Activity of sucrose hydrolysing enzymes and sugar accumulation during tomato fruit development

Relationships between the assimilate import rate and the activity of acid invertase and/or sucrose synthase have been investigated in the pericarp, locule and placenta of tomato fruit during development to establish the possible role of sucrose cleavage as the control step for the import of sucrose into these sink tissues. The rate of sucrose cleavage was estimated from the activities of these two enzymes as well as the ratio of hexoses to sucrose (i.e. the sucrose degradation index, SDI) in the tissues of the fruit, based on the assumption that the accumulation of hexoses is the consequence of imported sucrose being degraded by either or both of these two enzymes. The results showed that the change of sucrose synthase activity during fruit development was positively related to both the rate of dry matter accumulation in the fruit tissue and SDI. Although the role of acid invertase in regulating the rate of import during development remains uncertain, the actions of sucrose synthase on sucrose cleavage may regulate the import and compartmentation of sucrose in the early stage of tomato fruit development.     

Metabolism of carbohydrates during the development of seeds of the brazilian rubber tree [Hevea brasiliensis (Willd. Ex Adr. de Juss) Muell.-Arg.]

This work aimed at the assessment of the metabolism of carbohydrate during the development of the seeds of Brazilian rubber trees. The enzymatic activity of the acid invertase, neutral invertase and sucrose synthase (SuSy) and the levels of total soluble sugars (TSS), reducing sugars (RS) and sucrose were evaluated separately in each part of the fruit and seed—pericarp, seed coat, embryo and endosperm—on different days after the pollination (DAP). Based on the results obtained in this study, it is possible to conclude that in the beginning of the development of the rubber tree seeds, until 95 DAP, the endosperm presents high concentration of RS and low concentration of sucrose. After this period, the endosperm of the seed initiates starch accumulation and the concentration of RS decreases followed by the increase in the concentration of sucrose, presenting, after 120 DAP, an inversion of concentration of these two sugars. In the embryo, the levels of TSS, RS and sucrose show significant increase with the progress of the seed development. In the endosperm, the transition of the division phase and cell expansion for the storage of reserve material seem to occur around 120 DAP and is to be controlled mainly by the enzymes acid invertase and SuSy, while in the embryo, such transition seems to occur around 135 DAP and is to be controlled mainly by the enzymes acid and neutral invertases.     

Antisense inhibition of tomato fruit sucrose synthase decreases fruit setting and the sucrose unloading capacity of young fruit

The role of sucrose synthase (SuSy) in tomato fruit was studied in transgenic tomato (Lycopersicon esculentum) plants expressing an antisense fragment of fruit-specific SuSy RNA (TOMSSF) under the control of the cauliflower mosaic virus 35S promoter. Constitutive expression of the antisense RNA markedly inhibited SuSy activity in flowers and fruit pericarp tissues. However, inhibition was only slight in the endosperm and was undetectable in the embryo, shoot, petiole, and leaf tissues. The activity of sucrose phosphate synthase decreased in parallel with that of SuSy, but acid invertase activity did not increase in response to the reduced SuSy activity. The only effect on the carbohydrate content of young fruit was a slight reduction in starch accumulation. The in vitro sucrose import capacity of fruits was not reduced by SuSy inhibition at 23 days after anthesis, and the rate of starch synthesized from the imported sucrose was not lessened even when SuSy activity was decreased by 98%. However, the sucrose unloading capacity of 7-day-old fruit was substantially decreased in lines with low SuSy activity. In addition, the SuSy antisense fruit from the first week of flowering had a slower growth rate. A reduced fruit set, leading to markedly less fruit per plant at maturity, was observed for the plants with the least SuSy activity. These results suggest that SuSy participates in the control of sucrose import capacity of young tomato fruit, which is a determinant for fruit set and development.     

Ethylene biosynthesis in tissues of young and mature avocado fruits

Sitrit, Y., Blumenfeld, A. and Riov, J. 1987. Ethylene biosynthesis in tissues of young and mature avocado fruits.
Avocado (Persea americana Mill.) fruit tissues differ greatly in their capability to pro duce wound ethylene. In fruitlets, the endosperm lacks the ability to produce ethylene because no 1-aminocyclopropane-1-carboxylic acid (ACC) is synthesized and no activity of the ethylene-forming enzyme (EFE) is present. The cotyledons (embryo) do not produce significant amounts of ethylene at any of the developmental stages of the fruits, although in both young and mature fruits they contain a relatively high level of ACC synthase (EC 4.4.1.-) activity. Because of the very low EFE activity present in the cotyledons, most of the ACC formed in this tissue is conjugated. Of the various fruitlet tissues, the seed coat has the highest potential to produce ethylene. This is due to a high ACC synthase activity and particularly a high EFE activity. Also, the seed coat is very sensitive to the autocatalytic effect of ethylene. Fruitletpericarp possesses a lower potential to produce ethylene than the seed coat. Towardruit maturiy, the endosperm disappears and the seed coat shrivels and dies so that the pericarp and the cotyledons remain as the only active tissues in the mature fruit. At this stage, the pericarp is the only tissue producing ethylene. Mature precli macteric pericarp has a lower potential to produce ethylene than fruitlet pericarpThe role of ethylene in regulating various physiological processes at different stages of fruit maturation is discussed.     

Endogenous Gibberellin Profile During Christmas Rose (Helleborus niger L.) Flower and Fruit Development

Gibberellins (GAs) were identified and quantified during flower and fruit development in the Christmas rose (Helleborus niger L.), a native of southeastern Europe with a long international horticultural tradition. Physiologically, the plant differs from popular model species in two major respects: (1) following anthesis, the initially white or rose perianth (formed in this species by the sepals) turns green and persists until fruit ripening, and (2) the seed is shed with an immature embryo, a miniature endosperm, and a prominent perisperm as the main storage tissue. GA₁ and GA₄ were identified by full-scan mass spectra as the major bioactive GAs in sepals and fruit. LC-MS/MS system in accord with previously verified protocols also afforded analytical data on 12 precursors and metabolites of GAs. In the fruit, GA₄ peaked during rapid pericarp growth and embryo development and GA₁ peaked during the subsequent period of rapid nutrient accumulation in the seeds and continued pericarp enlargement. In the sepals, the flux through the GA biosynthetic pathway was highest prior to the light green stage when the photosynthetic system was induced. Unfertilized, depistillated, and deseeded flowers became less green than the seed-bearing controls; chlorophyll accumulation could be restored by applying GA₁, GA₄, and, less efficiently, GA₃ to the deseeded fruit. The sepals of unfertilized and depistillated flowers indeed contained very low levels of GA₄ and gradually decreasing levels of GA₁. However, the concentrations of their precursors and metabolites were less affected. These data suggest that a signal(s) from the fruit stimulates GA biosynthesis in the sepals resulting in greening. The fruit-derived GAs appear to be mainly involved in pericarp growth and seed development.     

Embryology in Trithuria submersa (Hydatellaceae) and relationships between embryo, endosperm, and perisperm in early-diverging flowering plants

? Premise of the study: Despite their highly reduced morphology, Hydatellaceae bear the unmistakable embryological signature of Nymphaeales, including a starch-rich maternal perisperm and a minute biparental endosperm and embryo. The co-occurrence of perisperm and endosperm in Nymphaeales and other lineages of flowering plants, and their respective functions during the course of seed development and embryo germination, remain enigmatic. ? Methods: Development of the embryo, endosperm, and perisperm was examined histologically from fertilization through germination in flowers and fruits of Trithuria submersa. ? Key results: The embryo of T. submersa initiates two cotyledons prior to seed maturity/dormancy, and their tips remain in contact with the endosperm throughout germination. The endosperm persists as a single layer of cells and serves as the interface between the embryo and the perisperm. The perisperm contains carbohydrates and proteins, and functions as the main storage tissue. The endosperm accumulates proteins and aleurone grains and functions as a transfer cell layer. ? Conclusions: In Nymphaeales, the multiple roles of a more typical endosperm have been separated into two different tissues and genetic entities: a maternal perisperm (nutrient acquisition, storage, mobilization) and a minute biparental endosperm (nutrient transfer to the embryo). The presence of perisperms among several other ancient lineages of angiosperms suggests a modest degree of developmental and functional lability for the nutrient storage tissue (perisperm or endosperm) within seeds during the early evolution of flowering plants. Finally, we examine the evolutionary developmental hypothesis that, contrary to longstanding assumptions, an embryo-nourishing perisperm along with a minute endosperm may represent the plesiomorphic condition for flowering plants.     

Sucrose Synthase,Starch Accumulation,and Tomato Fruit Sink Strength

Contrasting evidence has accumulated regarding the role of acid invertase and sucrose synthase in tomato fruit sink establishment and maintenance. In this work the relationships among the activities of sucrose synthase and acid invertase, Lycopersicon esculentum Mill cv UC-82B fruit growth, and starch accumulation were analyzed in fruit at 0 to 39 d after anthesis. Sucrose synthase, but not acid invertase, was found to be positively correlated with tomato fruit relative growth rate and with starch content in the pericarp tissue. A similar association between sucrose synthase activity and starch accumulation was also evident in the basal portion of the stem. Heat-shock treatments, which inhibited the increase in sucrose synthase activity at the beginning of the light period and had no effect on acid invertase activity, were used to examine the importance of sucrose synthase in relation to sucrose metabolism and starch synthesis. After the heat-shock treatment, concomitantly with the suppressed sucrose synthase activity relative to the controls, there was a reduction in sucrose cleavage and starch accumulation. These data substantiate the conclusion that, during the early phases of tomato fruit development, sucrose synthase rather than acid invertase is the dominant enzyme in metabolizing imported sucrose, which in turn plays a part in regulating the import of sucrose into the fruit.     

乙酰水杨酸在番茄果实发育期间对蔗糖代谢相关酶活性的影响

研究不同浓度乙酰水杨酸(ASA)对番茄品种‘辽园多丽’果实发育期间蔗糖代谢相关酶影响的结果表明:ASA可抑制果实的维管束和胶质胎座中酸性转化酶(AI)和中性转化酶(NI)活性,而提高蔗糖合成酶(SS)与蔗糖磷酸合成酶(SPS)活性;心室隔壁和中果肉中ASA的作用与此相反。ASA促进果实维管束中可溶性糖积累主要通过调控AI和NI活性实现,而在胶质胎座中主要通过调控SS活性实现;在中果肉和心室隔壁中主要通过调控SS和AI活性实现。     

Water Relations of Seed Development and Germination in Muskmelon (Cucumis melo L.) : IV. Characteristics of the Perisperm during Seed Development

We previously reported that an apparent water potential disequilibrium is maintained late in muskmelon (Cucumis melo L.) seed development between the embryo and the surrounding fruit tissue (mesocarp). To further investigate the basis of this phenomenon, the permeability characteristics of the tissues surrounding muskmelon embryos (the mucilaginous endocarp, the testa, a 2- to 4-cell-layered perisperm and a single cell layer of endosperm) were examined from 20 to 65 days after anthesis (DAA). Water passes readily through the perisperm envelope (endosperm + perisperm), testa, and endocarp at all stages of development. Electrolyte leakage (conductivity of imbibition solutions) of individual intact seeds, decoated seeds (testa removed), and embryos (testa and perisperm envelope removed) was measured during imbibition of freshly harvested seeds. The testa accounted for up to 80% of the total electrolyte leakage. Leakage from decoated seeds fell by 8- to 10-fold between 25 and 45 DAA. Presence of the perisperm envelope prior to 40 DAA had little effect on leakage, while in more mature seeds, it reduced leakage by 2- to 3-fold. In mature seeds, freezing, soaking in methanol, autoclaving, accelerated aging, and other treatments which killed the embryos had little effect on leakage of intact or decoated seeds, but caused osmotic swelling of the perisperm envelope due to the leakage of solutes from the embryo into the space between the embryo and perisperm. The semipermeability of the perisperm envelope of mature seeds did not depend upon cellular viability or lipid membrane integrity. After maximum seed dry weight is attained (35-40 DAA), the perisperm envelope prevents the diffusion of solutes, but not of water, between the embryo and the surrounding testa, endocarp, and mesocarp tissue.     

Development of maternal seed tissue in barley is mediated by regulated cell expansion and cell disintegration and coordinated with endosperm growth

After fertilization, filial grain organs are surrounded by the maternal nucellus embedded within the integuments and pericarp. Rapid early endosperm growth must be coordinated with maternal tissue development. Parameters of maternal tissue growth and development were analysed during early endosperm formation. In the pericarp, cell proliferation is accomplished around the time of fertilization, followed by cell elongation predominantly in longitudinal directions. The rapid cell expansion coincides with endosperm cellularization. Distribution of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling)-positive nuclei reveals distinct patterns starting in the nucellus at anthesis and followed later by the inner cell rows of the pericarp, then spreading to the whole pericarp. The pattern suggests timely and spatially regulated programmed cell death (PCD) processes in maternal seed tissues. When the endosperm is coenocytic, PCD events are only observed within the nucellus. Thereby, remobilization of nucellar storage compounds by PCD could nourish the early developing endosperm when functional interconnections are absent between maternal and filial seed organs. Specific proteases promote PCD events. Characterization of the barley vacuolar processing enzyme (VPE) gene family identified seven gene members specifically expressed in the developing grain. HvVPE2a (known as nucellain) together with closely similar HvVPE2b and HvVPE2d might be involved in nucellar PCD. HvVPE4 is strongly cell specific for pericarp parenchyma. Correlative evidence suggests that HvVPE4 plays a role in PCD events in the pericarp. Possible functions of PCD in the maternal tissues imply a potential nutritive role or the relief of a physical restraint for endosperm growth. PCD could also activate post-phloem transport functions.     

Fruit-localized phytochromes regulate lycopene accumulation independently of ethylene production in tomato

We show that phytochromes modulate differentially various facets of light-induced ripening of tomato fruit (Solanum lycopersicum L.). Northern analysis demonstrated that phytochrome A mRNA in fruit accumulates 11.4-fold during ripening. Spectroradiometric measurement of pericarp tissues revealed that the red to far-red ratio increases 4-fold in pericarp tissues during ripening from the immature-green to the red-ripe stage. Brief red-light treatment of harvested mature-green fruit stimulated lycopene accumulation 2. 3-fold during fruit development. This red-light-induced lycopene accumulation was reversed by subsequent treatment with far-red light, establishing that light-induced accumulation of lycopene in tomato is regulated by fruit-localized phytochromes. Red-light and red-light/far-red-light treatments during ripening did not influence ethylene production, indicating that the biosynthesis of this ripening hormone in these tissues is not regulated by fruit-localized phytochromes. Compression analysis of fruit treated with red light or red/far-red light indicated that phytochromes do not regulate the rate or extent of pericarp softening during ripening. Moreover, treatments with red or red/far-red light did not alter the concentrations of citrate, malate, fructose, glucose, or sucrose in fruit. These results are consistent with two conclusions: (a) fruit-localized phytochromes regulate light-induced lycopene accumulation independently of ethylene biosynthesis; and (b) fruit-localized phytochromes are not global regulators of ripening, but instead regulate one or more specific components of this developmental process.     

Cloning and characterization of a sucrose synthase-encoding gene from muskmelon

A full-length cDNA clone encoding sucrose synthase (SS; EC 2.4.1.13) was isolated from muskmelon (Cucumis melo L.) by RT-PCR and RACE. The clone, designated as CmSS1, contains 2,585 nucleotides with an open reading frame of 2,412 nucleotides. The deduced 804 amino acid sequence showed high identities with other plant sucrose synthase. Real time PCR analysis indicated that CmSS1 expression differed among root, stem, leaf, flower and fruit tissues. The analysis during fruit development indicated that CmSS1 mRNA showed its maximum level at 5 days after pollination (DAP) and decreased gradually during fruit development until its minimum level in mature fruit. The sucrose content was very low in fruit before 20 DAP but increased dramatically between 20 and 30 DAP during fruit development. However, SS activities in both direction of sucrose synthesis and sucrose cleavage were very low and changed little during fruit development, suggesting that SS may play little role in determining sucrose accumulation during muskmelon fruit.     

Sugar uptake by protoplasts isolated from tomato fruit tissues during various stages of fruit growth

The uptake of radioactive glucose and sucrose by protoplasts isolated from pericarp and placenta tissues of tomato ( Lycopersicon esculentum cv. Counter) fruit was investigated in relation to the dry matter accumulation rates of these tissues. Uptake of glucose by protoplasts isolated from pericarp tissue was highest in fruit of around 20 g fresh weight or 25 days after anthesis. Sucrose uptake by pericarp protoplasts was lower than that of glucose and did not show a peak of uptake. The maximum rate of glucose uptake by protoplasts from the pericarp was at the time when the tomato fruit was accumulating dry matter at the highest rate. Glucose uptake by placenta protoplasts was lower and at a similar level as sucrose.
Protoplast uptake of glucose, but not of sucrose, was partially inhibited by (1) p -chloromercuribenzene sulphonic acid, a sulphydryl group modifier; (2) erythrosin B, an H⁺-ATPase inhibitor; and (3) valinomycin, a K⁺-ionophore, suggesting that membrane transport of glucose by tomato fruit sink cells may be a carrier-mediated, energy-dependent process.
The main route of carbohydrate accumulation by tomato fruit during the period of rapid fruit growth may be by cleavage of sucrose by apoplastic acid invertase prior to hexose transport across the plasma membrane.     

Localization of sucrose synthase activity in developing maize kernels by in situ enzyme histochemistry

Sucrose synthase is usually localized by immunocytochemistry, but this method does not show the actual activity of the localized enzyme. A histochemical assay is presented here showing the activity of sucrose synthase by tetrazolium salt precipitation on sections of developing maize kernels. The advantages of the assay are a high sensitivity for low amounts of active sucrose synthase and the independence of specific antibodies.In this study the activity of endosperm sucrose synthase is shown to move gradually from the apical part of the endosperm to the basal endosperm during kernel development. This shift in sucrose synthase activity correlates well with the localization of starch synthesis during kernel development. The assay also shows the early loss of activity in the aleurone layer bordering the embryo, and a loss of activity in the apical aleurone during the final stage of kernel development while the enzyme was still found by immunocytochemistry. This is in contrast to a high sucrose synthase activity in the epithelium of the scutellum, where hardly any labelling was found with antibodies against maize sucrose synthase. Low sucrose synthase activities were found in the pericarp and pedicel parenchyma.Possible functions of the high and low activity patterns in the developing maize kernels and differences between the enzyme assay and immunocytochemistry are discussed.     

Sucrose-to-Starch Metabolism in Tomato Fruit Undergoing Transient Starch Accumulation

Immature green tomato (Lycopersicon esculentum) fruits undergo a period of transient starch accumulation characterized by developmental changes in the activities of key enzymes in the sucrose (Suc)-to-starch metabolic pathway. Activities of Suc synthase, fructokinase, ADP-glucose (Glc) pyrophosphorylase, and soluble and insoluble starch synthases decline dramatically in parallel to the decrease in starch levels in the developing fruit. Comparison of "maximal" in vitro activities of the enzymes in the Suc-to-starch pathway suggests that these same enzymes are limiting to the rate of starch accumulation. In contrast, activities of invertase, UDP-Glc pyrophosphorylase, nucleoside diphosphate kinase, phosphoglucoisomerase, and phosphoglucomutase do not exhibit dramatic decreases in activity and appear to be in excess of starch accumulation rates. Starch accumulation is spatially localized in the inner and radial pericarp and columella, whereas the outer pericarp and seed locule contain little starch. The seed locule is characterized by lower activities of Suc synthase, UDP-Glc pyrophosphorylase, phosphoglucomutase, ADP-Glc pyrophosphorylase, and soluble and insoluble starch synthases. The outer pericarp exhibits comparatively lower activities of ADP-Glc pyrophosphorylase and insoluble starch synthase only. These data are discussed in terms of the developmental and tissue-specific coordinated control of Suc-to-starch metabolism.