Regulation of ribulose-1,5-bisphosphate carboxylase activity by the activase system in lysed spinach chloroplasts

Ribulose-1,5-bisphosphate (RuBP) carboxylase in lysed spinach (Spinacia oleracea L. cv virtuosa) chloroplasts that had been partly inactivated at low CO₂ and Mg²⁺ by incubating in darkness with 4 millimolar partially purified RuBP was reactivated by light. If purified RuBP was used to inhibit dark activation of the enzyme, reactivation by light was not observed unless fructose-1,6-bisphosphate, ATP, or ADP plus inorganic phosphate were also added. Presumably, ADP plus inorganic phosphate acted as an ATP-generating system with a requirement for the generation of ΔpH across the thylakoid membrane. When the RuBP obtained from Sigma Chemical Co. was used, light did not reactivate the enzyme. There was no direct correlation between ΔpH and activation. Therefore, thylakoids are required in the ribulose-1,5-bisphosphate carboxylase activase system largely to synthesize ATP. Inactivation of RuBP carboxylase in isolated chloroplasts or in the lysed chloroplast system was not promoted simply by a transition from light to dark conditions but was caused by low CO₂ and Mg²⁺.     

Exchange Properties of the Activator CO(2) of Spinach Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

The exchange properties of the activator CO₂ of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase were characterized both in vitro with the purified enzyme, and in situ within isolated chloroplasts. Carboxyarabinitol-1,5-bisphosphate, a proposed reaction intermediate analog for the carboxylase activity of the enzyme, was used to trap the activator CO₂ on the enzyme both in vitro and in situ. Modulation of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in intact chloroplasts during a light/dark cycle was associated with a similar modulation in carboxyarabinitol-1,5-bisphosphate-trapped CO₂. The exchange kinetics of the activator CO₂ were monitored by activation of the enzyme to steady state in the presence of ¹²CO₂, followed by addition of ¹⁴CO₂ and determination of the amount of labeled CO₂ trapped on the enzyme by carboxyarabinitol-1,5-bisphosphate. Rate constants (K_obs) for exchange with both the purified enzyme (0.45 min⁻¹) and in illuminated chloroplasts (0.18 min⁻¹) were comparable to the observed rate constants for enzyme activation under the two conditions. A similar exchange of the activator CO₂ was not observed in chloroplasts in the dark. Kinetic analysis of the exchange properties of the purified enzyme were consistent with an equilibrium between active and inactive forms of the enzyme during steady state activation.     

Ribulose 1,5-bisphosphate and activation of the carboxylase in the chloroplast

Ribulose 1,5-bisphosphate in the chloroplast has been suggested to regulate the activity of the ribulose bisphosphate carboxylase/oxygenase. To generate high levels of ribulose bisphosphate, isolated and intact spinach chloroplasts were illuminated in the absence of CO₂. Under these conditions, chloroplasts generate internally up to 300 nanomoles ribulose 1,5-bisphosphate per milligram chlorophyll if O₂ is also absent. This is equivalent to 12 millimolar ribulose bisphosphate, while the enzyme, ribulose bisphosphate carboxylase, offers up to 3.0 millimolar binding sites for the bisphosphate in the chloroplast stroma. During illumination, the ribulose bisphosphate carboxylase is deactivated, due mostly to the absence of CO₂ required for activation. The rate of deactivation of the ribulose bisphosphate carboxylase was not affected by the chloroplast ribulose bisphosphate levels. Upon addition of CO₂, the carboxylase in the chloroplast was completely reactivated. Of interest, addition of 3-phosphoglycerate stopped deactivation of the carboxylase in the chloroplast while ribulose bisphosphate accumulated. With intact chloroplasts in light, no correlation between deactivation of the carboxylase and ribulose bisphosphate levels could be shown.     

The Association of d-Ribulose- 1,5-Bisphosphate Carboxylase/Oxygenase with Phosphoribulokinase

When Ribulose- 1,5-bisphosphate carboxylase/oxygenase was purified from spinach leaves (Spinacia oleracea) using precipitation with polyethylene glycol and MgCl₂ followed by DEAE cellulose chromatography, 75% of phosphoribulokinase and 7% of phosphoriboisomerase activities copurified with ribulose- 1,5-bisphosphate carboxylase/oxygenase. This enzyme preparation showed ribose-5-phosphate and ribulose-5-phosphate dependent carboxylase and oxygenase activities which were nearly equivalent to its corresponding ribulose- 1,5-bisphosphate dependent activity. The ribose-5-phosphate and ribulose-5-phosphate dependent reaction rates were stable and linear for much longer time periods than the ribulose- 1,5-bisphosphate dependent rates. When sucrose gradients were used to purify ribulose- 1,5-bisphosphate carboxylase/oxygenase from crude stromal extracts, phosphoribulokinase was found to cosediment with ribulose- 1,5-bisphosphate carboxylase. Under these conditions most of the phosphoriboisomerase activity remained with the slower sedimenting proteins. Ammonium sulfate precipitation resulted in separation of the ribulose- 1,5-bisphosphate carboxylase peak from phosphoribulokinase peak. Crude extracts of peas Pisum sativum and spinach contained 0.725 to 0.730 milligram of phosphoribulokinase per milligram of chlorophyll, respectively, based on an enzyme-linked immunosorbent assay.     

Histochemical Compartmentation of Photosynthesis in the Crassulacean Acid Metabolism Plant Crassula falcata

The succulent leaf of the obligate Crassulacean acid metabolism plant Crassula falcata comprises two distinct types of parenchyma. The peripheral tissue is dark green, whereas the central tissue is relatively colorless. We have investigated whether the conventional interpretation of Crassulacean acid metabolism—simply, temporal separation of light and dark CO₂ fixation within individual cells—is sufficient. Ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) and chlorophyll, indicating the photosynthetic-carbon-reduction pathway, were concentrated in peripheral tissue. Specific activities of P-enolpyruvate carboxylase (4.1.1.31) and of NAD⁺-malic enzyme (1.1.1.39), indicating capacity for dark CO₂ fixation and release, respectively, were high in both types of parenchyma. Measured directly as malic acid decline at the beginning of the photoperiod, CO₂ “storage” occurred in both tissues. These data indicate that there is a spatial component to Crassulacean acid metabolism in C. falcata.     

Interactions between ribulose-1,5-bisphosphate carboxylase and stromal metabolites. II. Corroboration of the role of this enzyme as a metabolite buffer

The capacity of ribulose-1,5-bisphosphate carboxylase to bind reversibly chloroplast metabolites which are the substrates for both thylakoid and stromal enzymes was assessed using spinach chloroplasts and chloroplast extracts and with pure wheat ribulose-1,5-bisphosphate carboxylase. Measurements of the rate of coupled electron flow to methyl viologen in ‘leaky’ chloroplasts (which retained the chloroplast envelope and stromal enzymes but which were permeable to metabolites) and also with broken chloroplasts and washed thylakoids were used to study the effects of binding ADP and inorganic phopshate to ribulose-1,5-bisphosphate carboxylase. The presence of ribulose-1,5-bisphosphate carboxylase significantly altered the values obtained for apparent K_m for inorganic phosphate and ADP of coupled electron transport. The K_m (P_i) in washed thylakoids was 60–80 μM, in ‘leaky’ chloroplasts it was increased to 180–200 μM, while in ‘leaky’ chloroplasts preincubated with KCN and ribulose 1,5-bisphosphate the value was decreased to 40–50 μM. Similarly, the K_m (ADP) of coupled electron transport in washed thylakoids was 60–70 μM, in ‘leaky’ chloroplasts it was 130–150 μM and with ‘leaky’ chloroplasts incubated in the presence of KCN and ribulose 1,5-bisphosphate a value of 45–50 μM was obtained. The ability of ribulose 1,5-bisphosphate carboxylase to reduce the levels of free glycerate 3-phosphate in the absence of ribulose 1,5-bisphosphate was examined using a chloroplast extract system by varying the concentrations of stromal protein or purified ribulose 1,5-bisphosphate carboxylase. The effect of binding glycerate 3-phosphate to ribulose-1,5-bisphosphate carboxylase on glycerate 3-phosphate reduction was to reduce both the rate an the amount of NADPH oxidation for a given amount of glycerate 3-phosphate added. The addition of ribulose 1,5-bisphosphate reinitiated NADPH oxidation but ATP or NADPH did not. Incubation of purified ribulose-1,5-bisphosphate carboxylase with carboxyarabinitolbisphosphate completely inhibited the catalytic activity of the enzyme and decreased inhibition of glycerate-3-phosphate reduction. Two binding sites with different affinities for glycerate 3-phosphate were observed with pure ribulose-1,5-bisphosphate carboxylase.     

Causes for the Disappearance of Photosynthetic CO(2) Fixation with Isolated Spinach Chloroplasts

When isolated spinach chloroplasts are illuminated, photosynthesis and CO₂ fixation die off within 30 to 90 minutes. Even when air levels of CO₂ are used which maintain high and rate-saturating amounts of ribulose 1,5-bisphosphate inside the plastids, CO₂ fixation declines. The decline begins with a drop in activity of the ribulose 1,5-bishosphate carboxylase/oxygenase, specifically loss of the enzyme-activator CO₂-Mg²⁺ form. Next, the light reactions cause gradual leakage of the carboxylase and other stromal proteins to the suspending medium. The chloroplast outer envelope appears to reseal and protect the thylakoids since there is little change in the ferricyanide-dependent Hill reaction. In the dark, under otherwise identical conditions, leakage of carboxylase does not occur.     

Photosynthesis of Ulva sp: III. O(2) Effects, Carboxylase Activities, and the CO(2) Incorporation Pattern

Ulva, a common green seaweed, performs at the biochemical level as a typical C₃ plant. Over 90% of label was found in glycerate 3-phosphate following a 3 second ¹⁴C pulse in the light, and the label was subsequently transferred to sugars. Also, the level of ribulose-1,5-bisphosphate carboxylase activity in crude extracts was about 10 times higher than that of phosphoenolpyruvate carboxylase. Concerning gas exchange, photosynthetic rates of Ulva showed no O₂ sensitivity, indicating that photorespiratory CO₂ losses are repressed as in C₄ plants. This apparent anomaly could be explained by the efficient HCO₃⁻ uptake system of Ulva which might concentrate CO₂ to the chloroplasts, thus suppressing the oxygenase activity of ribulose-1,5-bisphosphate carboxylase.     

Development and Characterization of Ribulose-1,5-bisphosphate Carboxylase : Evidence Indicating a Lack of Carboxylase Function in CO(2) Fixation in Endosperm of Germinating Castor Bean Seedlings

Ribulose-1,5-bisphosphate carboxylase activity was found in endosperm of germinating castor bean seed Ricinus communis and was localized in proplastids. The endosperm carboxylase has been extensively purified and is composed of two different subunits. The molecular weights of the native carboxylase and its subunits were 560,000, 55,000, and 15,000 daltons, respectively. The Michaelis-Menten constants, K_m, for the endosperm carboxylase with respect to ribulose 1,5-bisphosphate, bicarbonate, CO₂, and magnesium in millimolar are 0.54, 13.60, 0.92, and 0.57, respectively. The endosperm carboxylase was activated by Mg²⁺ and HCO₃⁻. The preincubation of the carboxylase with 1 millimolar HCO₃⁻ and 5 millimolar MgCl₂ resulted in activation by low and inhibition by high concentrations of 6-phosphogluconate.

In studies of dark ¹⁴CO₂ fixation by endosperm slices, [¹⁴C]malate and [¹⁴C]citrate were the predominantly labeled products after 30 seconds of exposure of the tissue to H¹⁴CO₃⁻. In pulse-chase experiments, 87% of the label is malate, and citrate was transferred to sugars after a 60-minute chase with a small amount of the label appearing in the incubation medium as ¹⁴CO₂. The minimal incorporation of the label from ¹⁴CO₂ into phosphoglyceric acid indicated a lack of the endosperm ribulose-1,5-bisphosphate carboxylase participation in the endosperm's CO₂ fixation system. The activities of key Calvin cycle enzymes were examined in the endosperms and cotyledons of dark-grown castor bean seedlings. Many of these autotrophic enzymes develop in the dark in these tissues. The synthesis of ribulose-1,5-bisphosphate carboxylase in the nonphotosynthetic endosperms is not repressed in the dark, and high levels of enzymic activity appear with germination. All of the Calvin cycle enzymes are present in the castor bean endosperm except NADP-linked glyceraldehyde 3-P dehydrogenase, and the absence of this dehydrogenase probably prevents the functioning of these series of reactions in dark CO₂ fixation.

     

Ribulosebisphosphate carboxylase activity and photosynthetic o(2) evolution rate in vicia guard-cell protoplasts

Activities of ribulose-1,5-bisphosphate carboxylase and rates of photosynthetic O₂ evolution were measured in guard-cell and mesophyll protoplasts from Vicia faba. The ribulose-1,5-bisphosphate carboxylase activity of guard-cell protoplasts was 30% of that of mesophyll protoplasts; however, the O₂ evolution rate was 3 times higher in guard-cell protoplasts than in mesophyll protoplasts on a chlorophyll basis. When the dark-adapted, guard-cell protoplasts were illuminated by red light, O₂ was evolved with an induction period, which became shorter when the protoplasts were reilluminated. High activity of irreversible NADP-glyceraldehyde-3-phosphate dehyrogenase was found in guard-cell protoplasts. Several lines of evidence revealed that there was virtually no contamination by mesophyll cells in guard-cell preparations. These results indicate that guard cells fix CO₂ photosynthetically and imply that the cells utilize a considerable proportion of reducing equivalents from water for reactions other than CO₂ fixation.     

Synthesis of proteins by chloroplasts from iron-deficient Euglena gracilis

Chloroplasts isolated from Euglena gracilis made iron deficient by growth on 0.5 μm iron show distinct qualitative and quantitative changes in their polypeptide composition in comparison with iron-sufficient (40 μm) chloroplasts. These changes were noted in the stromal, thylakoid, and envelope subfractions. Iron-deficient chloroplasts have a sedimentation behavior similar to that of iron-sufficient chloroplasts and also contain substantial amounts of ribulose-1,5-bisphosphate carboxylase. In addition, iron-deficient chloroplasts incorporate [³H]leucine into polypeptides at rates about one-third of those from control chloroplasts (40 μm Fe) on a per-microgram-chlorophyll basis. Incorporation of [³H]leucine into specific polypeptides, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, shows relatively normal synthesis of the large subunit of ribulose-1,5-bisphosphate carboxylase and two of the three major chloroplast-derived polypeptides of the thylakoids. No incorporation was detected, however, into a polypeptide of ca. 33 kd which is synthesized by normal plastids. Iron-deficient chloroplasts also synthesize a stromal polypeptide of ca. 85 kd not seen in chloroplasts from normal cells. This evidence is consistent with a direct or indirect role for iron in the regulation of synthesis of specific proteins in the chloroplast.     

Mass-spectrometric evidence for the double-carboxylation pathway of malate synthesis by Crassulacean acid metabolism plants in light

Phyllodia of the Crassulacean acid metabolism (CAM) plant Kalanchoë tubiflora were allowed to fix ¹³CO₂ in light and darkness during phase IV of the diurnal CAM cycle, and during prolongation of the regular light period. After ¹³CO₂ fixation in darkness, only singly labelled [¹³C]malate molecules were found. Fixation of ¹³CO₂ under illumination, however, produced singly labelled malate as well as malate molecules which carried label in two, three or four carbon atoms. When the irradiance during ¹³CO₂ fixation was increased, the proportion of singly labelled malate decreased in favour of plurally labelled malate. The irradiance, however, did not change either the ratio of labelled to unlabelled malate molecules found in the tissue after the ¹³CO₂ application, or the magnitude of malate accumulation during the treatment with label. The ability of the tissue to store malate and the labelling pattern changed throughout the duration of the prolonged light period. The results indicate that malate synthesis by CAM plants in light can proceed via a pathway containing two carboxylation steps, namely ribulose-1,5-bisphosphate-carboxylase/oxygenase (EC 4.1.1.39) and phosphoenolpyruvate carboxylase (EC 4.1.1.31) which operate in series and share common intermediates. It can be concluded that, in light, phosphoenolpyruvate carboxylase can also synthesize malate independently of the proceeding carboxylation step by ribulose-1,5-bisphosphate carboxylase/oxygenase.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase (EC 4.1.1.31) - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) - TMS trimethylsilyl     

Isolation of Intact and Functional Chloroplasts from Mesophyll and Bundle Sheath Protoplasts of the C(4) Plant Panicum miliaceum

A procedure is described for isolating and purifying mesophyll protoplasts and bundle sheath protoplasts of the C₄ plant Panicum miliaceum. Following enzymic digestion of leaf tissue, mesophyll protoplasts and bundle sheath protoplasts are released and purified by density centrifugation. The lower density of mesophyll protoplasts allowed rapid separation of the two protoplast types. Evidence for separation of mesophyll protoplasts and bundle sheath protoplasts (up to 95% purity) is provided from light microscopy (based on size difference in both chloroplasts and protoplasts), levels of marker enzymes in the preparations (i.e. pyruvate, Pi dikinase and phosphoenolpyruvate carboxylase for mesophyll and ribulose-1,5-bisphosphate carboxylase for bundle sheath), and differences in substrate-dependent O₂ evolution by chloroplasts isolated from protoplasts.     

Immuno-gold localization of cytochrome f,light-harvesting complex,ATP synthase and ribulose 1,5-bisphosphate carboxylase/oxygenase

The proteins ribulose 1,5-bisphosphate carboxylase/oxygenase, ATP synthase, light-harvesting chlorophyll a/b protein, and cytochrome f, have been localized in mesophyll chloroplasts of barley (Hordeum vulgare L.) by electron microscopy of immunogold-labelled sections. The light-harvesting chlorophyll a/b protein and cytochrome f are shown to be present in the grana, both within the stacks and at the margins, and in the stromal membranes. Although the absolute amount of labelling for these proteins is greater in the grana than in the stromal membranes, when expressed as label/membrane length the partitioning appears approximately equal between appressed and non-appressed membranes for both the light-harvesting chlorophyll a/b protein and cytochrome f. ATP synthase is restricted to the non-appressed thylakoid membranes, and ribulose 1,5-bisphosphate carboxylase/oxygenase is uniformly distributed through the stromal contents.Abbreviations CF₁ ATP synthase - LHCPII light-harvesting chlorophyll a/b protein - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase     

D-Ribulose-1,5-bisphosphate carboxylase/oxygenase from chemolithotrophically grown Rhizobium japonicum

Ribulose-1,5-bisphosphate carboxylase/oxygenase has been purified from chemolithotrophically grown Rhizobium japonicum SR and ribulose-5-phosphate kinase activity has also been detected in extracts of such cells. Electrophoretically homogeneous ribulosebisphosphate carboxylase/oxygenase purified in the presence of PMSF showed two types of large subunits of 55 000 and 53 000 daltons and small subunits of 14 200 daltons. The heterogeneity of large subunits was not observed when the enzyme was prepared in the presence of PMSF and DIFP. Ribulose-1,5-bisphosphate carboxylase from R. japonicum was inhibited by antibodies to this enzyme and a single precipitin band from the antibody-enzyme interaction was observed on double diffusion plates. Antibodies to R. japonicum enzyme did not cross-react on immunodiffusion plates with the ribulosebisphosphate carboxylase/oxygenases from wheat, spinach, soybean and tobacco.     

Characteristics of light-dependent inorganic carbon uptake by isolated spinach chloroplasts

The light-dependent accumulation of radioactively labeled inorganic carbon in isolated spinach (Spinacia oleracea L.) chloroplasts was determined by silicone oil filtering centrifugation. Intact chloroplasts, dark-incubated 60 seconds at pH 7.6 and 23°C with 0.5 millimolar sodium bicarbonate, contained 0.5 to 1.0 millimolar internal inorganic carbon. The stromal pool of inorganic carbon increased 5- to 7-fold after 2 to 3 minutes of light. The saturated internal bicarbonate concentration of illuminated spinach chloroplasts was 10- to 20-fold greater than that of the external medium. This ratio decreased at lower temperatures and with increasing external bicarbonate. Over one-half the inorganic carbon found in intact spinach chloroplasts after 2 minutes of light was retained during a subsequent 3-minute dark incubation at 5°C. Calculations of light-induced stromal alkalization based on the uptake of radioactively labeled bicarbonate were 0.4 to 0.5 pH units less than measurements performed with [¹⁴C]dimethyloxazolidine-dione. About one-third of the binding sites on the enzyme ribulose 1,5-bisphosphate carboxylase were radiolabeled when the enzyme was activated in situ and ¹⁴CO₂ bound to the activator site was trapped in the presence of carboxypentitol bisphosphates. Deleting orthophosphate from the incubation medium eliminated inorganic carbon accumulation in the stroma. Thus, bicarbonate ion distribution across the chloroplast envelope was not strictly pH dependent as predicted by the Henderson-Hasselbach formula. This finding is potentially explained by the presence of bound CO₂ in the chloroplast.     

Biochemical Changes that Occur during Senescence of Wheat Leaves : I. Basis for the Reduction of Photosynthesis

Changes in activities of photosynthetic enzymes and photochemical processes were followed with aging of vegetative and flag leaves of wheat (Triticum aestivum L. cv Roy). Activities of stromal enzymes began to decline prior to photochemical activities. In general, total soluble protein and the activities of ribulose-1,5-bisphosphate carboxylase and NADP-triose-phosphate dehydrogenase declined in parallel and at an earlier age than leaf chlorophyll (Chl), leaf photosynthesis, and photosynthetic electron transport activity. Leaves appeared to lose whole chloroplasts as opposed to a general degradation of all chloroplasts based on three lines of evidence: (a) electron transport activity calculated on an area basis declined much earlier than the same data expressed on a Chl basis; (b) Chl content per chloroplast was similar for mature and senescent tissue; and (c) the absorbance at 550 nanometers (light scattering) per unit of Chl remained essentially constant until the end of senescence. Chloroplasts did, however, undergo some modifications before they were lost (e.g. loss of stromal enzyme activities), but the reduction in leaf photosynthesis was apparently caused by a loss of whole chloroplasts.     

In silico,in vitro and in vivo approach in understanding the functional relationship between ergosterol and Rubisco

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) is one of the key enzymes involved in assimilation of CO₂ in chloroplasts. Phylloplane microfungi and their metabolites have been reported to affect the physiology of host plants, particularly, their photosynthesis. However, information is lacking on the effect of these microflora on the physiology of chloroplasts. The current study emphasized the impact of two dominant phylloplane fungi, Aspergillus niger and Fusarium oxysporum, on activity of Rubisco in tomato chloroplasts. Ergosterol, which is a component of only fungal cell membranes and is not synthesized by plants, have been demonstrated to elicit activity of Rubisco. In the present study, it was demonstrated through in silico, in vitro, and in vivo approaches. Results demonstrated that the fungal metabolites, which contained ergosterol, could double Rubisco activity. Maximum carboxylation rate of Rubisco increased also in ergosterol-treated plants. Michaelis-Menten constant of Rubisco was also slightly affected. Ergosterol was found also to influence and enhance the binding of CO₂ and ribulose-1,5-bisphosphate to Rubisco. Therefore we can postulate that the physiology of the chloroplast is probably influenced by phylloplane microfungi.     

Changes in Activity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Three Peroxisomal Enzymes during Tomato Fruit Development and Ripening

Ribulose-1,5-bisphosphate carboxylase/oxygenase, catalase, glycolate oxidase, and hydroxypyruvate reductase activities on a protein and fresh weight basis were measured over seven stages of tomato fruit development and ripening. Ribulose-1,5-bisphosphate carboxylase decreased steadily during fruit development from 23 ± 8 nmoles per minute per milligram protein at the mature green stage to 13.4 ± 2 at the table ripe stage. There was no change in partially purified preparations of the enzyme in the ratio of carboxylase to oxygenase activity, which was about 10. Catalase activity reached a maximum during the climacteric, simultaneously with increased ethylene and CO₂ formation. Glycolate oxidase activity decreased during early stages of development and was barely detectable at the climacteric. Hydroxypyruvate reductase, associated with serine formation by the glycerate pathway, increased in specific activity during early stages of tomato fruit ripening. In the fruit of the rin tomato mutant, which does not ripen normally, none of these changes in enzyme activity occurred.