Identification and modulation of the key amino acid residue responsible for the pH sensitivity of neoculin, a taste-modifying protein

Neoculin occurring in the tropical fruit of Curculigo latifolia is currently the only protein that possesses both a sweet taste and a taste-modifying activity of converting sourness into sweetness. Structurally, this protein is a heterodimer consisting of a neoculin acidic subunit (NAS) and a neoculin basic subunit (NBS). Recently, we found that a neoculin variant in which all five histidine residues are replaced with alanine elicits intense sweetness at both neutral and acidic pH but has no taste-modifying activity. To identify the critical histidine residue(s) responsible for this activity, we produced a series of His-to-Ala neoculin variants and evaluated their sweetness levels using cell-based calcium imaging and a human sensory test. Our results suggest that NBS His11 functions as a primary pH sensor for neoculin to elicit taste modification. Neoculin variants with substitutions other than His-to-Ala were further analyzed to clarify the role of the NBS position 11 in the taste-modifying activity. We found that the aromatic character of the amino acid side chain is necessary to elicit the pH-dependent sweetness. Interestingly, since the His-to-Tyr variant is a novel taste-modifying protein with alternative pH sensitivity, the position 11 in NBS can be critical to modulate the pH-dependent activity of neoculin. These findings are important for understanding the pH-sensitive functional changes in proteinaceous ligands in general and the interaction of taste receptor-taste substance in particular.     

Neoculin as a new taste-modifying protein occurring in the fruit of Curculigo latifolia

A unique taste-modifying activity that converts the sense of sourness to the sense of sweetness occurs in the fruit of the plant Curculigo latifolia, intrinsic to West Malaysia. The active component, known as curculin, is a protein consisting of two identical subunits. We have found a new taste-modifying protein, named neoculin, of the same origin. Both chemical analysis and cDNA cloning characterized neoculin as a heterodimeric protein consisting of an acidic, glycosylated subunit of 113 amino acid residues and a basic subunit that is the monomeric curculin itself.     

Crystal structure of neoculin: insights into its sweetness and taste-modifying activity

Although the majority of sweet compounds are of low molecular mass, several proteins are known to elicit sweet taste responses in humans. The fruit of Curculigo latifolia contains a heterodimeric protein, neoculin, which has both sweetness and a taste-modifying activity that converts sourness to sweetness. Here, we report the crystal structure of neoculin at 2.76A resolution. This is the first well-defined tertiary structure of a taste-modifying protein of this kind. The overall structure is quite similar to those of monocot mannose-binding lectins. However, crucial topological differences are observed in the C-terminal regions of both subunits. In both subunits of neoculin, the C-terminal tails turn up to form loops fixed by inter-subunit disulfide bonds that are not observed in the lectins. Indeed, the corresponding regions of the lectins stretch straight over the surface of another subunit. Such a C-terminal structural feature as is observed in neoculin results in a decrease in subunit-subunit interactions. Moreover, distribution of electrostatic potential on the surface of neoculin is unique and significantly different from those of the lectins, particularly in the basic subunit (NBS). We have found that there is a large cluster composed of six basic residues on the surface of NBS, and speculate that it might be involved in the elicitation of sweetness and/or taste-modifying activity of neoculin. Molecular dynamics simulation based on the crystallography results suggests that neoculin may adopt a widely "open" conformation at acidic pH, while unprotonated neoculin at neutral pH is in a "closed" conformation. Based on these simulations and the generation of a docking model between neoculin and the sweet-taste receptor, T1R2-T1R3, we propose the hypothesis that neoculin is in dynamic equilibrium between open and closed states, and that the addition of an acid shifts the equilibrium to the open state, allowing ligand-receptor interaction.     

Peroxisomes from pepper fruits (Capsicum annuum L.): purification, characterisation and antioxidant activity

Pepper is a vegetable of importance in human nutrition. Currently, one of the most interesting properties of natural products is their antioxidant content. In this work, the purification and characterisation of peroxisomes from fruits of a higher plant was carried out, and their antioxidative enzymatic and non-enzymatic content was investigated. Green and red pepper fruits (Capsicum annuum L., type Lamuyo) were used in this study. The analysis by electron microscopy showed that peroxisomes from both types of fruits contained crystalline cores which varied in shape and size, and the presence of chloroplasts and chromoplasts in green and red pepper fruits, respectively, was confirmed.

Peroxisomes were purified by differential and sucrose density-gradient centrifugations. In the peroxisomal fractions, the activity of the photorespiration, β-oxidation and glyoxylate cycle enzymes, and the ROS-related enzymes catalase, superoxide dismutase, xanthine oxidase, glutathione reductase and NADP⁺-dehydrogenases, was determined. Most enzymes studied had higher specific activity and protein content in green than in red fruits. By native PAGE and western blot analysis, the localisation of a Mn-SOD in fruit peroxisomes was demonstrated. The ascorbate and glutathione levels were also determined in crude extracts and in peroxisomes purified from both green and red peppers. The total ascorbate content (200-220 mg per 100 g FW) was similar in crude extracts from the two types of fruits, but higher in peroxisomes from red peppers. The glutathione concentration was 2-fold greater in green pepper crude extracts than in red fruits, whereas peroxisomes from both tissues showed similar values. The presence in pepper peroxisomes of different antioxidative enzymes and their corresponding metabolites implies that these organelles might be an important pool of antioxidants in fruit cells, where these enzymes could also act as modulators of signal molecules (O₂^˙-, H₂O₂) during fruit maturation.     

Morphology and phenology of seeds and whole fruit eaten by Milne-Edwards’ sifaka,Propithecus diadema edwardsi,in Ranomafana National Park,Madagascar

Although it is an anatomical folivore, the diet of the Milne-Edwards’ sifaka (Propithecus diadema edwardsiA. Grandidier, 1871) in Ranomafana National Park contained 35% seeds, 30% whole fruit, and 28% leaves. Plant species used as seed sources differed from those used as whole fruit sources in terms of temporal variation in consumption, taxonomic affiliation, morphology, and phenology. Although seeds were destroyed in both exploitation styles used by the sifakas—seed and whole fruit-eating—the gross morphology of species used as seed sources conformed to the complex of traits typical for fruits experiencing seed predation, while species used as whole fruit sources conformed to traits typical for fruits that do not experience predispersal predation. Many of the 19 plant species from which the seed was extracted and eaten contained a single seed with moderate testa thickness, and fruits containing this type of seed were medium-sized with dry or fibrous flesh, moderate skin thickness, and a dull color. In contrast, brightly colored, juicy fruits with minimally protected seeds were characteristic of the 38 plant species from which both pericarp and seed were eaten. Compared to transectwide measures of fruit availability or patterns restricted to whole fruit sources, fewer species of seed sources produced fruit per month and fruiting activity was more seasonal.     

Biochemical aspects of the developing and ripening banana

The peel and pulp of the banana fruit and the pseudostem were examined for glutamate-oxaloacetate transaminase (GOT), glutamate-pyruvate transaminase (GPT) and aldolase activities and protein, phenolics, chlorophyll and starch. The peel-pulp ratio at various stages of fruit development on the plant and in detached fruits showing incipient ripening were used as an index of the physiological age of the fruit. The enzymes exhibited maximum activity at a stage corresponding to the initiation of the climacteric. GPT level at this stage was higher than that of GOT. An initial increase in the protein content was followed by a decline in both peel and pulp, the level reaching a minimum in climacteric fruits. Astringency, measured in terms of total phenolics, decreased with development; in mature fruits, peel contained 4–5 × as much phenolics as pulp. Chlorophyll in mature fruits was 10 × higher than in young fruits and decreased in ripe fruits. The onset of ripening was attended with a pronounced decrease in the starch. The various analyses were carried out also on the pseudostem removed from the plant soon after flower formation.     

Sucrose Synthase in Wild Tomato, Lycopersicon chmielewskii, and Tomato Fruit Sink Strength

Here it is reported that sucrose synthase can be readily measured in growing wild tomato fruits (Lycopersicon chmielewskii) when suitable methods are adopted during fruit extraction. The enzyme also was present in fruit pericarp tissues, in seeds, and in flowers. To check for novel characteristics, the wild tomato fruit sucrose synthase was purified, by (NH₄)₂SO₄ fraction and chromatography with DE-32, Sephadex G-200, and PBA-60, to one major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The following characteristics were obtained: native protein relative molecular weight 380,000; subunit relative molecular weight 89,000; K_m values with: sucrose 53 millimolar, UDP 18.9 micromolar, UDP-glucose 88 micromolar, fructose 8.4 millimolar; pH optima between 6.2 to 7.3 for sucrose breakdown and 7 to 9 for synthesis; and temperature optima near 50°C. The enzyme exhibited a high affinity and a preference for uridylates. The enzyme showed more sensitivity to divalent cations in the synthesis of sucrose than in its breakdown. Sink strength in tomato fruits also was investigated in regard to sucrose breakdown enzyme activities versus fruit weight gain. Sucrose synthase activity was consistently related to increases in fruit weight (sink strength) in both wild and commercial tomatoes. Acid and neutral invertases were not, because the published invertase activity values were too variable for quantitative analyses regarding the roles of invertases in tomato fruit development. In rapidly growing fruits of both wild and commercially developed tomato plants, the activity of sucrose synthase per growing fruit, i.e. sucrose synthase peak activity X fruit size, was linearly related to final fruit size; and the activity exceeded fruit growth and carbon import rates by at least 10-fold. In mature, nongrowing fruits, sucrose synthase activities approached nil values. Therefore, sucrose synthase can serve as an indicator of sink strength in growing tomato fruits.     

The beta subunit of tomato fruit polygalacturonase isoenzyme 1: isolation, characterization, and identification of unique structural features.

We have purified and isolated cDNAs encoding the beta subunit of tomato fruit polygalacturonase isoenzyme 1 (PG1), a cell wall protein that associates with, and apparently regulates, the catalytic PG2 polypeptides. Expression of the beta subunit is fruit specific and temporally separated from the expression of PG2 during fruit development. The 37- to 39-kD beta subunit is encoded as a 69-kD precursor protein containing a signal sequence and two propeptide domains. The mature protein is composed almost entirely of the novel 14-amino acid motif FTNYGxxGNGGxxx in which many of the phenylalanine residues are post-translationally modified. The unique structural features of the motif suggest an important role in the function of the protein and hence in the activity of PG1. The beta subunit may represent a class of bifunctional plant proteins that interact both with structural components of the cell wall and catalytic proteins to localize and/or regulate metabolic activities within the cell wall.     

Effect of assimilate supply on the growth of individual cucumber fruits

The effects of assimilate supply on the growth of individual fruits during different stages of fruit development were analysed in cucumber ( Cucumis sativus L. cv. Corona). The assimilate supply was varied by maintaining different numbers of fruits per plant or maintaining different irradiances. The growth rate of a cucumber fruit strongly increased with increasing assimilate supply, but its growing period was not noticeably affected. At a low assimilate supply both cell number and cell size were reduced. Increasing the assimilate supply at different stages of fruit development showed that the early development of a cucumber fruit was not crucial for setting its growth potential. A small number of cells, due to a low assimilate supply, during early fruit development, was to a great extent compensated by an increased expansion rate of individual cells. It is concluded that cell number is not an important determinant of fruit size in cucumber, although fruit size is often positively correlated with cell number. In the early stages of fruit development the effect of irradiance on the fruit growth rate depends on the presence of an earlier developed fruit because of dom-inance between fruits. In later stages of fruit development, a decrease in irradiance reduced the growth rate of all fruits relatively to the same extent independent of age or presence of other fruits.     

Temporally extended gene expression of the ADP-Glc pyrophosphorylase large subunit (AgpL1) leads to increased enzyme activity in developing tomato fruit

Tomato plants (Solanum lycopersicum) harboring the allele for the AGPase large subunit (AgpL1) derived from the wild species Solanum habrochaites (AgpL1 ^H ) are characterized by higher AGPase activity and increased starch content in the immature fruit, as well as higher soluble solids in the mature fruit following the breakdown of the transient starch, as compared to fruits from plants harboring the cultivated tomato allele (AgpL1 ^E ). Comparisons of AGPase subunit gene expression and protein levels during fruit development indicate that the increase in AGPase activity correlates with a prolonged expression of the AgpL1 gene in the AgpL1 ^H high starch line, leading to an extended presence of the L1 protein. The S1 (small subunit) protein also remained for an extended period of fruit development in the AgpL1 ^H fruit, linked to the presence of the L1 protein. There were no discernible differences between the kinetic characteristics of the partially purified AGPase-L1^E and AGPase-L1^H enzymes. The results indicate that the increased activity of AGPase in the AgpL1 ^H tomatoes is due to the extended expression of the regulatory L1 and to the subsequent stability of the heterotetramer in the presence of the L1 protein, implying a role for the large subunit not only in the allosteric control of AGPase activity but also in the stability of the AGPase L1–S1 heterotetramer. The introgression line of S. lycopersicum containing the wild species AgpL1 ^H allele is a novel example of transgressive heterosis in which the hybrid multimeric enzyme shows higher activity due to a modulated temporal expression of one of the subunits.     

Effects of below- and above-ground herbivores on plant growth,flower visitation and seed set

Separate and combined effects of root and leaf herbivores on plant growth, flower visitation and seed set were tested in a factorial experiment using potted mustard, Sinapis arvensis, at an old fallow field. A 50% leaf removal by cabbageworms (Pieris rapae) when the seedlings had their first four leaves reduced plant height and shoot mass, and delayed the onset of flowering. Root herbivory by two wireworms (Agriotes sp.) over the whole experiment changed flower visitation; the number of flower visitors per plant was higher in plants with root herbivores than in plants without root herbivores. Combined leaf and root herbivory affected flowering period, number of fruits per plant and number of seeds per fruit. Plants attacked by leaf and root herbivores had a shorter flowering period and produced fewer fruits per plant than plants with root herbivores only. Although the experimental plants faced major herbivore-induced growth changes, plant reproduction (seed set and weight per plant) was similar in all treatments, documenting their ability to effectively compensate for leaf and root herbivory.     

Fruit size, crop mass, and plant height explain differential fruit choice of primates and birds

Seed dispersal by animals is an important ecological process shaping plant regeneration. In general, seed dispersers are highly variable and often opportunistic in their fruit choice. Despite much research, the factors that can explain patterns of fruit consumption among different animal groups remain contentious. Here, we analysed the interactions between 81 animal species feeding on the fruits of 30 plant species in Kakamega Forest, Kenya, during 840 h of observations. Our aim was to determine whether plant characteristics, fruit morphology, fruit colours and/or fruit compounds such as water, sugar, phenols and tannins explained the relative importance of fruit consumption by the two most important consumer groups, primates and birds. We found significant differences in fruit choice between both groups. Primates fed on larger fruits and on higher trees that had larger fruit crops, whereas birds were observed feeding on smaller fruits and on smaller plants producing fewer fruits. Fruit colours did not differ between fruits consumed by primates and those consumed by birds. However, differences in the fruit choice among frugivorous birds were associated with differences in fruit colours. Smaller plants with smaller fruits produced red fruits which contrasted strongly with the background; these fruits were dispersed by a distinct set of bird species. The contents of water, sugar, phenols and tannins did not differ between fruits eaten by primates and those eaten by birds. Some phylogenetic patterns were apparent; primates fed preferentially on a phylogenetically restricted subsample of large plants with large fruits of the subclass Rosidae. We discuss why the observed primate dispersal syndrome is most likely explained by a process of ecological fitting.     

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.     

Molluscicidal activity of various solvent extracts from Solanum nigrum var. villosum L. aerial parts against Galba truncatula

Molluscicidal activity of Solanum nigrum var. villosum (morelle velue) extracts and their fractions were tested against the mollusca gastropoda Galba truncatula intermediate host of Fasciola hepatica. The results indicated that the hydro-methanol (MeOH-H2O) immature fruit extract possess the highest molluscicidal activity (LC50 = 3.96 mg/L) against Galba truncatula compared with other tested compounds. After acido-basic treatment, the methanolic extract fraction isolated from the immature fruits and the richest in alkaloids was the most toxic (LC50 = 1.65 mg/L). The fractions richest in saponosides obtained from the hydromethanolic and methanolic extracts of immature fruits showed interesting molluscicidal activities (LC50 = 6.15 mg/L and LC50 = 7.91 mg/L, respectively). The observed molluscicide activity could be attributed to the presence of alkaloids or saponosides. So, the immature fruits of Solanum nigrum var. villosum could be substrates of choice for molluscicide activity. In addition, total alkaloids and saponosides present in this plant deserve further investigations in order to identify the active principles and demonstrate their activities on mollusks in their natural habitat. According to the World Health Organization's guidelines on screening for plant molluscicides, use of these fractions may add to the arsenal of methods to control snail transmitting fasciolosis in tropical and Third World countries where fasciolosis is a common disease.     

Accumulation of Capsaicin in Seed, Pericarp and Placenta of Capsicum annuum L Fruit

The accumulation of capsaicin in different parts of fruit viz, placenta, pericarp and seeds of Capsicum annuum L cv Punjab Lal was compared with the activities of first four enzymes of capsaicin biosynthetic pathway at various physiological stages. Capsaicin accumulation (mg g^?1 DW) was about ten fold higher in placenta (63.96), than in pericarp (7.12) and seeds (5.06) in ripe fruits. Capsaicin accumulation was 5.79 mg g^?1 DW at 28 DAF in whole fruit. The specific activity of PAL was also ten times higher in placenta, whereas the specific activities of Ca4H, Ca3H and CaOMT were about two times higher in placenta than in other parts of fruit. The trend CaOMT > PAL > Ca4H > Ca3H was observed with peak activity at 28 DAF for Ca3H and CaOMT and at 35 DAF for PAL and Ca4H in placenta. These four enzymes showed low activity during the period up to 21 DAF, and peak activities for these enzymes were obtatined at the time of maximum growth of fruit in length and thereafter.     

Taste-modifying sweet protein, neoculin, is received at human T1R3 amino terminal domain

This study examines taste reception of neoculin, a Curculigo latifolia sweet protein with taste-modifying activity which converts sourness to sweetness. Neoculin tastes sweet to humans, but not to mice, and is received by the human sweet taste receptor hT1R2-hT1R3. In the present study with calcium imaging analysis of HEK cells expressing human and mouse T1Rs, we demonstrated that hT1R3 is required for the reception of neoculin. Further experiments using human/mouse chimeric T1R3s revealed that the extracellular amino terminal domain (ATD) of hT1R3 is essential for the reception of neoculin. Although T1R2-T1R3 is known to have multiple potential ligand-binding sites to receive a wide variety of sweeteners, the present study is apparently the first to identify the ATD of hT1R3 as a new sweetener-binding region.     

Early Application of Ethrel Extends Tomato Fruit Cell Division and Increases Fruit Size and Yield with Ripening Delay

Flowers of tomato (Lycopersicon esculentum Mill.) plants cv. Castle Rock were sprayed with 100 ppm of ethrel, 0.5 mm aminooxyacetic acid (AOA), or water (control) 2 days after anthesis. The fruit period of cell division was extended up to 16–18 days after anthesis with the application of ethrel but reduced from 10–12 days (control) down to only 6–8 days with the application of AOA. In a trend opposite to AOA application, fruits that received ethrel treatment were of higher ethylene and 1-aminocyclopropane-1-carboxylic acid (ACC) levels than control. This was noticed not only during the first 2 weeks after anthesis but also during the fruit climacteric phase. Mesocarp cells of ethrel-treated fruits were greater in number/mm² but smaller in size than control; an opposite trend was obtained with the application of AOA. This was observed for a period of 18 days after anthesis, but by that time or at earlier ages, fruits of AOA treatment were larger in size and heavier in weight than control, and both were larger and heavier than ethrel-treated ones. At 5 weeks after anthesis and thereafter, the fruit response to all treatments was totally reversed because early ethrel-treated fruits became significantly larger in size and heavier in weight with a ripening delay of about 10 and 15 days compared with those of control and AOA-treated ones, respectively. When the same treatments were applied to the whole plant, similar results were obtained because the early application of ethrel increased the fruit yield by about 15% over control with a pronounced ripening delay; an opposite trend was obtained with the application of AOA. No significant differences were found among all treatments in terms of flower or fruit abscission or fruit number/plant. The data suggest that ethylene regulates tomato fruit transmission from cell division to cell enlargement. In addition, fruit cell division is terminated only when endogenous ethylene decreases to its basal level, allowing cell enlargement to dominate and proceed as in the case of the early application of AOA. The ripening delay of ethrel-treated fruits may be caused by the longer time required for the increased cell number to reach maturation. A low level of ethrel application at the tomato early fruiting stage may be used for increasing fruit yield by increasing fruit size and consequently its quality. Received June 1, 1998; accepted December 7, 1998     

Reduction of tomato polygalacturonase beta subunit expression affects pectin solubilization and degradation during fruit ripening.

The developmental changes that accompany tomato fruit ripening include increased solubilization and depolymerization of pectins due to the action of polygalacturonase (PG). Two PG isoenzymes can be extracted from ripe fruit: PG2, which is a single catalytic PG polypeptide, and PG1, which is composed of PG2 tightly associated with a second noncatalytic protein, the beta subunit. Previous studies have correlated ripening-associated increases in pectin solubilization and depolymerization with the presence of extractable PG1 activity, prior to the appearance of PG2, suggesting a functional role for the beta subunit and PG1 in pectin metabolism. To assess the function of the beta subunit, we produced and characterized transgenic tomatoes constitutively expressing a beta subunit antisense gene. Fruit from antisense lines had greatly reduced levels of beta subunit mRNA and protein and accumulated < 1% of their total extractable PG activity in ripe fruit as PG1, as compared with 25% for wild type. Inhibition of beta subunit expression resulted in significantly elevated levels of EDTA-soluble polyuronides at all stages of fruit ripening and a significantly higher degree of depolymerization at later ripening stages. Decreased beta subunit protein and extractable PG1 enzyme activity and increased pectin solubility and depolymerization all cosegregated with the beta subunit antisense transgene in T2 progeny. These results indicate (1) that PG2 is responsible for pectin solubilization and depolymerization in vivo and (2) that the beta subunit protein is not required for PG2 activity in vivo but (3) does play a significant role in regulating pectin metabolism in wild-type fruit by limiting the extent of pectin solubilization and depolymerization that can occur during ripening. Whether this occurs by direct interaction of the beta subunit with PG2 or indirectly by interaction of the beta subunit with the pectic substrate remains to be determined.