Rapid Enrichment of CHAPS-Solubilized UDP-Glucose: (1,3)-beta-Glucan (Callose) Synthase from Beta vulgaris L. by Product Entrapment : Entrapment Mechanisms and Polypeptide Characterization

Rapid enrichment of CHAPS-solubilized UDP-glucose:(1,3)-β-glucan (callose) synthase from storage tissue of red beet (Beta vulgaris L.) is obtained when the preparation is incubated with an enzyme assay mixture, then centrifuged and the enzyme released from the callose pellet with a buffer containing EDTA and CHAPS (20-fold purification relative to microsomes). When centrifuged at high speed (80,000g), the enzyme can also be pelleted in the absence of substrate (UDP-Glc) or synthesis of callose, due to nonspecific aggregation of proteins caused by excess cations and insufficient detergent in the assay buffer. True time-dependent and substrate-dependent product-entrapment of callose synthase is obtained by low-speed centrifugation (7,000-11,000g) of enzyme incubated in reaction mixtures containing low levels of cations (0.5 millimolar Mg²⁺, 1 millimolar Ca²⁺) and sufficient detergent (0.02% digitonin, 0.12% CHAPS), together with cellobiose, buffer, and UDP-Glc. Entrapment conditions, therefore, are a compromise between preventing nonspecific precipitation of proteins and permitting sufficient enzyme activity for callose synthesis. Further enrichment of the enzyme released from the callose pellet was not obtained by rate-zonal glycerol gradient centrifugation, although its sedimentation rate was greatly enhanced by inclusion of divalent cations in the gradient. Preparations were markedly cleaner when product-entrapment was conducted on enzyme solubilized from plasma membranes isolated by aqueous two-phase partitioning rather than by gradient centrifugation. Product-entrapped preparations consistently contained polypeptides or groups of closely-migrating polypeptides at molecular masses of 92, 83, 70, 57, 43, 35, 31/29, and 27 kilodaltons. This polypeptide profile is in accordance with the findings of other callose synthase enrichment studies using a variety of tissue sources, and is consistent with the existence of a multi-subunit enzyme complex.     

Cloning of 9-cis-epoxycarotenoid dioxygenase (NCED) gene and the role of ABA on fruit ripening

In order to understand more details about the role of abscisic acid (ABA) in fruit ripening and senescence, six 740 bp cDNAs (LeNCED1, LeNCED2, PpNCED1, VVNCED1, DKNCED1 and CMNCED1) which encode 9-cis-epoxycarotenoid dioxygenase (NCED) as a key enzyme in ABA biosynthesis, were cloned from fruits of tomato, peach, grape, persimmon and melon using an RT-PCR approach. A Blast homology search revealed a similarity of amino acid 85.76% between the NCEDs. A relationship between ABA and ethylene during ripening was also investigated. At the mature green stage, exogenous ABA treatment increased ABA content in flesh, and promoting ethylene synthesis and fruit ripening, while treatment with nordihydroguaiaretic acid (NDGA), inhibited them, delayed fruit ripening and softening. However, ABA inhibited the ethylene synthesis obviously while NDGA promoted them when treated the immature fruit with these chemicals. At the breaker, NDGA treatment cannot block ABA accumulation and ethylene synthesis. Based on the results obtained in this study, it was concluded that ABA plays different role in ethylene synthesis system in different stages of tomato fruit ripening.Key words: tomato, NCED gene, ABA, ethylene, fruit ripening, peach, grape, persimmon, melon     

Expression and regulation of pear 1-aminocyclopropane-1-carboxylic acid synthase gene (PpACS1a) during fruit ripening,under salicylic acid and indole-3-acetic acid treatment,and in diseased fruit

In plants, the level of ethylene is determined by the activity of the key enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS). A gene encoding an ACC synthase protein was isolated from pear (Pyrus pyrifolia). This gene designated PpACS1a (GenBank accession no. KC632526) was 1488 bp in length with an open reading frame (ORF) encoding a protein of 495 amino acids that shared high similarity with other pear ACC synthase proteins. The PpACS1a was grouped into type-1 subfamily of plant ACS based on its conserved domain and phylogenetic status. Real-time quantitative PCR indicated that PpACS1a was differentially expressed in pear tissues and predominantly expressed in anthers. The expression signal of PpACS1a was also detected in fruit and leaves, but no signal was detected in shoots and petals. Furthermore, the PpACS1a expression was regulated during fruit ripening. In addition, the PpACS1a gene expression was regulated by salicylic acid (SA) and indole-3-acetic acid (IAA) in fruit. Moreover, the expression of the PpACS1a was up-regulated in diseased pear fruit. These results indicated that PpACS1a might be involved in fruit ripening and response to SA, IAA and disease.     

Sucrose metabolism and fruit growth in parthenocarpic vs seeded blueberry (Vaccinium ashei) fruits

Although fruit set and development are induced by applications of gibberellins, final fruit weight of gibberellin-induced parthenocarpic fruit is often less than that of pollinated fruit. We examined changes in the activities of sucrose-metabolizing enzymes and sugar accumulation in developing fruits of cultivated blueberry (Vaccinium ashei Reade) and their correlation with fruit growth upon pollination or exogenous applications of gibberellic acid (GA₃). The objective was to determine if differences in fruit growth could be attributed to differences in enzyme activities and subsequent sugar accumulation in fruits. The fruit development period of GA₃-treated fruits was 15 days longer than that of pollinated fruits. At maturity, GA₃-treated fruit accumulated an average of 180 mg dry weight while pollinated fruit accumulated 390 mg dry weight. Dry weight accumulation in nonpollinated fruits was negligible and these fruits abscised by 45 days after bloom (DAB). The total carbon (C) cost (dry weight C + respiratory C) for fruit development was 109 and 244 mg C fruit^-1 for GA₃-treated and pollinated fruits, respectively. Hexose concentration increased to 100 mg (g fresh weight)^-1 at ripening in both GA₃-treated and pollinated fruits. Nonpollinated fruits reached a maximum hexose concentration at 45 DAB. Sucrose phosphate synthase (EC 2.4.1.14) and sucrose synthase (EC 2.4.1.13) activities reached a maximum of ≤5.0 μmol (g fresh weight)^-1 h^-1 in both GA₃-treated and pollinated fruits. Soluble acid invertase (EC 3.2.1.26) activity increased to about 60 μmol (g fresh weight)^-1 h^-1 in both GA₃-treated and pollinated fruits at ripening, while in nonpollinated fruits, a maximum soluble acid invertase activity of 0.12 μmol (g fresh weight)^-1 h^-1 was measured at 24 DAB. Insoluble acid invertase activity declined during the early stages of fruit growth and remained relatively low throughout fruit development. Neutral invertase activity was low throughout development, increasing to 5 μmol (g fresh weight)^-1 h^-1 at ripening in GA₃-treated and pollinated fruits. Our studies demonstrate that blueberry fruit development does not appear to be limited by sucrose metabolizing enzyme activity and/or the ability to accumulate sugars in either GA₃-treated or pollinated fruits.     

Role of sucrose phosphate synthase in sucrose biosynthesis in ripening bananas and its relationship to the respiratory climacteric

During ripening of bananas (Musa spp. [AAA group, Cavendish subgroup]), there is a massive conversion of starch to sucrose. Also during ripening there is a rise in respiration known as the respiratory climacteric. In this study changes in carbohydrate content, activities of starch and sucrose metabolizing enzymes, and respiration were measured to assess their potential interrelationships. Sucrose phosphate synthase activity increased dramatically during the first 4 days after initiation of ripening by ethylene treatment. Starch concentration decreased and sucrose concentration increased during this time period. Developmental changes in sucrose phosphate synthase activity were measured with limiting substrate (plus Pi) and saturating substrate concentrations. Activities were not parallel under the two assay conditions, providing tentative evidence that kinetically different forms of the enzyme may exist at different stages of ripening. Sucrose accumulation rate was most highly correlated with sucrose phosphate synthase activity assayed with limiting substrate concentrations (plus Pi). The cumulative amount of CO₂ respired during ripening was positively correlated with sugar accumulation (R² = 0.97). From this linear regression it was calculated that a constant 0.605 millimoles of CO₂ was evolved per mole of sucrose formed throughout ripening. Using this quantity, the percentage of the total respiratory ATP produced which was required for the conversion of starch to sucrose was calculated assuming different models for carbon export from the amyloplast. The results suggest that sucrose biosynthesis during ripening constitutes a significant sink for respiratory ATP.     

Senescense: association of synthesis of Acid phosphatase with banana ripening

During ripening of banana (Musa sapientum L., var. Gros Michel or Valery) acid phosphatase activity increases 13-to 26-fold in the precipitate and 2- to 4-fold in the supernatant fraction of tissue homogenates. These increases are closely correlated with the onset and peak of the climacteric. The precipitate enzyme may be extracted with Triton X-100, CaCl₂ or NaCl; about 80% of it is in a 500g precipitate. Studies on effect of tonicity of the grinding medium indicate that the precipitate enzyme is desorbed from membrane or cell wall surfaces, and is not released as a result of lysis of membranes. The development of acid phosphatase during aging of tissue slices is the same as in intact fruit. Short term studies of tissue slices with cycloheximide and actinomycin D indicate that the increase in activity is owed to new enzyme synthesis, which is dependent upon synthesis of RNA. The possible effects of the increase in acid phosphatase on ripening are discussed.     

Banana Ripening: Implications of Changes in Internal Ethylene and CO(2) Concentrations, Pulp Fructose 2,6-Bisphosphate Concentration, and Activity of Some Glycolytic Enzymes

In ripening banana (Musa acuminata L. [AAA group, Cavandish subgroup] cv. Valery) fruit, the steady state concentration of the glycolytic regulator fructose 2,6-bisphosphate (Fru 2,6-P₂) underwent a transient increase 2 to 3 hours before the respiratory rise, but coincident with the increase in ethylene synthesis. Fru 2,6-P₂ concentration subsequently decreased, but increased again approximately one day after initiation of the respiratory climacteric. This second rise in Fru 2,6-P₂ continued as ripening proceeded, reaching approximately five times preclimacteric concentration. Pyrophosphate-dependent phosphofructokinase glycolytic activity exhibited a transitory rise during the early stages of the respiratory climacteric, then declined slightly with further ripening. Cytosolic fructose 1,6-bisphosphatase activity did not change appreciably during ripening. The activity of ATP-dependent phosphofructokinase increased approximately 1.6-fold concurrent with the respiratory rise. A balance in the simultaneous glycolytic and gluconeogenic carbon flow in ripening banana fruit appears to be maintained through changes in substrate levels, relative activities of glycolytic enzymes and steady state levels of Fru 2,6-P₂.     

Identification, cloning and expression analysis of strawberry (Fragaria x ananassa) mitochondrial citrate synthase and mitochondrial malate dehydrogenase

Salt-extractable proteins from the cell walls of immature and ripe strawberry ( Fragaria  ×  ananassa Duch. cv. Elsanta) fruit were separated using two-dimensional polyacrylamide gel electrophoresis. Seven polypeptides (enzymes) were characterized from their N-terminal sequences: (1) glyceraldhyde-3-phosphate dehydrogenase (EC 1.2.1.12); (2) triose phosphate isomerase (TPI; EC 5.3.1.1); (3) mitochondrial malate dehydrogenase (mMDH; EC 1.1.1.37); (4) NADH glutamate dehydrogenase (EC 1.4.1.3); (5) chalcone synthase (ChS; EC 2.3.1.74); (6) mitochondrial citrate synthase (mCS; EC 4.1.3.7); and (7) UDP glucose:flavonoid 3- O -glucosyltransferase (UDPG:FGT; EC 2.4.1.91). The sequenced polypeptides identified only cytosolic proteins, two of which (ChS and UDPG:FGT) had already been identified as being up-regulated in ripening (strawberry) fruit and important contributors to ripe fruit character. Our focus was therefore diverted to the enzymes mMDH and mCS for further molecular characterization as potentially important determinants of fruit flavour via regulation of the sugar : acid balance. Citrate synthase (CS) and malate dehydrogenase (MDH) enzyme activities increased substantially during ripening, as did citrate and malate contents. The increase in CS activity is supported by western blot analysis. One strawberry mCS ( Fa-mCS-I ) and two mMDH ( Fa-mMDH-I and -II ) cDNAs were cloned that were 77, 82 and 53% identical (respectively) to sequences from other plant sources. Northern analysis showed that CS and MDH expression did not correlate with enzyme activities and these findings are discussed.     

Inhibition and labeling of red beet uridine 5'diphospho-glucose: (1,3)-β-glucan (callose) synthase by chemical modification with formaldehyde and uridine 5'diphospho-pyridoxal

The effects of the lysine-reactive chemical modification reagents, uridine 5’ diphospho (UDP)-pyridoxal and formaldehyde (HCHO), on the activity of membrane-bound and solubilized UDP-Glc: (1,3)-β-D-glucan synthase (callose synthase) from red beet (Beta vulgaris L.) storage tissue were compared. Exposure to micromolar levels of UDP-pyridoxal, or millimolar levels of HCHO in the presence of NaCNBH₃, resulted in complete enzyme inactivation. Conditions for inhibition of membrane-bound enzyme activity by the two reagents were markedly similar; divalent cations were required for inactivation, and complete protection of activity was obtained with EDTA or EGTA. The substrate, UDP-Glc, protected membrane-bound callose synthase against inactivation by UDP-pyridoxal or HCHO, but protected the solubilized enzyme only against inhibition by UDP-pyridoxal, suggesting that the lysine residue modified by both these reagents is at the enzyme active site, and that the site is more open or has a certain conformational flexibility in the solubilized enzyme. Potential UDP-Glc-binding polypeptides of callose synthase were identified by a two-step labeling procedure. First, nonessential lysine residues were blocked by irreversible modification reaction with HCHO or UDP-pyridoxal in the presence of UDP-Glc to protect lysines at UDP-Glc-binding sites. In the second step, proteins were recovered, reacted with [¹⁴C]-HCHO in the absence of UDP-Glc, and polypeptide labeling patterns analyzed by SDS-polyacrylamide gel electrophoresis and fluorography. This procedure reduced incorporation of label by 5- to 8-fold compared to a procedure omitting the preblocking step, and with enzyme partially purified by solubilization in CHAPS followed by product entrapment, labeling was limited to a small set of polypeptides. Taken together with the results of other studies, the data suggest that one or more polypeptides migrating in the 54–57 kDa region are good candidates for the UDP-Glc-binding components of callose synthase.     

UDP-Glucose: (1-->3)-beta-Glucan Synthases from Mung Bean and Cotton: Differential Effects of Ca and Mg on Enzyme Properties and on Macromolecular Structure of the Glucan Product

A re-examination of the kinetic properties of UDP-glucose: (1→3)-β-glucan (callose) synthases from mung bean seedlings (Vigna radiata) and cotton fibers (Gossypium hirsutum) shows that these enzymes have a complex interaction with UDP-glucose and various effectors. Stimulation of activity by micromolar concentrations of Ca²⁺ and millimolar concentrations of β-glucosides or other polyols is highest at low (<100 micromolar) UDP-glucose concentrations. These effectors act both by raising the V_max of the enzyme, and by lowering the apparent K_m for UDP-glucose from >1 millimolar to 0.2 to 0.3 millimolar. Mg²⁺ markedly enhances the affinity of the mung bean enzyme for Ca²⁺ but not for β-glucoside; with saturating Ca²⁺, Mg²⁺ only slightly stimulates further production of glucan. However, the presence of Mg²⁺ during synthesis, or NaBH₄ treatment after synthesis, changes the nature of the product from dispersed, alkali-soluble fibrils to highly aggregated, alkali-insoluble fibrils. Callose synthesized in vitro by the Ca²⁺, β-glucoside-activated cotton fiber enzyme, with or without Mg²⁺, is very similar in size to callose isolated from cotton fibers, but is a linear (1→3)-β-glucan lacking the small amount of branches at C-0-6 found in vivo. We conclude that the high degree of aggregation of the fibrils synthesized with Mg²⁺in vitro is caused either by an alteration of the glucan at the reducing end or, indirectly, by an effect of Mg²⁺ on the conformation of the enzyme. Rate-zonal centrifugation of the solubilized mung bean callose synthase confirms that divalent cations can affect the size or conformation of this enzyme.     

beta-Furfuryl-beta-Glucoside: An Endogenous Activator of Higher Plant UDP-Glucose:(1-3)-beta-Glucan Synthase : Biological Activity, Distribution, and in Vitro Synthesis

In a recent paper (P Ohana, DP Delmer, JC Steffens, DE Matthews, R Mayer, M Benziman [1991] J Biol Chem 266: 13472-13475), we described the purification and structural characterization of β-furfuryl-β-glucoside (FG), an endogenous activator of plant UDP-glucose:(1→3)-β-glucan (callose) synthase. In the present report, we provide evidence that FG specifically stimulates callose synthase. The effects of FG on the kinetic properties of callose synthase were studied, and we ascertained that FG, or at least a very similar compound, is present in other plant systems. Chemically synthesized α-furfuryl-β-glucoside also stimulates callose synthase, exhibiting a slightly higher K_a of 80 micromolar, compared with 50 micromolar for FG. In addition, we have identified and partially characterized an enzyme that catalyzes the synthesis of FG using β-furfuryl alcohol and UDP-glucose as substrates. A model for the regulation of callose synthesis in vivo, involving changes in intracellular compartmentation of FG and Ca²⁺, is proposed.     

Cell Wall Changes in Nectarines (Prunus persica) : Solubilization and Depolymerization of Pectic and Neutral Polymers during Ripening and in Mealy Fruit

Nectarine fruit (Prunus persica L. Batsch var nectarina [Ait] maxim) cultivar Fantasia were either ripened immediately after harvest at 20°C or stored for 5 weeks at 2°C prior to ripening. Fruit ripened after 5 weeks of storage did not soften to the same extent as normally ripened fruit, they lacked juice, and had a dry, mealy texture. Pectic and hemicellulosic polysaccharides were solubilized from the mesocarp of the fruit using phenol:acetic acid:water (PAW) treatment to yield PAW-soluble material and cell wall material (CWM). The carbohydrate composition and relative molecular weight (M_r) of polysaccharide fractions released from the CWM by sequential treatment with cyclohexane-trans-1,2-diamine tetra-acetate, 0.05 m Na₂CO₃, 6 m guanidinium thiocyanate, and 4 m KOH were determined. Normal ripening of nectarines resulted in solubilization of pectic polymers of high M_r from CWM during the first 2 d at ripening temperatures. Concurrently, galactan side chains were removed from pectic polymers. Solubilized pectic polymers were depolymerized to lower M_r species during the latter stages of ripening. Upon removal from cool storage, fruit had undergone some pectic polymer solubilization, and after ripening, pectins were not depolymerized and were of high M_r. Side chains did not appear to be removed from insoluble pectic polymers and branched pectins accumulated in the CWM. The molecular weight profiles obtained by gel filtration of the hemicellulosic fractions from normally ripening and mealy fruit were similar. The results suggest that mealiness results as a consequence of altered pectic polymer breakdown, including that associated with neutral side chains.     

Changes in NADP+-linked isocitrate dehydrogenase during tomato fruit ripening

The activity of NADP⁺-specific isocitrate dehydrogenase (NADP⁺-IDH, EC 1.1.1.42) was investigated during the ripening of tomato (Lycopersicon esculentum Mill.) fruit. In the breaker stage, NADP⁺-IDH activity declined but a substantial recovery was observed in the late ripening stages when most lycopene synthesis occurs. These changes resulted in higher NADP⁺-IDH activity and specific polypeptide abundance in ripe than in green fruit pericarp. Most of the enzyme corresponded to the predominant cytosolic isoform which was purified from both green and ripe fruits. Fruit NADP⁺-IDH seems to be a dimeric enzyme having a subunit size of 48 kDa. The K _m values of the enzymes from green and ripe pericarp for NADP⁺, isocitrate and Mg²⁺ were not significantly different. The similar molecular and kinetic properties and chromatographic behaviour of the enzymes from the two kinds of tissue strongly suggest that the ripening process is not accompanied by a change in isoenzyme complement. The increase in NADP⁺-IDH in the late stage of ripening also suggests that this enzyme is involved in the metabolism of C₆ organic acids and in glutamate accumulation in ripe tissues.     

Expression of xyloglucan endotransglucosylase/hydrolase (XTH) genes and XET activity in ethylene treated apple and tomato fruits

Xyloglucan endotransglucosylase/hydrolase (XTHs: EC 2.4.1.207 and/or EC 3.2.1.151), a xyloglucan modifying enzyme, has been proposed to have a role during tomato and apple fruit ripening by loosening the cell wall. Since the ripening of climacteric fruits is controlled by endogenous ethylene biosynthesis, we wanted to study whether XET activity was ethylene-regulated, and if so, which specific genes encoding ripening-regulated XTH genes were indeed ethylene-regulated. XET specific activity in tomato and apple fruits was significantly increased by the ethylene treatment, as compared with the control fruits, suggesting an increase in the XTH gene expression induced by ethylene. The 25 SlXTH protein sequences of tomato and the 11 sequences MdXTH of apple were phylogenetically analyzed and grouped into three major clades. The SlXTHs genes with highest expression during ripening were SlXTH5 and SlXTH8 from Group III-B, and in apple MdXTH2, from Group II, and MdXTH10, and MdXTH11 from Group III-B. Ethylene was involved in the regulation of the expression of different SlXTH and MdXTH genes during ripening. In tomato fruit fifteen different SlXTH genes showed an increase in expression after ethylene treatment, and the SlXTHs that were ripening associated were also ethylene dependent, and belong to Group III-B (SlXTH5 and SlXTH8). In apple fruit, three MdXTH showed an increase in expression after the ethylene treatment and the only MdXTH that was ripening associated and ethylene dependent was MdXTH10 from Group III-B. The results indicate that XTH may play an important role in fruit ripening and a possible relationship between XTHs from Group III-B and fruit ripening, and ethylene regulation is suggested.     

Influence of avocado (Persea americana) Cx-cellulase on the structural features of avocado cellulose

Avocado (Persea americana Mill.) fruit produce copious quantities of the enzyme Cx-cellulase (EC 3.2.1.4) during ripening. The possibility that Cx-cellulase is able to disrupt cellulose microfibril oranization was investigated using molecular weight (M_r), x-ray diffraction, and ultrastructural analyses of cell walls from unripe avocado fruit incubated with the purified enzyme. Results indicate that Cx-cellulase causes a downshift in the M_r of unbranched cell-wall polymers in the M_r range of 10⁶–10⁷ Da. There is an increase in the proportion of crystalline cellulose, and cellulose fibrils appear to lose cohesiveness in response to enzyme activity. We propose that Cx-cellulase attacks avocado cellulose at accessible sites in the peripheral and integral noncrystalline regions of the microfibril, resulting in a loss of cohesiveness within the fibril structure and an alteration in the binding of associated cell-wall matrix polysaccharides. The initial loss of avocado mesocarp firmness during fruit ripening may be linked to the onset of Cx-cellulase activity.Abbreviations CMC carboxymethylcellulose - DMAC dimethylacetamide - DS developmental stage - M molecular weight - XG xyloglucan     

Susceptibility of UDP-Glucose:(1,3)-beta-Glucan Synthase to Inactivation by Phospholipases and Trypsin

UDP-glucose:(1,3)-β-glucan synthase from Beta vulgaris L. was rapidly inactivated by treatment with phospholipases C, D, and A₂. Enzyme activity could not be restored to the phospholipase-treated enzyme by the addition of phosphatidylethanolamine or other phospholipids. Membrane-bound and solubilized glucan synthase were also trypsin-labile with inactivation rates equal in the presence or absence of divalent cations or chelators. Gradual activity declines were observed in membranes incubated with divalent cations, but not with chelators.     

The effect of reduced activity of phytoene synthase on isoprenoid levels in tomato pericarp during fruit development and ripening

Carotenoids, gibberellins (GAs), sterols, abscisic acid and -amyrins were analysed in tomato (Lycopersicon esculentum Mill.) pericarp during fruit development and ripening. The contents of these isoprenoids in wild-type (cv. Ailsa Craig) fruit were compared with those in fruit of the carotenoid-deficient R-mutant and a transgenic plant containing antisense RNA to a phytoene synthase gene. In both carotenoid-deficient genotypes, a 14-fold reduction in carotene and twofold decrease in xanthophyll content, compared to the wild type, was found in ripe fruit. Immature green fruit from wild type and R-mutant plants contained similar amounts of the C₁₉-GAs, GA₁, and GA₂₀, and their C₂₀ precursor, GA₁₉. Immature fruit from the transgenic plants contained three- to fivefold higher contents of these GAs. In wild-type fruit at the mature green stage the contents of these GAs had decreased to < 10% of the levels in immature fruit. A similar decrease in GA₁₉ content occurred in the other genotypes. However, the contents of GA₁ and GA₂₀ in fruit from phytoene synthase antisense plants decreased only to 30% between the immature and mature green stages and did not decrease at all in R-mutant fruit. At the breaker and ripe stages, the contents of each GA were much reduced for all genotypes. The amount of abscisic acid was the same in immature fruit from all three genotypes, but, on ripening, the levels of this hormone in antisense and R-mutant fruit were ca. 50% of those in the wild type. Quantitative differences in the amounts of the triterpenoid -amyrins, total sterols, as well as individual sterols, such as campesterol, stigmasterol and sitosterol, were apparent between all three genotypes during development. Amounts of free sterols of wild type and antisense fruit were greatest during development and decreased during ripening, whereas the opposite was found in the R-mutant. This genotype also possessed less free sterol and more bound sterol in comparison to the other varieties. These data provide experimental evidence to support the concept of an integrated metabolic relationship amongst the isoprenoids.Abbreviations ABA abscisic acid - dpb days post breaker - FDP farnesyl diphosphate - GA gibberellin - GGDP geranyl-geranyl diphosphate We thank Mr. Paul Gaskin (Long Ashton Research Station) for the qualitative GC-MS of triterpenoids and Dr. R. Horgan (University of Wales, Aberystwyth) for a gift of [6-³H₂]ABA. The work was supported by a research grant (No. PG111/617) to P.M.B. from the Agricultural and Food Research Council to whom we express our thanks.     

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.     

Differential effects of senescence on the molecular organization of membranes in ripening tomato fruit

Changes in the molecular organization of membranes in pericarp cells of ripening tomato fruit were examined by fluorescence depolarization after labeling with fluorescent lipid-soluble probes. The fluorescent labels were partitioned into isolated protoplasts and purified plastids from fruit at various stages of senescence. Values for steady-state anisotropy (r_ss) of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labeled protoplasts rose progressively during the early stages of ripening over a time frame that overlapped the climacteric rise in ethylene production. This can be interpreted as reflecting a decrease in the lipid fluidity of primarily plasma membrane. By contrast, there was no significant change during ripening in r_ss for plastid membranes labeled with DPH, 1-[4-trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), and cis- or trans-parinaric acid. Nor was there any change during ripening in the limiting fluorescence anisotropy (r_oo) and order parameter (S) for plastids labeled with DPH or TMA-DPH, parameters that are corrected for any differences in lifetime. Some degree of lifetime heterogeneity, possibly reflecting structurally distinct domains, was discerned in both young and senescent plastids that had been labeled with DPH or TMA-DPH, but this also did not change as ripening progressed. Thus membranes of the pericarp cells sustain different fates as the tomato fruit ripens, implying that there are distinguishable mechanisms of membrane deterioration in senescing tissues.