values were estimated directly according to the formula of R.O. Erickson and F. Michelini (1957, Am. J. Bot. 44, 297-305), and indirectly through a linear model estimating the exponential rates of leaf elongation (r) and the ln of the plastochron ratios (q). The P values were obtained as P=q/r, and with one exception values obtained with both methods were comparable. Low temperature significantly decreased r in all genotypes, but the extent of this reduction depended on the genotype. The hybrid exhibited the least reduction, followed by the high-elevation L. hirsutum, L. esculentum and the lowelevation L. hirsutum. While the q values of the L. hirsutum accessions were significantly reduced by low temperature, those of L. esculentum and the hybrid were not. With the exception of the low-altitude L. hirsutum, low temperature significantly increased P, however the extent of the increase was significantly greater in L. esculentum. Analysis of temperature dependent changes of r, q and P indicate that L. esculentum extended its P by approximately the same factor its r was reduced. On the other hand, the L. hirsutum accessions increased P to a lesser extent, therefore having the ability to produce, comparatively, more leaves at lower temperatures than the cultivated tomato. The linear model of the plastochron is proposed as a tool for comparative studies of environmental growth responses of different genotypes. Plant size was reduced by low temperature. Considering plant size attained at high temperature as 100%, at low temperature sizes were reduced to 73% for the hybrid, 61% for the high-altitude L. hirsutum, 39% for L. esculentum and 30% for the low-altitude L. hirsutum. The low-temperature regime delayed flowering by two, three and nine plastochrons in the hybrid, the high-altitude L. hirsutum and L. esculentum, respectively, while the low-altitude L. hirsutum did not flower for the duration of the experiment. When artificially pollinated, L. esculentum yielded parthenocarpic fruits, while the high-altitude L. hirsutum and the hybrid produced fruits with viable seeds.

Simultaneous measurement of metabolic heat rate, CO2 production, and O2 consumption by microcalorimetry.

This study describes methods and equipment for measurement of metabolic heat rates of cells and tissues under conditions that provide simultaneous determinations of the flux rates of both O2 and CO2. Isothermal measurement of metabolic heats are conducted in a sealed ampule. A trapping solution is employed to absorb metabolic CO2. Absorption of CO2 produces heat at a rate proportional to the rate of CO2 production. Under these conditions, O2 consumption by the tissue results in a decrease in the partial pressure of O2 within the sealed ampule. The decrease in pressure can be monitored with a pressure sensor and related to O2 consumption rates. The combined measurements of heat rates, CO2, and O2 fluxes provide important information on bioenergetic efficiency of cell metabolism. These data can also suggest possible shifts in metabolic pathways or substrate sources as cells develop, or are exposed to effectors, inhibitors, and environmental factors.