Purification and partial characterization of β-glucosidase from papaya fruit

β-Glucosidase (I) was isolated from Carica papaya fruit pulp and purified ca 1000-fold to electrophoretic homogeneity. The procedure used ammonium sulphate fractionation followed by chromatography on Phenyl-Sepharose CL-4B and Sephacryl S-200 to separate α-mannosidase (II) and, in part, β-galactosidase (III) from (I). Final separation of (III) from (I) was achieved by preparative isoelectric focusing (PIEF). The glycosidases had pI of 5.2 (I), 4.9 (II) and 6.9 (III). M,s of 54 000 (I), 260 000 (II) and 67 000 (III) were determined by gel filtration. The M, of (I) estimated by SDS-PAGE was 27 000 suggesting that (I) consisted of two subunits. The optimum pH and optimum temperature of (I) were 5.0 and 50°, respectively, and the enzyme followed typical Michaelis kinetics with K_m and V_max of 1.1 × 10⁻⁴ M and 1.8 × 10⁻⁶ mol/hr, respectively, for p-nitrophenyl-β-d-glucoside (40°).     

Foliar application of aminoethoxyvinylglycine (AVG) delays fruit ripening and reduces pre-harvest fruit drop and ethylene production of bagged “Kogetsu” apples

Covering apple fruits with double layer waterproof bags to enhance fruit quality and evenness of blush colour is typical on many cultivars in Korea and Japan. Aminoethoxyvinylglycine (AVG) applied to unbagged apple fruits at 3–4 weeks before commercial harvest reduces ethylene production in the fruit, delays fruit ripening and reduces pre-harvest fruit drop. Spray application of AVG to trees of bagged apples should have no effect on apple ripening as there is␣no direct contact with the fruit and the translocation of AVG in apple trees is regarded as negligible. However, preliminary experiments suggested that AVG applied to trees of bagged apples reduced pre-harvest fruit drop in “Kotgetsu” apples. This study investigated the effect of spray treatments of 125 ppm of AVG on fruit drop, fruit ripening (firmness, starch conversion and soluble solids) and ethylene production to whole trees with bagged or unbagged “Kogetsu” fruit, as well as sprays of only the bagged or unbagged fruit on trees on two orchards. AVG applied to whole trees with unbagged apples reduced fruit drop from an average of 58.9% to 10.4%, delayed starch conversion and decreased ethylene production. AVG applied to whole trees with bagged fruit was equally effective in reducing pre-harvest drop, delaying fruit ripening and reducing ethylene production. Application of AVG to unbagged fruit only was nearly as effective as application to whole trees with unbagged fruit but application to bagged fruit only had no effect on fruit ripening or ethylene production. Application of AVG to bagged fruit only did reduce fruit drop to an average of 42.5% but this was not as effective as spraying unbagged fruit only or whole trees with bagged fruit. Possible mechanisms for this effect are discussed.     

Accumulation of the β-subunit of polygalacturonase 1 in normal and mutant tomato fruit

Polyclonal antiserum raised against the native PG1 isoform of tomato fruit (Lycopersicon esculentum Mill.) polygalacturonase [poly(1,4--d-galacturonide) glycanohydrolase, EC 3.2.1.15] bound to each of the subunits of the protein and also to a range of other fruit proteins. Affinity purification was used to remove antibody molecules that bound to the native form of the PG2 isoform. The resulting serum bound to native PG1, denatured PG2 and -subunits of PG1 but not to native PG2 or other fruit proteins. This anti-PG1 serum was used to monitor the occurrence of the PG1 -subunit and PG2 in detergent extracts of tomato tissues. The -subunit polypeptide was detected in pericarp but not locule tissue of fruit, including fruit of the rin and nor mutants. It increased in amount in the pericarp tissues from an early stage to the mature green stage, clearly prior to any appreciable accumulation of the PG2 subunit. The -subunit polypeptide was not detected in stem or leaf tissues. A PG2-specific antiserum was used to study the interaction of PG2 with the isolated -subunit. The PG2 isoform was bound to the -subunit over a wide range of salt concentrations and pH; the interaction was independent of the presence of reducing agents. It is concluded that strong non-covalent forces are involved in the interaction. The results are consistent with a model in which the -subunit is positioned in the cell wall structure and provides a specific binding site for the active PG2 subunit when this is synthesised during ripening.Abbreviations B breaker - MG mature green - M_r relative molecular mass - nor non-ripening mutant - PAGE polyacrylamide gel electrophoresis - PG polygalacturonase - rin ripening inhibitor mutant - SDS sodium dodecyl sulphate     

Inheritance of unique fruit and foliage color mutation in NuMex piñata

The inheritance of mature fruit color in peppers (Capsicum spp.) is controlled by several genes. However, the inheritance of the transition of colors the fruit undergo during ripening has not been described extensively. The authors describe the inheritance of a unique gene which affects foliage color and fruit color transition occurring in the jalape?o cultivar NuMex Pi?ata. The gene responsible is designated the tra gene.     

Biochemical changes involved in stress response and ripening behaviour of γ-irradiated mango fruit

The stress response of ripening mango fruits treated in the pre-climacteric phase with radiation doses of 0.75, 1.25 and 1.75 kGy was investigated. L-Phenylalanine ammonialyase, polyphenol oxidase, peroxidase and catalase activities were determined and significant differences were observed. Differences in the patterns of total phenolics, flavanols and proteins were also observed. Malic enzyme activity was used as an indicator of the ripening stage (climacteric rise, climacteric peak and post-climacteric phase) as well as a measure of the effect of γ-irradiation on fruit ripening. It seems that radiation treatment causes a stress condition in the fruit which, depending on the dose, may lead to browning of the tissue or necrotic decay.     

Peroxidase and IAA oxidase activities and peroxidase isoenzymes in the pericarp of seeded and seedless “Redhaven” peach fruit

Parthenocarpic peach fruit (Prunus persica L. Batsch., cv. Redhaven) were induced with 1-(3-chlorophthalimide)-cyclohexane carboxamide (AC 94377). The activities of soluble, and ionically and covalently bound peroxidase and indole-3-acetic acid (IAA) oxidase in the pericarp of both seeded and parthenocarpic fruit were determined from 21–43 days after anthesis. Seedless fruit grew faster during early stage I and ceased growth earlier than seeded fruit. Total peroxidase and IAA oxidase activities increased with development on both types of fruit, but higher values were found in seedless fruit. The ionic fraction showed the greatest increase for both enzyme activities. Isoperoxidase profile showed new cationic isoenzymes and higher levels of the less anionic isoenzymes in the pericarp of seedless fruit, whereas the seeded fruit contained higher levels of the more acidic isoperoxidases.     

Maturation characteristics of Rubus pennsylvanicus fruit: are black and red the same?

Summary Fruit of the blackberry, Rubus pennsylvanicus Poir. (Rosaceae), were examined to determine variation in maturation characteristics. Maturation timing and rate varied greatly among individual fruits, resulting in a bi-colored fruiting display comprised largely of two maturation stages, pre-ripe (salmon and scarlet) and ripe (dark brown and black). While ripe fruit were generally larger and heavier than pre-ripe fruit, exhibiting greater fresh and dry fruit weight, diameter, water content, and total seed weight, no significant differences were found in energy content, i.e. numbers of calories per gram pulp, or in pulp:seed ratio. The differences between ripe and pre-ripe fruit appear to be due largely to an increase in water content and seed weight with maturity. The fact that little energetic benefit accrues to the preferential selection of ripe fruit suggests that bi-colored Rubus displays may be considered to be unicolored.     

Ethylene action blockade and cold storage affect ripening of ‘Golden’ papaya fruit

The purpose of this work was to evaluate the effects of ethylene action blockade and cold storage on the ripening of ‘Golden’ papaya fruit. Papayas harvested at maturity stage 1 (up to 15% yellow skin) were evaluated. Half of the fruits, whether treated or not treated with 100 nL L⁻¹ of 1-methylcyclopropene (1-MCP), were stored at 23°C, while the other half were stored at 11°C for 20 days prior to being stored at 23°C. Non-refrigerated fruits receiving 1-MCP application presented a reduction in respiratory activity, ethylene production, skin color development and pectinmethylesterase activity. Even with a gradual increase in ethylene production at 23°C, fruits treated with 1-MCP maintained a high firmness, but presented a loss of green skin color. Cold storage caused a decrease in ethylene production when fruits were transferred to 23°C. The results suggest that pulp softening is more dependent on ethylene than skin color development, and that some processes responsible for loss of firmness do not depend on ethylene.     

Are possums important dispersers of large-seeded fruit?

The claim by Dungan et al. (2002) that “in many areas possums may be the only potential dispersal vector for large-seeded native species” is unsubstantiated. There is little evidence possums excrete viable seed of large-seeded fruit greater than 10 mm diameter, and seeds up to this size are dispersed by a suite of bird species. Nowhere in New Zealand are there likely to be possums in the absence of this suite of bird species.     

Effect of simultaneous down-regulation of pectate lyase and endo-β-1,4-glucanase genes on strawberry fruit softening

Strawberry is a soft fruit with a short postharvest shelf-life. The loss of fruit firmness during ripening is mainly due to the disassembly of parenchyma cell walls mediated by the expression of genes encoding enzymes acting on pectins, such as pectate lyase, or hemicellulose, e.g. endo-β-1,4-glucanase. To determine if the simultaneous down-regulation of FaplC and FaEG3 genes, encoding a pectate lyase and a endo-β-1,4-glucanase, respectively, exerted an additive effect on strawberry softening, transgenic plants expressing tandem antisense sequences of both genes under the control of the constitutive promoter CaMV35S were generated. Fifteen independent transgenic lines were obtained and fruit yields and several quality parameters of transgenic ripe fruit were recorded during two consecutive years. Fruit yield was reduced in most of the lines, especially in the first evaluation period, and five out of 15 lines (33 %) did not set fruit. The expression of FaplC and FaEG3 genes was measured in ripe fruits from six selected lines showing the highest fruit yields. All selected lines showed a high level of FaplC gene silencing, ranging from 97 to 71 %; however, FaEG3 gene expression was only significantly down-regulated in two lines. Fruit colour and soluble solids contents were similar in control and transgenic ripe fruits, while fruit weight was slightly lower than control in some of the lines. In all lines, transgenic fruits were significantly firmer than control, with an increase in firmness ranging from 19 to 32 %. The reduction of fruit softening in transgenic fruits was not correlated with the suppression of FaEG3 gene expression, and lines with the highest simultaneous down-regulation of FaplC and FaEG3 showed similar fruit firmness to lines where only FaplC was suppressed. These results indicate that pectate lyase and endo-β-1,4-glucanase do not act in an additive or synergistic way during strawberry softening, and question the role of glucanases in this process.     

Bateman’s principle and plant reproduction: The role of pollen limitation in fruit and seed set

Bateman’s principle states that male fitness is usually limited by the number of matings achieved, while female fitness is usually limited by the resources available for reproduction. When applied to flowering plants this principle leads to the expectation that pollen limitation of fruit and seed set will be uncommon. However, if male searching for mates (including pollen dissemination via external agents) is not sufficiently successful, then the reproductive success of both sexes (or both sex functions in hermaphroditic plants) will be limited by number of matings rather than by resources, and Bateman’s principle cannot be expected to apply. Limitation of female success due to inadequate pollen receipt appears to be a common phenomenon in plants. Using published data on 258 species in which fecundity was reported for natural pollination and hand pollination with outcross pollen, I found significant pollen limitation at some times or in some sites in 159 of the 258 species (62%). When experiments were performed multiple times within a growing season, or in multiple sites or years, the statistical significance of pollen limitation commonly varied among times, sites or years, indicating that the pollination environment is not constant. There is some indication that, across species, supplemental pollen leads to increased fruit set more often than increased seed set within fruits, pointing to the importance of gamete packaging strategies in plant reproduction. Species that are highly self-incompatible obtain a greater benefit relative to natural pollination from artificial application of excess outcross pollen than do self-compatible species. This suggests that inadequate pollen receipt is a primary cause of low fecundity rates in perennial plants, which are often self-incompatible. Because flowering plants often allocate considerable resources to pollinator attraction, both export and receipt of pollen could be limited primarily by resource investment in floral advertisement and rewards. But whatever investment is made is attraction, pollinator behavioral stochasticity usually produces wide variation among flowers in reproductive success through both male and female functions. In such circumstances the optimal deployment of resources among megaspores, microspores, and pollinator attraction may often require more flowers or more ovules per flower than will usually be fertilized, in order to benefit from chance fluctuations that bring in large number of pollen grains. Maximizing seed set for the entire plant in a stochastic pollination environment might thus entail a packaging strategy for flower number or ovule number per flower that makes pollen limitation of fruit or seed set likely. Pollen availability may limit female success in individual flowers, entire plants (in a season or over a lifetime), or populations. The appropriate level must be distinguished depending on the nature of the question being addressed.     

The polyphenolic content of fruit and vegetables and their antioxidant activities. What does a serving constitute?

Analysis of the major flavone, flavonol, anthocyanidin and hydroxycinnamic acid constituents (and their glycosides) of onion, tomato, egg plant and apple has been undertaken and the antioxidant activities of the phenolic extracts determined. The major phenolic antioxidant components of egg plant are chlorogenic acid in the flesh and a delphinidin conjugate in the skin. In the case of apple, the major phenolic antioxidants detected are chlorogenic acid, procyanidins/catechin compounds, rutin and phloridzin. Quercetin glycosides are well-known to be the major phenolic components of onion. Assessment of the antioxidant activities of a serving of 100 g fresh weight fruit, vegetable and comparison with previously reported findings for 150 ml beverage (500 ml portion in the case of beer), expressed in μmol Trolox equivalents show that the antioxidant activities of 1 glass (150 ml) red wine ≡ 12 glasses white wine ≡ 2 cups of tea ≡ 4 apples ≡ 5 portions of onion ≡ 5.5 portions egg plant ≡ 3.5 glasses of blackcurrant juice ≡ 3.5 (500 ml) glasses of beer ≡ 7 glasses of orange juice ≡ 20 glasses of apple juice (long life).     

Genotype–environment interactions affect flower and fruit herbivory and plant chemistry of Arabidopsis thaliana in a transplant experiment

Large differences exist in flower and fruit herbivory between dune and inland populations of plants of Arabidopsis thaliana (Brassicaceae). Two specialist weevils Ceutorhynchus atomus and C. contractus (Curculionidae) and their larvae are responsible for this pattern in herbivory. We test, by means of a reciprocal transplant experiment, whether these differences reflect environmental influences or genetic variation in plant defense level. All plants suffered more damage after being transplanted to the dune site than after being transplanted to the inland site. Plants of inland origin suffered more flower and fruit herbivory than plants of dune origin when grown at the dune transplant site, but differences were much smaller at the inland site. Both flower damage by adult weevils and fruit damage by their larvae were subject to significant genotype × environment interactions. The observed pattern in herbivory is a strong indication for local adaption of plant defense to the level of herbivory by Ceutorhynchus. In order to identify the mechanism of defense, a quantitative analysis of glucosinolates was performed on the seeds with HPLC. Highly significant differences were found in glucosinolate types and total concentration. These patterns were mainly determined by the origin of the plants (dune or inland) and by a genotype × environment interaction. Herbivory was not significantly correlated to the concentration of glucosinolates in seeds. We therefore analyzed the total metabolic composition of seeds, using NMR spectroscopy and multivariate data analysis. Major differences in chemical composition were found in the water–methanol fractions: more glucosinolate and sucrose in the dune and more fatty acids, lipids and sinapoylmalate in the inland populations. We discuss which of these chemical factors could explain the marked differences in damage between populations.     

Changes in γ-Methyleneglutamic acid, γ-Methyleneglutamine and other amino acids and amides during fruit growth of Tribulus terrestris

Abstract

Distribution and metabolism of γ-methyleneglutamic acid, γ-methyleneglutamine and other amino acids and amides has been studied during fruit growth of Tribulus terrestris. The largest concentration of free amino acids and amides has been observed in fruit stage 1. The marked decline in the amount of γ-methyleneglutamic acid and γ-methyleneglutamine after fruit stage 1 may indicate their rapid utilization along with asparagine and glutamine during fruit growth. In leaf and in different fruit growth stages, γ-methyleneglutamic acid dominated over γ-methyleneglutamine.     

QTLs mapping for fruit size and shape in chromosomes 2 and 4 in pepper and a comparison of the pepper QTL map with that of tomato

Quantitative trait locus (QTL) mapping for fruit weight and shape in pepper (Capsicum spp.) was performed using C. chinense and C. frutescens introgression lines of chromosomes 2 and 4. In chromosome 2, a single major fruit-weight QTL, fw2.1, was detected in both populations that explained 62% of the trait variation. This QTL, as well as a fruit-shape QTL, fs2.1, which had a more minor effect, were localized to the tomato fruit-shape gene ovate. The cloned tomato fruit-weight QTL, fw2.2, did not play a major role in controlling fruit size variations in pepper. In chromosome 4, two fruit-weight QTLs, fw4.1 and fw4.2, were detected in the same genomic regions in both mapping populations. In addition, a single fruit-shape QTL was detected in each of the mapping populations that co-localized with one of the fruit-weight QTLs, suggesting pleiotropy or close linkage of the genes controlling size and shape. fw2.1 and fw4.2 represent major fruit-weight QTLs that are conserved in the three Capsicum species analyzed to date for fruit-size variations. Co-localization of the pepper QTLs with QTLs identified for similar traits in tomato suggests that the pepper and tomato QTLs are orthologous. Compared to fruit-shape QTLs, fruit-weight QTLs were more often conserved between pepper and tomato. This implies that different modes of selection were employed for these traits during domestication of the two Solanaceae species.S. Zygier and A. Ben Chaim contributed equally to this work.     

Development of internal COI primers to improve and extend barcoding of fruit flies (Diptera: Tephritidae: Dacini)

Accurate species-level identifications underpin many aspects of basic and applied biology;however,identifications can be hampered by a lack of discriminating morphological characters,taxonomic expertise or time.Molecular approaches,such as DNA"barcoding"of the cytochrome c oxidase(COI)gene,are argued to overcome these issues.However,nuclear encoding of mitochondrial genes(numts)and poor amplification success of suboptimally preserved specimens can lead to erroneous identifications.One insect group for which these molecular and morphological problems are significant are the dacine fruit flies(Diptera:Tephritidae:Dacini).We addressed these issues associated with COI barcoding in the dacines by first assessing several"universal"COI primers against public mitochondrial genome and numt sequences for dacine taxa.We then modified a set of four primers that more closely matched true dacine COI sequence and amplified two overlapping portions of the COI barcode region.Our new primers were tested alongside universal primers on a selection of dacine species,including both fresh preserved and decades-old dry specimens.Additionally,Bactrocera tiyoni mitochondrial and nuclear genomes were compared to identify putative numts.Four numt clades were identified,three of which were amplified using existing universal primers.In contrast,our new primers preferentially amplified the"true"mitochondrial COI barcode in all dacine species tested.The new primers also successfully amplified partial barcodes from dry specimens for which full length barcodes were unobtainable.Thus we recommend these new primers be incorporated into the suites of primers used by diagnosticians and quarantine labs for the accurate identification of dacine species.     

Effect of soil cadmium application and pH on growth and cadmium accumulation in roots, leaves and fruit of strawberry plants (Fragaria × ananassa Duch.)

Three strawberry (Fragaria × ananassa Duch.) cultivars Rainier, Totem and Selva were grown under greenhouse conditions in a Parkhill sandy loam soil with a background DTPA-extractable Cd concentration of 0.18 mg kg^-1 and a pH of 5.1. Experimental treatments included combinations of 4 Cd applications (0, 15, 30 and 60 mg Cd kg^-1 soil) applied as CdSO₄ and 2 soil pH values 5.1 and 6.8. Both the application of Cd and pH of the soil significantly affected plant growth, yield and Cd accumulation in plant tissue anf fruit. Although roots accumulated the highest concentrations of Cd of all plant parts investigated, increased soil Cd application reduced leaf weight more than root weight. In general, yield of strawberries was decreased by an increase in amount of soil-applied Cd, however the yield response varied among cultivars. At 60 mg Cd kg^-1 soil, yield of Rainier cultivar was reduced to 17.6% of control plants. Over 90% of total Cd taken up by plants grown in Cd-treated soil accumulated in roots, regardless of the Cd level in the soil. Root Cd concentrations ranged from 2.6 mg kg^-1 (control plants) to 505.7 mg kg^-1 (Totem plants grown in soil at highest Cd and a soil pH 5.1) and were directly related to soil Cd concentrations. Cd translocation from roots to leaves and fruit was very limited, resulting in a maximum Cd concentration in root leaf tissue of 10.2 mg kg^-1. Accumulation of Cd in fruit was found to correlate well with leaf Cd, although even at the highest amount of applied Cd, fruit Cd concentration did not exceed 700 g kg^-1 of fresh weight.Contribution no. 951     

Morphological characteristics of leaf epidermis and size variation of leaf,flower and fruit in different ploidy levels in Buddleja macrostachya （Buddlejaceae）

Buddleja macrostachya （Buddlejaceae） is a widespread shrub native to the Sino-Himalayan mountains and beyond. It has been found to occur at two ploidy levels, hexaploid, 2n=6x=114 and dodecaploid, 2n= 12x=228. To determine if morphological characters might be used as indicators of ploidy levels, we measured floral and fruit length, relative and absolute leaf size, trichome density on both leaf surfaces, and stomatal density and length in different populations orB. macrostachya. In general, flower and fruit length, absolute leaf size, and stomatal length in,eased with an increase at ploidy level （P〈0.01）, whereas adaxial cell and stomatal density decreased with an increase at ploidy level （P〈0.01）. We found no conspicuous differences in relative leaf size （P〉0.05） in different populations. Other characters studied such as trichome type, cuticular membrane and ornamentation of stomata, cell and stomatal shape, and anticlinal wall pattern were quite constant in this species. Thus it appears that flower and fruit length, absolute leaf size, and stomatal frequency and length can be used to distinguish hexaptoid from dodecaploid cytotypes either in the field or in herbarium specimens.