RuBPCase Activity Research Articles

Regulation of Autotrophic and Heterotrophic Metabolism in Pseudomonas oxalaticus OX1. Growth on Fructose and on Mixtures of Fructose and Formate in Batch and Continuous Cultures

In Pseudomonas oxalaticus the synthesis of enzymes involved in autotrophic CO2 fixation via the Calvin cycle is regulated by repression/derepression. During growth of the organism on fructose alone, the synthesis of ribulosebisphosphate carboxylase (RuBPCase) remained fully repressed, both in batch culture and in fructose-limited continuous cultures at various dilution rates. Growth in batch culture on a mixture of fructose and formate resulted in the simultaneous utilization of both substrates. Under these conditions we observed synthesis of RuBPCase up to high levels, indicating that formate did not merely function as an ancillary energy source in the metabolism of fructose, but stimulated autotrophic CO2 fixation via the Calvin cycle. In subsequent experiments growth of P. oxalaticus on mixtures of fructose and formate was studied in carbon source-limited continuous cultures. In these experiments further evidence was obtained that fructose is a poor source of (co-)repressor molecules for the synthesis of RuBPCase in the presence of formate. Thus, addition of formate to the medium reservoir of a fructose-limited continuous culture resulted in derepression of RuBPCase synthesis at (relatively) high ratios of fructose over formate. In the reverse experiment the specific activity of RuBPCase decreased with increasing concentrations of fructose in the medium reservoir. However, it can be calculated that the total capacity of RuBPCase in the culture to fix CO2 remained constant. In these experiments the dry weight produced on the various mixtures equalled the sum of the dry weight values obtained during growth on the same amounts of the two substrates separately. This indicated that, once RuBPCase was present, autotrophic and heterotrophic carbon assimilation pathways functioned simultaneously and independently of each other. Possible explanations for the low repressing effect of fructose on autotrophic CO2 fixation in P. oxalaticus are discussed.

Effects of Light and Elevated Atmospheric CO2 on the Ribulose Bisphosphate Carboxylase Activity and Ribulose Bisphosphate Level of Soybean Leaves

Soybean (Glycine max L. Merr. cv Bragg) was grown throughout its life cycle at 330, 450, and 800 microliters CO(2) per liter in outdoor controlled-environment chambers under solar irradiance. Leaf ribulose-1,5-bisphosphate carboxylase (RuBPCase) activities and ribulose-1,5-bisphosphate (RuBP) levels were measured at selected times after planting. Growth under the high CO(2) levels reduced the extractable RuBPCase activity by up to 22%, but increased the daytime RuBP levels by up to 20%.Diurnal measurements of RuBPCase (expressed in micromoles CO(2) per milligram chlorophyll per hour) showed that the enzyme values were low (230) when sampled before sunrise, even when activated in vitro with saturating HCO(3) (-) and Mg(2+), but increased to 590 during the day as the solar quantum irradiance (photosynthetically active radiation or PAR, in micromoles per square meter per second) rose to 600. The nonactivated RuBPCase values, which averaged 20% lower than the corresponding HCO(3) (-) and Mg(2+)-activated values, increased in a similar manner with increasing solar PAR. The per cent RuBPCase activation (the ratio of nonactivated to maximum-activated values) increased from 40% before dawn to 80% during the day. Leaf RuBP levels (expressed in nanomoles per milligram chlorophyll) were close to zero before sunrise but increased to a maximum of 220 as the solar PAR rose beyond 1200. In a chamber kept dark throughout the morning, leaf RuBPCase activities and RuBP levels remained at the predawn values. Upon removal of the cover at noon, the HCO(3) (-) and Mg(2+)-activated RuBPCase values and the RuBP levels rose to 465 and 122, respectively, after only 5 minutes of leaf exposure to solar PAR at 1500.These results indicate that, in soybean leaves, light may exert a regulatory effect on extractable RuBPCase in addition to the well-established activation by CO(2) and Mg(2+).

Ribulose Bisphosphate Carboxylase Activity in Anther-Derived Plants of Saintpaulia ionantha Wendl. Shag

Plants obtained from anther culture of the African violet, Saintpaulia ionantha Wendl. ;Shag' and vegetatively cloned copies of the parent anther donor plant were examined for their ploidy and ribulose-1,5-biphosphate carboxylase (RuBPcase) activity. The cloned parent plants were all diploid and did not vary much in their nuclear DNA, chlorophyll, and RuBPcase activity. Some of the anther-derived plants were similar to the parent plants while others were not. Different levels of ploidy were observed among the androgenetic plants. RuBPcase activities higher than that of the parent plants were found in some anther-derived plants. However, there was no direct correlation between ploidy and RuBPcase activity. Expression of nuclear genes from a single parent in the anther-derived plants and it's diploidization or plastid changes during early stages of microsporogenesis or androgenesis are suggested as possible reasons for the variations observed among them. This could be a useful technique to obtain physiological variants which could be agronomically desirable.

Primary production of marine snow during and after an upwelling event1

Marine snow (flocculent macroscopic particles) collected during an upwelling event near Santa Barbara, California, was recently formed and inhabited by phytoplankton physiologically similar to those free‐living in the water. At least 20% of all primary production, Chl a, and phytoplankton occurred on this marine snow, suggesting that a significant fraction of new organic carbon entering the upwelling food chain is available to large‐particle feeders or passes directly through the detrital food chain on particles. In contrast, marine snow collected 2 weeks after the upwelling event was inhabited by a physiologically distinct community of aging, senescent phytoplankton; <1% of total primary production and Chl a occurred on this marine snow. High dark fixation rates, high RUBPCase activity on photosynthetically depressed snow, and high ammonium concentrations within this snow suggest that chemoautotrophic bacteria, possibly nitrifying bacteria, may be particularly active on these macroscopic particles.

RuBP carboxylase determination by enzymic estimation of D-3-PGA formed

RuBPcarboxylase activity was measured in extracts of barley (Hordeum Vulgare L., cv. HOP) seedlings both with the standard radiometric method and by measuring D-3-phosphoglyceric acid formed enzymically in a two stage assay. In the different conditions used, characterized by different NaHCO3 concentrations, different pH and the presence and absence of oxygen, essentially the same ratio of D-3-PGA formed per (14)CO2 fixed was obtained. This ratio respected the known stoichiometry of two molecules of D-3-PGA formed per CO2 fixed.It is suggested that measurement of D-3-PGA enzymically in a two stage assay can be routinely used for the determination of RuBP case activity instead of the radiometric method. The advantages and the validity of the method are discussed.

Fluoride, hydrogen, and formate activate ribulosebisphosphate carboxylase formation in Alcaligenes eutrophus.

Alcaligenes eutrophus formed ribulosebisphosphate carboxylase (RuBPCase; EC 4.1.1.39) when grown on fructose. Addition of sodium fluoride (NaF) to fructose minimal medium resulted in a slightly decreased growth rate and a rapid fivefold increase in RuBPCase specific activity. With citrate, a glucogenic carbon source, RuBPCase was also formed, However, addition of NaF to cells growing on citrate resulted in a 50% decrease in RuBPCase specific activity. Among the enzymes of fructose catabolism, NaF (10 mM) inhibited enolase in vitro by 98% and gluconate 6-phosphate dehydratase by 87%. Inhibition of the dehydratase by NaF was insignificant in vivo, as determined with a mutant defective in phosphoglycerate mutase activity. Growth of this mutant on fructose was not inhibited by NaF, and only a minor increase in RuBPCase activity was observed. From these results, we concluded that the product of the enolase reaction, phosphoenolpyruvate, played a role in RuBPCase formation. Addition of H2 or formate to the wild type growing on fructose or citrate did not affect the growth rate but resulted in rapid formation of RuBPCase activity. Mutants impaired in H2 metabolism formed RuBPCase at a low rate during growth on fructose plus H2 but at a high rate on formate. Apparently, additional reductant from H2 or formate metabolism induced RuBPCase formation in A. eutrophus.

Metabolic Changes in Senescing Soybean Leaves of Similar Plant Ontogeny1

Delaying the onset or reducing the rate of leaf senescence may be one means of increasing crop productivity. But before senescence can be controlled, it must first be characterized. We compared the onset of leaf senescence, defined as the time at which apparent photosynthesis began to decline irreversibly, with ribulose‐l,5‐bisphosphate carboxylase (RuBPCase) activity, chlorophyll content, total protein content, leaf diffusive resistance, and dark respiration in outdoorgrown soybean [Glycine max (L.) Merr. ‘Amsoy 71’] leaves that had emerged during plant reproduction. We sought to determine which parameter(s) was associated with the onset of senescence. Apparent photosynthesis, as estimated by infrared gas analysis, began to decline about 85 days after planting in leaves at both nodes 12 and 15. There was no change in diffusive resistance, protein content, or dark respiration until about 3 weeks later. On the other hand, RuBPCase activity and chlorophyll content declined concomitantly with photosynthesis. This pattern was similar in both leaves. Thus, we conclude that RuBPCase activity and chlorophyll were best associated with the onset of senescence and that a similar mechanism seems to be operating in both leaves.

RuBP carboxylase determination by enzymic estimation of D-3-PGA formed

RuBPcarboxylase activity was measured in extracts of barley (Hordeum Vulgare L., cv. HOP) seedlings both with the standard radiometric method and by measuring D-3-phosphoglyceric acid formed enzymically in a two stage assay. In the different conditions used, characterized by different NaHCO3 concentrations, different pH and the presence and absence of oxygen, essentially the same ratio of D-3-PGA formed per (14)CO2 fixed was obtained. This ratio respected the known stoichiometry of two molecules of D-3-PGA formed per CO2 fixed.It is suggested that measurement of D-3-PGA enzymically in a two stage assay can be routinely used for the determination of RuBP case activity instead of the radiometric method. The advantages and the validity of the method are discussed.

Ploidy Effects in Isogenic Populations of Alfalfa

The influence of polyploidization on ribulose-1,5-bisphosphate carboxylase (RuBPCase), buffer-soluble protein (BSP), chlorophyll (Chl), and DNA was examined in fully expanded leaves of isogenic diploid-tetraploid (DDC 2X-4X) and tetraploid-octoploid (IC 4X-8X) sets of alfalfa (Medicago sativa L.). The concentration of RuBPCase in leaf extracts was determined by rocket immunoelectrophoresis. Activities of RuBPCase, expressed per milligram protein or per milligram Chl, and leaf tissue concentrations of RuBPCase, BSP, Chl, and DNA were similar between ploidy levels of the DDC 2X-4X set. Tetraploids and octoploids were similar in RuBPCase activities, expressed per milligram protein or per milligram Chl, and in leaf tissue concentrations of RuBPCase and DNA. Octoploids were significantly lower than tetraploids in concentrations of Chl and BSP.When compared on a per leaf basis, tetraploids were 80% higher in BSP and essentially double comparable diploids in fresh weight, RuBPCase, Chl, and DNA. The observation that leaves of the DDC tetraploid population contain twice as much DNA as comparable diploids suggests that leaves of both ploidy levels contain similar numbers of cells. Leaves of the octoploid population were 33% to 80% higher than corresponding tetraploids in BSP, fresh weight, RuBPCase, Chl, and DNA. Ratios of RuBPCase to DNA and Chl to DNA were similar across ploidy levels of both isogenic sets suggesting that cellular content of Chl and RuBPCase increases proportionately with the amount of DNA per cell.

Effects of UV-B radiation (280–320 nm) on photosynthetic constituents and processes in expanding leaves of soybean ( Glycine max (L.) Merr.)

Soybean plants, Glycine max (L.) Merr. cv. Bragg, were grown and exposed to UV-B radiation (280–320 nm) under greenhouse conditions. Concentrations of chlorophyll, carotenoids and soluble protein, net CO 2 uptake rates, RuBP case activity, and leaf fresh weight, area and specific weight of the first trifoliate leaves (numbered acropetally above the cotyledonary node) were analysed at selected times during the period from 8 to 43 days after leaf emergence. Plants were irradiated for 6 hr daily with UV-B irradiance which was provided by two Westinghouse FS-40 sun lamps filtered with 0.127-mm cellulose acetate film (UV-B treatments) or 0.127-mm Mylar® S (control). Two UV-B radiation levels were used: 1.36 UV-B seu (treatment T 1) and 1.83 UV-B seu (treatment T 2) where 1 UV-B seu equals 15.98 mW m −2 of weighted solar UV-B irradiance, designed to simulate the DNA absorption spectrum by the weighting function EXP-[(λ-265)/21] 2. These UV-B levels corresponded to calculated 21 and 36% decreases in ozone concentration for T 1 and T 2, respectively. Continuous exposure of soybean plants to UV-B radiation caused significant depressions in content of chlorophyll and carotenoids. Leaf soluble protein, except at 8 days (for both T 1 and T 2) and 11 days (for T 2), was always lower in the two UV-B treatments than in thhe Mylar control. Activity of RuBPcase extracted from leaves of the T 2 plants was always significantly lower than that of the control. Differences in RuBPcase activity between T 1 and control were observed at 8 days, 25 days, and 36 days. Net CO 2 uptake rates which were measured at 15 days after leaf emergence were significantly lower in both UV-B-exposed treatments when compared with those of the Mylar control. At 15 days after emergence, fresh weight and area of leaves were smaller whereas specific leaf weight was greater in the T 2 treatment. These data show the potential of excessive solar UV-B radiation to reduce photosynthetic pigments, net photosynthetic rates, soluble protein, RuBPcase activity, and leaf weight and area that could lead to decreased vegetative growth and reduced yields of UV-B-sensitive soybean plants.

Changes in the Activities of Ribulose 1,5-Bisphosphate and Phosphoenolpyruvate Carboxylases in Submersed Aquatic Angiosperms during Aging

Changes in the activities of ribulose-1,5-bisphosphate carboxylase (RuBPcase) and phosphoenolpyruvate carboxylase (PEPcase) during aging and senescence of leaves of submersed aquatic angiosperms, Potamogeton pectinatus L., Vallisneria spiralis L., and Hydrilla verticillata (L.f.) Royle were studied. The activity of RuBPcase decreased with both aging and senescence in three species. PEPcase activity increased from young to mature leaves in all three plants and also in old leaves of Hydrilla. The ratio of RuBPcase to PEPcase activity was lowest in mature and highest in old leaves, and increased with aging of isolated mature leaves in Potamogeton and Vallisneria, but decreased markedly in Hydrilla with aging and senescence. Kinetin treatment (0.23 mm) increased RuBPcase activity in three species and PEPcase activity in Potamogeton and Vallisneria, but decreased PEPcase activity in Hydrilla. Treatments with 2-chloroethylphosphonic acid (Ethrel, 0.69 mm) and abscisic acid (ABA, 0.075 mm) showed almost an opposite trend.

Photosynthetic and respiratory consequences of hydrogen chloride gas exposures of Phaseolus vulgaris L. and Spinacea oleracea L.

The effects of supra-acute exposure to gaseous HCl on Phaseolus vulgaris L. and Spinacea oleracea L. have been examined with respect to chlorophyll, ultrastructure, photosynthesis, respiration and RubPCase activity. Changes in ultrastructure and chlorophyll levels were noted in HCl-exposed plants. Photosynthetic and respiratory rates of leaf discs sampled from treated plants were: reduced directly proportional to the severity of injury; more depressed following HCl exposure than 24 h after exposure and enhanced in plants which exhibited minimal or no visual necrosis injury. Similar results were obtained using isolated chloroplasts. The rate of photosynthesis in chloroplasts isolated from spinach was shown to decrease with increasing incubation medium acidity resulting from the addition of HNO 3, H 2SO 4 or HCl. Enzyme extracts from control and HCl-exposed Phaseolus vulgaris L. were prepared and RubPCase was measured. Enzyme activity was stimulated in plants sustaining little or no visual injury and was depressed as either injury or concentration of HCl increased. Results suggest that exposure to HCl causes tissue acidification.

Light-shade adaptation by the oceanic dinoflagellates Pyrocystis noctiluca and P. fusiformis

Species-specific rates of photosynthetic carbon uptake (P), chlorophyll a content and P versus irradiance (P-I), have been measured for cells of Pyrocystis noctiluca and P. fusiformis isolated from natural populations collected in the euphotic zone within and below the surface mixed layer in the Sargasso Sea. These same measurements and the assay for ribulose bis-phosphate carboxylase (RuBP-Case), have been made for cultures of P. noctiluca in a 12 h L: 12 h D photoperiod at 9 different constant or at changing light intensities. In nature chl a cell-1 was constant throughout the euphotic zone. The photosynthetic capacity (Pmax), of cells captured below the surface mixed layer was lower by a factor of 10 compared with cells collected from the surface mixed layer. The Pmax for P. noctiluca collected and incubated within the surface mixed layer was the same as for cell cultures grown under high light, non nutrient-limiting conditions, suggesting that photosynthesis in the natural system was not nutrient limited. In laboratory cultures under constant low light intensities, chl a cell-1 increased by a factor of 5 while both Pmax and RuBPCase activity decreased by a factor of ca 4 compared with high light intensities. In changing light intensities both Pmax and RuBPCase activities were decreased by factors of 4 during low light intervals while chl a cell-1 approached a constant intermediate value. The change in chl a cell-1 in response to prolonged exposure to constant low light intensities was first order with a rate constant of 0.33 d-1. For all irradiance conditions in culture, the P-I dependence could be described by the simple Michaelis-Menten formula. The ratio of Pmax to KI, (the light intensity where P=Pmax/2) was a constant with a Coefficient of Variation of 12%: The constancy of this ratio, the parallel changes in RuBPCase activity with Pmax and the constant chl a cell-1 in the Sargasso Sea imply that for P. noctiluca and presumably P. fusiformis in nature, a dark enzymatic step rather than changes in photosynthetic pigment concentrations may regulate the photosynthetic capacity in the changing photic environment.

Temperature adaptation in phaeodactylum tricornutum Bohlin: Photosynthetic rate compensation and capacity

Two views of temperature adaptation in marine phytoplankton have been proposed. The first emphasizes that adaptation is manifest as a compensation of photosynthetic rate (some time after the initial chemical kinetic response to temperature shift) so that eventually cells are able to perform more or less at the same level over a wide temperature range. The second emphasizes that adaptation is manifest as a change in the maximum photosynthetic ability. In order to examine these two concepts concurrently, short-term light saturated photosynthetic rates per cell ( P m ) were examined as a function of temperature for Phaeodactylum tricornutum Bohlin grown at various temperatures in turbidostat cultures. Within the range from 10 to 25°C, when cells grown at a high temperature were transferred to a low temperature, P m decreased according to Arrhenius-type kinetics in the short term. However, prolonged exposure at the lower temperature led eventually to compensation which partly restored P m to its original value. Within the same temperature range, growth at lower temperatures resulted in higher maximum photosynthetic abilities (i.e. P m measured at the optimal assay temperature) which were positively correlated with in vitro activities of ribulose-l,5-bisphosphate carboxylase (RuBPCase). Within this temperature range, the amount of total protein per cell did not change but there was an increase in the amount of chlorophyll α per cell with temperature. It is suggested that the regulation of RuBPCase activity may perhaps be associated with the two manifestations of temperature adaptation exhibited by P. tricornutum: partial compensation of P m and change in maximum photosynthetic ability.

Photosynthesis in Polyploid Tall Fescue

Net photosynthesis on a leaf area and leaf weight basis increased significantly with ploidy in a 4X, 6X, 8X and 10X allopolyploid series of tail fescue (Festuca arundinacea Schreb.). Total protein did not increase significantly with ploidy. Rocket immunoelectrophoresis was used to quantitate ribulose-1, 5-bisphosphate carboxylase (RuBPCase) protein. RuBPCase content, expressed on both a concentration basis and as a percentage of total protein increased significantly with ploidy in both field and greenhouse experiments. The range of RuBPCase content was 16 to 73% of total protein and 2.8 and 6.5 mg/ml of extract. Specific activity of RuBPCase did not increase significantly with ploidy. Chlorophyll concentration increased as a quadratic function of ploidy, with the mean for 8X genotypes representing maximal chlorophyll content. Evidence is presented that increasing concentrations of RuBPCase are associated with higher net photosynthesis rates in tall fescue. This suggests that RuBPCase may represent a marker for increased net photosynthesis. RuBPCase was extracted in a partially active state or inhibited state and must be fully activated by Mg(2+) and HCO(3) (-) to measure maximal activities. Polyploidization appeared to increase selectively the allocation of total protein for synthesis of RuBPCase; however, there was also a range for carboxylase content among the genotypes within a given ploidy level.

Quantification and intracellular distribution of ribulose-1,5-bisphosphate carboxylase in Thiobacillus neapolitanus, as related to possible functions of carboxysomes

Ribulose-1,5-bisphosphate carboxylase (RuBPCase) has been quantified by immunological methods in Thiobacillus neapolitanus cultivated under various growth conditions in the chemostat at a fixed dilution rate of 0.07 h-1. RuBPCase was a major protein in T. neapolitanus accounting for a maximum of 17% of the total protein during CO2 limitation and for a minimum of 4% during either ammonium- or thiosulfate limitation in the presence of 5% CO2 (v/v) in the gasphase. The soluble RuBPCase (i.e. in the cytosol) and the particulate RuBPCase (i.e. in the carboxysomes) were shown to be immunologically identical. The intracellular distribution of RuBPCase protein between carboxysomes and cytosol was quantified by rocket immunoelectrophoresis. The particulate RuBPCase content, which correlated with the volume density of carboxysomes, was minimal during ammonium limitation (1.3% of the total protein) and maximal during CO2 limitation (6.8% of the total protein). A protein storage function of carboxysomes is doubtful since nitrogen starvation did not result in degradation of particulate RuBPCase within 24 h. Proteolysis of RuBPCase was not detected. Carboxysomes, on the other hand, were degraded rapidly (50% within 1 h) after change-over from CO2 limitation to thiosulfate limitation with excess CO2. Particulate RuBPCase protein became soluble during this degradation of carboxysomes, but this did not result in an increase in soluble RuBPCase activity. Modification of RuBPCase resulting in a lower true specific activity was suggested to explain this phenomenon. The true specific activity was very similar for soluble and particulate RuBPCase during various steady state growth conditions (about 700 nmol/min·mg RuBPCase protein), with the exception of CO2-limited growth when the true specific activity of the soluble RuBPCase was extremely low (260 nmol/min ·mg protein). When chemostat cultures of T. neapolitanus were exposed to different oxygen tensions, neither the intracellular distribution of RuBPCase nor the content of RuBPCase were affected. Short-term labelling experiments showed that during CO2 limitation, when carboxysomes were most abundant, CO2 is fixed via the Calvin cycle. The data are assessed in terms of possible functions of carboxysomes.

Regulation of Ribulose Bisphosphate Carboxylase Activity in Intact Wheat Leaves by Light, CO2, and Temperature

The activity of the enzyme ribulose bisphosphate carboxylase (RuBPCase) was estimated after rapidly extracting it from intact wheat leaves pretreated under different light and C02 levels. No HCOj was added to the extraction buffer since it is shown to inhibit RuBPCase. The activity increased as light intensity or C02 concentration during pretreatment was increased. Enzyme activity increased as temperature during pretreatment was decreased. Light activation did not affect the affinity of RuBPCase for C02. A Km of 30 //M C02 under air level 02 was determined. C02, light and temperature are three main limiting factors of photosynthesis. It seems that the activity of RuBPCase is regulated by these factors according to the requirements for C02 fixation.

Physiological observations of extranuclear temperature-sensitive lethality in Nicotiana tabacum L.

Ultrastructural observations of extranuclear temperature-sensitive induced lethality in a mutant of Nicotiana tabacum L. have shown that chloroplasts in this mutant gradually degenerate in plants grown at reduced temperatures, while mitochondria remain intact (Nessler and Wernsman, 1979). To further elucidate the organellar site of temperature sensitivity in this same tobacco mutant, leaf chlorophyll content, rates of oxygen exchange and ribulose 1,5 bisphosphate carboxylase (RuBPCase) activities were examined in temperature sensitive and control genotypes at varying time intervals during 35 days exposure to lethal temperatures (13°C day/8°C night). Rates of photosynthetic oxygen evolution by leaf disks rapidly declined in mutant plants after one week at low temperatures and remained significantly lower than rates in controls. Total chlorophylls in both mutant and control plants gradually decreased with the increased time at reduced temperatures. This decrease in chlorophyll content of temperature sensitive plants was not sufficiently rapid to account for the sharp drop observed in photosynthetic rates and probably resulted from the disruption of chloroplast membranes. Mitochondrial function in mutant plants was apparently unaffected by prolonged exposure to lethal temperatures as evidenced by the almost constant rates of respiratory oxygen uptake seen throughout the experiment. RuBPCase activities on a leaf area basis were similar in mutant and control plants over the last four sampling dates (days 14–35); thus this enzyme does not appear to be the site of the temperature-sensitive lesion. The loss of chlorophylls, rapid decline in photosynthetic oxygen evolution and continued respiratory oxygen uptake reported here for mutant plants grown at lethal temperatures supports the concept that the chloroplast is the site of extranuclear temperature-sensitivity in this tobacco genotype.

Photosynthesis, Leaf Resistances, and Ribulose-1,5-Bisphosphate Carboxylase Degradation in Senescing Barley Leaves

The relationship between loss of ribulose-1,5-bisphosphate carboxylase (RuBPCase) and the decline in photosynthesis during the senescence of barley primary leaves was assessed. Loss of RuBPCase accounted for about 85% of the decrease in soluble protein. RuBPCase was highly correlated with in vitro RuBPCase activity (r = 0.95) and gross photosynthesis (r = 0.96). However, the rate of photosynthesis per milligram RuBPCase increased during the early stages of leaf senescence. The concentration of nonreducing sugars was negatively correlated (1% level) with photosynthesis. Free alpha-amino N, in contrast to nonreducing sugars, declined markedly during senescence. A decrease in chlorophyll and an increase in in vitro protease activity was observed, but these changes did not appear to be closely related to the decline in photosynthesis and RuBPCase. Mesophyll resistance increased at the same rate that photosynthesis and RuBPCase declined. Stomatal resistance increased more rapidly than mesophyll resistance and accounted for about 24% of the total increase in resistance to CO(2) diffusion. The concentration of CO(2) in the intercellular air spaces decreased during the last stage of senescence. Although loss of RuBPCase probably is the primary event responsible for the decline in photosynthesis during leaf senescence, other factors such as in vivo regulation and stomatal aperture must also be considered.

Changes in Photosynthesis, Ribulose Bisphosphate Carboxylase, Proteolytic Activity, and Ultrastructure of Soybean Leaves during Senescence1

Changes in photosynthesis, rlbulose bisphosphate carboxylase (RuBPCase), and proteolytic activity were followed in the leaves of field‐grown soybeans [Glycine max (L.) Merr. cv. Kent] from flowering through senescence. These parameters were followed in relation to changes in leaf resistance, chlorophyll, protein, starch, total N levels, and seed development. In addition, changes in leaf ultrastructure were observed. The initial symptoms of senescence (evident 3 to 4 weeks after flowering) were a decline in photosynthesis, chlorophyll, and total leaf N and an increase in proteolytic activity. Preceding these changes there was a swelling of the chloroplasts and a disorientation of the chloroplast lamellae, possibly resulting from the apparent increase in starch deposition. Also, large numbers of osmiophilic granules appeared within the chloroplasts. These changes were evident prior to the time the seed entered its most rapid period of growth which was 4 to 7 weeks after flowering, The initial decline in photosynthesis did not appear to be due to an increase in leaf resistance or a decline in RuBPCase activity or level. The decline in protein levels began between 5 and 6 weeks after flowering and was paralleled by the decline in carboxylase activity and level. Associated with these changes were an increase in the size of the osmiophilic granules within the chloroplasts, a decrease in the number of chloroplasts with a corresponding increase in the apparent cellular breakdown products, and a dissolution of the vacuoles. No large increase in leaf resistance or change in specific activity of carboxylase was observed until late in senescence.