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Background The phytohormone ethylene controls many processes in plant development and acts as a key signaling molecule in response to biotic and abiotic stresses: it is rapidly induced by flooding, wounding, drought, and pathogen attack as well as during abscission and fruit ripening. In kiwifruit (Actinidia spp.), fruit ripening is characterized by two distinct phases: an early phase of system-1 ethylene biosynthesis characterized by absence of autocatalytic ethylene, followed by a late burst of autocatalytic (system-2) ethylene accompanied by aroma production and further ripening. Progress has been made in understanding the transcriptional regulation of kiwifruit fruit ripening but the regulation of system-1 ethylene biosynthesis remains largely unknown. The aim of this work is to better understand the transcriptional regulation of both systems of ethylene biosynthesis in contrasting kiwifruit organs: fruit and leaves. Results A detailed molecular study in kiwifruit (A. chinensis) revealed that ethylene biosynthesis was regulated differently between leaf and fruit after mechanical wounding. In fruit, wound ethylene biosynthesis was accompanied by transcriptional increases in 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS), ACC oxidase (ACO) and members of the NAC class of transcription factors (TFs). However, in kiwifruit leaves, wound-specific transcriptional increases were largely absent, despite a more rapid induction of ethylene production compared to fruit, suggesting that post-transcriptional control mechanisms in kiwifruit leaves are more important. One ACS member, AcACS1, appears to fulfil a dominant double role; controlling both fruit wound (system-1) and autocatalytic ripening (system-2) ethylene biosynthesis. In kiwifruit, transcriptional regulation of both system-1 and -2 ethylene in fruit appears to be controlled by temporal up-regulation of four NAC (NAM, ATAF1/2, CUC2) TFs (AcNAC1–4) that induce AcACS1 expression by directly binding to the AcACS1 promoter as shown using gel-shift (EMSA) and by activation of the AcACS1 promoter in planta as shown by gene activation assays combined with promoter deletion analysis. Conclusions Our results indicate that in kiwifruit the NAC TFs AcNAC2–4 regulate both system-1 and -2 ethylene biosynthesis in fruit during wounding and ripening through control of AcACS1 expression levels but not in leaves where post-transcriptional/translational regulatory mechanisms may prevail.

Despite recent progress in its treatment, Multiple Myeloma (MM) remains incurable and its associated bone disease persists even after complete remission. Thus, identification of new therapeutic agents that simultaneously suppress MM growth and protect bone is an unmet need. Herein, we examined the effects of Aplidin, a novel anti-cancer marine-derived compound, on MM and bone cells. In vitro, Aplidin potently inhibited MM cell growth and induced apoptosis, effects that were enhanced by dexamethasone (Dex) and bortezomib (Btz). Aplidin modestly reduced osteocyte/osteoblast viability and decreased osteoblast mineralization, effects that were enhanced by Dex and partially prevented by Btz. Further, Aplidin markedly decreased osteoclast precursor numbers and differentiation, and reduced mature osteoclast number and resorption activity. Moreover, Aplidin reduced Dex-induced osteoclast differentiation and further decreased osteoclast number when combined with Btz. Lastly, Aplidin alone, or suboptimal doses of Aplidin combined with Dex or Btz, decreased tumor growth and bone resorption in ex vivo bone organ cultures that reproduce the 3D-organization and the cellular diversity of the MM/bone marrow niche. These results demonstrate that Aplidin has potent anti-myeloma and anti-resorptive properties, and enhances proteasome inhibitors blockade of MM growth and bone destruction.

Epidemics of obesity and type 2 diabetes drive strong consumer interest in plant-based low-calorie sweeteners. Trilobatin is a sweetener found at high concentrations in the leaves of a range of crabapple (Malus) species, but not in domesticated apple (Malus × domestica) leaves, which contain trilobatin’s bitter positional isomer phloridzin. Variation in trilobatin content was mapped to the Trilobatin locus on LG 7 in a segregating population developed from a cross between domesticated apples and crabapples. Phloretin glycosyltransferase2 (PGT2) was identified by activity-directed protein purification and differential gene expression analysis in samples high in trilobatin but low in phloridzin. Markers developed for PGT2 cosegregated strictly with the Trilobatin locus. Biochemical analysis showed PGT2 efficiently catalyzed 4′-o-glycosylation of phloretin to trilobatin as well as 3-hydroxyphloretin to sieboldin. Transient expression of double bond reductase, chalcone synthase, and PGT2 genes reconstituted the apple pathway for trilobatin production in Nicotiana benthamiana. Transgenic M. × domestica plants overexpressing PGT2 produced high concentrations of trilobatin in young leaves. Transgenic plants were phenotypically normal, and no differences in disease susceptibility were observed compared to wild-type plants grown under simulated field conditions. Sensory analysis indicated that apple leaf teas from PGT2 transgenics were readily discriminated from control leaf teas and were perceived as significantly sweeter. Identification of PGT2 allows marker-aided selection to be developed to breed apples containing trilobatin, and for high amounts of this natural low-calorie sweetener to be produced via biopharming and metabolic engineering in yeast.

Fruit softening is controlled by hormonal and developmental cues, causing an upregulation of cell wall-associated enzymes that break down the complex sugar matrices in the cell wall. The regulation of this process is complex, with different genotypes demonstrating quite different softening patterns, even when they are closely related. Currently, little is known about the relationship between cell wall structure and the rate of fruit softening. To address this question, the softening of two Actinidia chinensis var. chinensis (kiwifruit) genotypes (a fast 'AC-F' and a slow 'AC-S' softening genotype) was examined using a range of compositional, biochemical, structural, and molecular techniques. Throughout softening, the cell wall structure of the two genotypes was fundamentally different at identical firmness stages. In the hemicellulose domain, xyloglucanase enzyme activity was higher in 'AC-F' at the firm unripe stage, a finding supported by differential expression of xyloglucan transglycosylase/hydrolase genes during softening. In the pectin domain, differences in pectin solubilization and location of methyl-esterified homogalacturonan in the cell wall between 'AC-S' and 'AC-F' were shown. Side chain analyses and molecular weight elution profiles of polyuronides and xyloglucans of cell wall extracts revealed fundamental differences between the genotypes, pointing towards a weakening of the structural integrity of cell walls in the fast softening 'AC-F' genotype even at the firm, unripe stage. As a consequence, the polysaccharides in the cell walls of 'AC-F' may be easier to access and hence more susceptible to enzymatic degradation than in 'AC-S', resulting in faster softening. Together these results suggest that the different rates of softening between 'AC-F' and 'AC-S' are not due to changes in enzyme activities alone, but that fundamental differences in the cell wall structure are likely to influence the rates of softening through differential modification and accessibility of specific cell wall polysaccharides during ripening.

Terpene volatiles are found in many important fruit crops, but their relationship to flavor is poorly understood. Here, we demonstrate using sensory descriptive and discriminant analysis that 1,8-cineole contributes a key floral/eucalyptus note to the aroma of ripe 9Hort16A’ kiwifruit (Actinidia chinensis). Two quantitative trait loci (QTLs) for 1,8-cineole production were identified on linkage groups 27 and 29a in a segregating A. chinensis population, with the QTL on LG29a colocating with a complex cluster of putative terpene synthase (TPS)-encoding genes. Transient expression in Nicotiana benthamiana and analysis of recombinant proteins expressed in Escherichia coli showed four genes in the cluster (AcTPS1a–AcTPS1d) encoded functional TPS enzymes, which produced predominantly sabinene, 1,8-cineole, geraniol, and springene, respectively. The terpene profile produced by AcTPS1b closely resembled the terpenes detected in red-fleshed A. chinensis. AcTPS1b expression correlated with 1,8-cineole content in developing/ripening fruit and also showed a positive correlation with 1,8-cineole content in the mapping population, indicating the basis for segregation is an expression QTL. Transient overexpression of AcTPS1b in Actinidia eriantha fruit confirmed this gene produced 1,8-cineole in Actinidia. Structure-function analysis showed AcTPS1a and AcTPS1b are natural variants at key TPS catalytic site residues previously shown to change enzyme specificity in vitro. Together, our results indicate that AcTPS1b is a key gene for production of the signature flavor terpene 1,8-cineole in ripe kiwifruit. Using a sensory-directed strategy for compound identification provides a rational approach for applying marker-aided selection to improving flavor in kiwifruit as well as other fruits.

Deletion of connexin (Cx) 37 in mice leads to increased cancellous bone mass due to defective osteoclast differentiation. Paradoxically; however, Cx37-deficient mice exhibit reduced cortical thickness accompanied by higher bone strength, suggesting a contribution of Cx37 to bone matrix composition. Thus, we investigated whether global deletion of Cx37 alters the composition of organic bone extracellular matrix. Five-month-old Cx37-/- mice exhibited increased marrow cavity area, and periosteal and endocortical bone surface resulting in higher total area in tibia compared to Cx37+/+ control mice. Deletion of Cx37 increased genes involved in collagen maturation (loxl3 and loxl4) and glycosaminoglycans- (chsy1, chpf and has3) proteoglycans- associated genes (biglycan and decorin). In addition, expression of type II collagen assessed by immunostaining was increased by 82% whereas collagen maturity by picrosirius-polarizarion tended to be reduced (p=0.071). Expression of glycosaminoglycans by histochemistry was decreased, whereas immunostaining revealed that biglycan was unchanged and decorin was slightly increased in Cx37-/- bone sections. Consistent with these in vivo findings, MLO-Y4 osteocytic cells silenced for Cx37 gene exhibited increased mRNA levels for collagen synthesis (col1a1 and col3a1) and collagen maturation (lox, loxl1 and loxl2 genes). Furthermore, mechanistic studies showed Wnt/β-catenin activation in MLO-Y4 osteocytic cells, L5 vertebra, and authentic calvaria-derived osteocytes isolated by fluorescent-activated cell sorter. Our findings demonstrate that altered profile of the bone matrix components in Cx37-deficient mice acts in favor of higher resistance to fracture in long bones.

Apples (Malus spp.) accumulate significant quantities of the dihydrochalcone glycoside, phloridzin, whilst pears (Pyrus spp.) do not. To explain this difference, we hypothesized that a metabolic bottleneck in the phenylpropanoid pathway might exist in apple. Expression analysis indicated that transcript levels of early phenylpropanoid pathway genes in apple and pear leaves were similar, except for chalcone isomerase (CHI), which was much lower in apple. Apples also showed very low CHI activity compared with pear. To relieve the bottleneck at CHI, transgenic apple plants overexpressing the Arabidopsis AtCHI gene were produced. Unlike other transgenic apples where phenylpropanoid flux was manipulated, AtCHI overexpression (CHIox) plants were phenotypically indistinguishable from wild-type, except for an increase in red pigmentation in expanding leaves. CHIox plants accumulated slightly increased levels of flavanols and flavan-3-ols in the leaves, but the major change was a 2.8- to 19-fold drop in phloridzin concentrations compared with wild-type. The impact of these phytochemical changes on insect preference was studied using a two-choice leaf assay with the polyphagous apple pest, the two-spotted spider mite (Tetranychus urticae Koch). Transgenic CHIox leaves were more susceptible to herbivory, an effect that could be reversed (complemented) by application of phloridzin to transgenic leaves. Taken together, these findings shed new light on phenylpropanoid biosynthesis in apple and suggest a new physiological role for phloridzin as an antifeedant in leaves.

In apple (Malus×domestica) fruit, the different layers of the exocarp (cuticle, epidermis, and hypodermis) protect and maintain fruit integrity, and resist the turgor-driven expansion of the underlying thin-walled cortical cells during growth. Using in situ immunolocalization and size exclusion epitope detection chromatography, distinct cell type differences in cell wall composition in the exocarp were revealed during apple fruit development. Epidermal cell walls lacked pectic (1→4)-β-d-galactan (associated with rigidity), whereas linear (1→5)-α-l-arabinan (associated with flexibility) was exclusively present in the epidermal cell walls in expanding fruit and then appeared in all cell types during ripening. Branched (1→5)-α-l-arabinan was uniformly distributed between cell types. Laser capture microdissection and RNA sequencing (RNA-seq) were used to explore transcriptomic differences controlling cell type-specific wall modification. The RNA-seq data indicate that the control of cell wall composition is achieved through cell-specific gene expression of hydrolases. In epidermal cells, this results in the degradation of galactan side chains by possibly five β-galactosidases (BGAL2, BGAL7, BGAL10, BGAL11, and BGAL103) and debranching of arabinans by α-arabinofuranosidases AF1 and AF2. Together, these results demonstrate that flexibility and rigidity of the different cell layers in apple fruit during development and ripening are determined, at least in part, by the control of cell wall pectin remodelling.

Pannexins (Panxs), glycoproteins that oligomerize to form hemichannels on the cell membrane, are topologically similar to connexins, but do not form cell-to-cell gap junction channels. There are 3 members of the family, 1–3, with Panx1 being the most abundant. All Panxs are expressed in bone, but their role in bone cell biology is not completely understood. We now report that osteocytic Panx1 deletion (Panx1Δot) alters bone mass and strength in female mice. Bone mineral density after reaching skeletal maturity is higher in female Panx1Δot mice than in control Panx1fl/fl mice. Further, osteocytic Panx1 deletion partially prevented aging effects on cortical bone structure and mechanical properties. Young 4-month-old female Panx1Δot mice exhibited increased lean body mass, even though pannexin levels in skeletal muscle were not affected; whereas no difference in lean body mass was detected in male mice. Furthermore, female Panx1-deficient mice exhibited increased muscle mass without changes in strength, whereas Panx1Δot males showed unchanged muscle mass and decreased in vivo maximum plantarflexion torque, indicating reduced muscle strength. Our results suggest that osteocytic Panx1 deletion increases bone mass in young and old female mice and muscle mass in young female mice, but has deleterious effects on muscle strength only in males.

Terpenes are important compounds in plant trophic interactions. A meta-analysis of GC-MS data from a diverse range of apple (Malus × domestica) genotypes revealed that apple fruit produces a range of terpene volatiles, with the predominant terpene being the acyclic branched sesquiterpene (E,E)-α-farnesene. Four quantitative trait loci (QTLs) for α-farnesene production in ripe fruit were identified in a segregating 'Royal Gala' (RG) × 'Granny Smith' (GS) population with one major QTL on linkage group 10 co-locating with the MdAFS1 (α-farnesene synthase-1) gene. Three of the four QTLs were derived from the GS parent, which was consistent with GC-MS analysis of headspace and solvent-extracted terpenes showing that cold-treated GS apples produced higher levels of (E,E)-α-farnesene than RG. Transgenic RG fruit downregulated for MdAFS1 expression produced significantly lower levels of (E,E)-α-farnesene. To evaluate the role of (E,E)-α-farnesene in fungal pathogenesis, MdAFS1 RNA interference transgenic fruit and RG controls were inoculated with three important apple post-harvest pathogens [Colletotrichum acutatum, Penicillium expansum and Neofabraea alba (synonym Phlyctema vagabunda)]. From results obtained over four seasons, we demonstrate that reduced (E,E)-α-farnesene is associated with decreased disease initiation rates of all three pathogens. In each case, the infection rate was significantly reduced 7 days post-inoculation, although the size of successful lesions was comparable with infections on control fruit. These results indicate that (E,E)-α-farnesene production is likely to be an important factor involved in fungal pathogenesis in apple fruit.

Phenylpropenes, such as eugenol and trans-anethole, are important aromatic compounds that determine flavour and aroma in many herbs and spices. Some apple varieties produce fruit with a highly desirable spicy/aromatic flavour that has been attributed to the production of estragole, a methylated phenylpropene. To elucidate the molecular basis for estragole production and its contribution to ripe apple flavour and aroma we characterised a segregating population from a Royal Gala (RG, estragole producer) × Granny Smith (GS, non-producer) apple cross. Two quantitative trait loci (QTLs; accounting for 9.2 and 24.8% of the variation) on linkage group (LG) 1 and LG2 were identified that co-located with seven candidate genes for phenylpropene O-methyltransferases (MdoOMT1-7). Of these genes, only expression of MdoOMT1 on LG1 increased strongly with ethylene and could be correlated with increasing estragole production in ripening RG fruit. Transient over-expression in tobacco showed that MdoOMT1 utilised a range of phenylpropene substrates and catalysed the conversion of chavicol to estragole. Royal Gala carried two alleles (MdoOMT1a, MdoOMT1b) whilst GS appeared to be homozygous for MdoOMT1b. MdoOMT1a showed a higher affinity and catalytic efficiency towards chavicol than MdoOMT1b, which could account for the phenotypic variation at the LG1 QTL. Multiple transgenic RG lines with reduced MdoOMT1 expression produced lower levels of methylated phenylpropenes, including estragole and methyleugenol. Differences in fruit aroma could be perceived in these fruit, compared with controls, by sensory analysis. Together these results indicate that MdoOMT1 is required for the production of methylated phenylpropenes in apple and that phenylpropenes including estragole may contribute to ripe apple fruit aroma.

The current study estimates the radiation flux emitted from hot extended gas clouds characteristic of vapour cloud explosions along with the corresponding level of irradiance posed on particles suspended in the unburnt part of the cloud ahead of an advancing flame front. The data presented permits an assessment of the plausibility of combustion initiation by such particles due to forward thermal radiation. The thermal radiation will depend on the emissivity of the burned volume, which relates to the concentration of gaseous and particulate combustion products. A sensitivity analysis has been carried out to account for variations in the equivalence ratio, mixture pressure and radiative heat losses. The spatial distribution of irradiance ahead of the flame front has been computed by introducing appropriate geometrical factors to explore the impact of cloud size. Using fuel rich ethylene-air mixtures it has been shown that high flame emissivities can be achieved at path lengths of order 1 m even in the presence of very low soot volume fractions. The emissivity of gas-soot mixtures will hence be mainly determined by the soot concentration and to a lesser extent by the mixture temperature. Our analysis suggests that the role of forward thermal radiation as a contributing factor to flame propagation in large scale vapour cloud explosions can not currently be ruled out.

Background Unlike in abscission or dehiscence, fruit of kiwifruit Actinidia eriantha develop the ability for peel detachment when they are ripe and soft in the absence of a morphologically identifiable abscission zone. Two closely-related genotypes with contrasting detachment behaviour have been identified. The ‘good-peeling’ genotype has detachment with clean debonding of cells, and a peel tissue that does not tear. The ‘poor-peeling’ genotype has poor detachability, with cells that rupture upon debonding, and peel tissue that fragments easily. Results Structural studies indicated that peel detachability in both genotypes occurred in the outer pericarp beneath the hypodermis. Immunolabelling showed differences in methylesterification of pectin, where the interface of labelling coincided with the location of detachment in the good-peeling genotype, whereas in the poor-peeling genotype, no such interface existed. This zone of difference in methylesterification was enhanced by differential cell wall changes between the peel and outer pericarp tissue. Although both genotypes expressed two polygalacturonase genes, no enzyme activity was detected in the good-peeling genotype, suggesting limited pectin breakdown, keeping cell walls strong without tearing or fragmentation of the peel and flesh upon detachment. Differences in location and amounts of wall-stiffening galactan in the peel of the good-peeling genotype possibly contributed to this phenotype. Hemicellulose-acting transglycosylases were more active in the good-peeling genotype, suggesting an influence on peel flexibility by remodelling their substrates during development of detachability. High xyloglucanase activity in the peel of the good-peeling genotype may contribute by having a strengthening effect on the cellulose-xyloglucan network. Conclusions In fruit of A. eriantha, peel detachability is due to the establishment of a zone of discontinuity created by differential cell wall changes in peel and outer pericarp tissues that lead to changes in mechanical properties of the peel. During ripening, the peel becomes flexible and the cells continue to adhere strongly to each other, preventing breakage, whereas the underlying outer pericarp loses cell wall strength as softening proceeds. Together these results reveal a novel and interesting mechanism for enabling cell separation. Electronic supplementary material The online version of this article (doi:10.1186/s12870-017-1034-2) contains supplementary material, which is available to authorized users.

Summary The ‘fruity’ attributes of ripe apples (Malus × domestica) arise from our perception of a combination of volatile ester compounds. Phenotypic variability in ester production was investigated using a segregating population from a ‘Royal Gala’ (RG; high ester production) × ‘Granny Smith’ (GS; low ester production) cross, as well as in transgenic RG plants in which expression of the alcohol acyl transferase 1 (AAT1) gene was reduced. In the RG × GS population, 46 quantitative trait loci (QTLs) for the production of esters and alcohols were identified on 15 linkage groups (LGs). The major QTL for 35 individual compounds was positioned on LG2 and co-located with AAT1. Multiple AAT1 gene variants were identified in RG and GS, but only two (AAT1-RGa and AAT1-GSa) were functional. AAT1-RGa and AAT1-GSa were both highly expressed in the cortex and skin of ripe fruit, but AAT1 protein was observed mainly in the skin. Transgenic RG specifically reduced in AAT1 expression showed reduced levels of most key esters in ripe fruit. Differences in the ripe fruit aroma could be perceived by sensory analysis. The transgenic lines also showed altered ratios of biosynthetic precursor alcohols and aldehydes, and expression of a number of ester biosynthetic genes increased, presumably in response to the increased substrate pool. These results indicate that the AAT1 locus is critical for the biosynthesis of esters contributing to a ‘ripe apple’ flavour.

Tomato, melon, grape, peach, and strawberry primarily accumulate soluble sugars during fruit development. In contrast, kiwifruit (Actinidia Lindl. spp.) and banana store a large amount of starch that is released as soluble sugars only after the fruit has reached maturity. By integrating metabolites measured by gas chromatography–mass spectrometry, enzyme activities measured by a robot-based platform, and transcript data sets during fruit development of Actinidia deliciosa genotypes contrasting in starch concentration and size, this study identified the metabolic changes occurring during kiwifruit development, including the metabolic hallmarks of starch accumulation and turnover. At cell division, a rise in glucose (Glc) concentration was associated with neutral invertase (NI) activity, and the decline of both Glc and NI activity defined the transition to the cell expansion and starch accumulation phase. The high transcript levels of β -amylase 9 (BAM9) during cell division, prior to net starch accumulation, and the correlation between sucrose phosphate synthase (SPS) activity and sucrose suggest the occurrence of sucrose cycling and starch turnover. ADP-Glc pyrophosphorylase (AGPase) is identified as a key enzyme for starch accumulation in kiwifruit berries, as high-starch genotypes had 2- to 5-fold higher AGPase activity, which was maintained over a longer period of time and was also associated with enhanced and extended transcription of the AGPase large subunit 4 (APL4). The data also revealed that SPS and galactinol might affect kiwifruit starch accumulation, and suggest that phloem unloading into kiwifruit is symplastic. These results are relevant to the genetic improvement of quality traits such as sweetness and sugar/acid balance in a range of fruit species.

Summary Fruit accumulate a diverse set of volatiles including esters and phenylpropenes. Volatile esters are synthesised via fatty acid degradation or from amino acid precursors, with the final step being catalysed by alcohol acyl transferases (AATs). Phenylpropenes are produced as a side branch of the general phenylpropanoid pathway. Major quantitative trait loci (QTLs) on apple (Malus × domestica) linkage group (LG)2 for production of the phenylpropene estragole and volatile esters (including 2-methylbutyl acetate and hexyl acetate) both co-located with the MdAAT1 gene. MdAAT1 has previously been shown to be required for volatile ester production in apple (Plant J., 2014, https://doi.org/10.1111/tpj.12518), and here we show it is also required to produce p-hydroxycinnamyl acetates that serve as substrates for a bifunctional chavicol/eugenol synthase (MdoPhR5) in ripe apple fruit. Fruit from transgenic ‘Royal Gala’ MdAAT1 knockdown lines produced significantly reduced phenylpropene levels, whilst manipulation of the phenylpropanoid pathway using MdCHS (chalcone synthase) knockout and MdMYB10 over-expression lines increased phenylpropene production. Transient expression of MdAAT1, MdoPhR5 and MdoOMT1 (O-methyltransferase) genes reconstituted the apple pathway to estragole production in tobacco. AATs from ripe strawberry (SAAT1) and tomato (SlAAT1) fruit can also utilise p-coumaryl and coniferyl alcohols, indicating that ripening-related AATs are likely to link volatile ester and phenylpropene production in many different fruit.

Summary The polyphenol profile of apple (Malus x domestica) is dominated by the dihydrochalcone glycoside phloridzin, however, its physiological role is yet to be elucidated. Biosynthesis of phloridzin occurs as a side-branch of the main phenylpropanoid pathway with the final step mediated by the phloretin-specific glycosyltransferase UGT88F1. Unexpectedly, given that UGTs are sometimes viewed as ‘decorating enzymes’, UGT88F1 knockdown lines were severely dwarfed with greatly reduced internode lengths, narrow lanceolate leaves, and changes in leaf and fruit cellular morphology. These changes suggested that auxin transport had been altered in the knockdown lines, which was confirmed in assays showing that auxin flux from the shoot apex was increased in the transgenic lines. Metabolite analysis revealed no accumulation of the phloretin aglycone, as well as decreases in many non-target phenylpropanoid compounds. This decreased accumulation of metabolites appeared to be mediated by repression of the phenylpropanoid pathway via reduction in key transcript levels (e.g. phenylalanine ammonia lyase, PAL) and enzyme activities (PAL and chalcone synthase). Application of exogenous phloridzin to the UGT88F1 knockdown lines in tissue culture enhanced axial leaf growth and partially restored some aspects of ‘normal’ apple leaf growth. Together, our results strongly implicate dihydrochalcones as critical compounds in modulating phenylpropanoid pathway flux and establishing auxin patterning early in apple development. This article is protected by copyright. All rights reserved.

Kiwifruit is appreciated for its distinctive flavor . Green-fleshed Actinidia chinensis var. deliciosa kiwifruit have a tangy-sweet flavor with green notes of citrus and melon. Actinidia chinensis var. chinensis fruit have a more tropical flavor, with hints of mango and berry fruit. The flavors of kiwifruit result from a subtle mixture of volatile organic compounds (VOCs) balanced with both good sweetness and acidity. VOCs have been studied extensively in a range of cultivars by analytical means including GC-MS (gas chromatography–mass spectrometry) and GC-olfactometry analysis, and the major compounds and important odorants have been identified. In most kiwifruit species, esters such as methyl and ethyl butanoates are responsible for the fruity notes, while aldehydes such as hexanal and (E)-2-hexenal contribute green/grassy notes. Species-specific compounds such as monoterpenes and strawberry furanone have also been found, as well as certain sulfur compounds that are responsible for tropical notes. Sequestered volatiles may be an important pool of unreleased aroma potential in certain fruit. A detailed account of the genetics, gene regulation, and biosynthesis of these major aroma classes in kiwifruit is presented in the following review.

Terpenes are specialized plant metabolites that act as attractants to pollinators and as defensive compounds against pathogens and herbivores, but they also play an important role in determining the quality of horticultural food products. We show that the genome of cultivated apple (Malus domestica) contains 55 putative terpene synthase (TPS) genes, of which only 10 are predicted to be functional. This low number of predicted functional TPS genes compared with other plant species was supported by the identification of only eight potentially functional TPS enzymes in apple ‘Royal Gala’ expressed sequence tag databases, including the previously characterized apple (E,E)-α-farnesene synthase. In planta functional characterization of these TPS enzymes showed that they could account for the majority of terpene volatiles produced in cv Royal Gala, including the sesquiterpenes germacrene-D and (E)-β-caryophyllene, the monoterpenes linalool and α-pinene, and the homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene. Relative expression analysis of the TPS genes indicated that floral and vegetative tissues were the primary sites of terpene production in cv Royal Gala. However, production of cv Royal Gala floral-specific terpenes and TPS genes was observed in the fruit of some heritage apple cultivars. Our results suggest that the apple TPS gene family has been shaped by a combination of ancestral and more recent genome-wide duplication events. The relatively small number of functional enzymes suggests that the remaining terpenes produced in floral and vegetative and fruit tissues are maintained under a positive selective pressure, while the small number of terpenes found in the fruit of modern cultivars may be related to commercial breeding strategies.

Heteroxylans in the plant cell wall have been proposed to have a role analogous to that of xyloglucans or heteromannans, forming growth-restraining networks by interlocking cellulose microfibrils. A xylan endotransglycosylase has been identified that can transglycosylate heteroxylan polysaccharides in the presence of xylan-derived oligosaccharides. High activity was detected in ripe fruit of papaya (Carica papaya), but activity was also found in a range of other fruits, imbibed seeds and rapidly growing seedlings of cereals. Xylan endotransglycosylase from ripe papaya fruit used a range of heteroxylans, such as wheat arabinoxylan, birchwood glucuronoxylan and various heteroxylans from dicotyledonous primary cell walls purified from tomato and papaya fruit, as donor molecules. As acceptor molecules, the enzyme preferentially used xylopentaitol over xylohexaitol or shorter-length acceptors. Xylan endotransglycosylase was active over a broad pH range and could perform transglycosylation reactions up to 55 °C. Xylan endotransglycosylase activity was purified from ripe papaya fruit by ultrafiltration and cation exchange chromatography. Highest endotransglycosylase activity was identified in fractions that also contained high xylan hydrolase activity and correlated with the presence of the endoxylanase CpaEXY1. Recombinant CpaEXY1 protein transiently over-expressed in Nicotiana benthamiana leaves showed both endoxylanase and xylan endotransglycosylase activities in vitro, suggesting that CpaEXY1 is a single enzyme with dual activity in planta. Purified native CpaEXY1 showed two- to fourfold higher endoxylanase than endotransglycosylase activity, suggesting that CpaEXY1 may act primarily as a hydrolase. We propose that xylan endotransglycosylase activity (like xyloglucan and mannan endotransglycosylase activities) could be involved in remodelling or re-arrangement of heteroxylans of the cellulose-non-cellulosic cell wall framework.

Cell walls of tomato fruit contain hemicellulosic mannans that may fulfill a structural role. Two populations were purified from cell walls of red ripe tomato tissue and named galactoglucomannan-glucuronoxylan I and II (GGM-GX I and II), respectively. Both polysaccharides not only consisted of mannose, glucose and galactose, indicating the presence of GGM, but also contained xylose and glucuronic acid, indicating the presence of GX. Treatment of both polysaccharides with xylanase or endo-β-mannanase showed that the GX and the GGM were associated in a complex. The composition of GGM-GX II changed slightly during tomato ripening, but both GGM-GX I and II showed no change in molecular weight, indicating that they were not hydrolyzed during ripening. Ripe tomato fruit also possess an endo-β-mannanase, an enzyme that in vitro was capable of either hydrolyzing GGM-GX I and II (endo-β-mannanase activity), or transglycosylating them in the presence of mannan oligosaccharides (mannan transglycosylase activity). The lack of evidence for hydrolysis of these potential substrates in vivo suggests either that the enzyme and potential substrates are not accessible to each other for some reason, or that the main activity of endo-β-mannanase is not hydrolysis but transglycosylation, a reaction in which polysaccharide substrates and end-products are indistinguishable. Transglycosylation would remodel rather than weaken the cell wall and allow the fruit epidermis to possibly retain flexibility and plasticity to resist cracking and infection when the fruit is ripe.

Red-fleshed fruit occur in a few taxa of the genus Actinidia. In such taxa, anthocyanins can accumulate in different parts of the fruit including the skin, the whole pericarp or only the inner pericarp. Differences in the relative amounts of cyanidin- and delphinidin-based anthocyanins account for the different shades of red observed. Red-fleshed fruit of A. chinensis accumulate mainly cyanidin 3-Oxylo-galactoside and cyanidin 3-O-galactoside, which usually are restricted to the inner pericarp of the fruit, creating a characteristic and colourful pattern in mature fruit. In order to understand the process of anthocyanin synthesis in Actinidia fruit, we have studied the expression of the anthocyanin biosynthetic genes identified in our Actinidia EST database. Of the different genes of the pathway, only the expression of one glycosyltransferase (F3GT1), confirmed functionally to be cyanidin 3-O-galactosyltransferase, was strongly correlated with red pigmentation of the fruit. However, inheritance of the gene could not be associated with the segregation of the red phenotype of the fruit, suggesting that other factors are able to regulate the biosynthetic pathway. We show that the F3GT1 gene is regulated by a MYB-bHLH mechanism; allelic variation on these genes is likely to account for the expression of the different phenotype.

During climacteric fruit ripening, autocatalytic (Type II) ethylene production initiates a transcriptional cascade that controls the production of many important fruit quality traits including flavour production and softening. The last step in ethylene biosynthesis is the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene by the enzyme ACC oxidase (ACO). Ten independent kiwifruit (Actinidia chinensis) lines were generated targeting suppression of fruit ripening-related ACO genes and the fruit from one of these lines (TK2) did not produce detectable levels of climacteric ethylene. Ripening behaviour in a population of kiwifruit at harvest is asynchronous, so a short burst of exogenous ethylene was used to synchronize ripening in TK2 and control fruit. Following such a treatment, TK2 and control fruit softened to an 'eating-ripe' firmness. Control fruit produced climacteric ethylene and softened beyond eating-ripe by 5 d. In contrast, TK2 fruit maintained an eating-ripe firmness for >25 d and total volatile production was dramatically reduced. Application of continuous exogenous ethylene to the ripening-arrested TK2 fruit re-initiated fruit softening and typical ripe fruit volatiles were detected. A 17 500 gene microarray identified 401 genes that changed after ethylene treatment, including a polygalacturonase and a pectate lyase involved in cell wall breakdown, and a quinone oxidoreductase potentially involved in volatile production. Many of the gene changes were consistent with the softening and flavour changes observed after ethylene treatment. However, a surprisingly large number of genes of unknown function were also observed, which could account for the unique flavour and textural properties of ripe kiwifruit.

The volatile compounds that constitute the fruit aroma of ripe tomato (Solanum lycopersicum) are often sequestered in glycosylated form. A homology-based screen was used to identify the gene SlUGT5, which is a member of UDP-glycosyltransferase 72 family and shows specificity towards a range of substrates, including flavonoid, flavanols, hydroquinone, xenobiotics and chlorinated pollutants. SlUGT5 was shown to be expressed primarily in ripening fruit and flowers, and mapped to chromosome I in a region containing a QTL that affected the content of guaiacol and eugenol in tomato crosses. Recombinant SlUGT5 protein demonstrated significant activity towards guaiacol and eugenol, as well as benzyl alcohol and methyl salicylate; however, the highest in vitro activity and affinity was shown for hydroquinone and salicyl alcohol. NMR analysis identified isosalicin as the only product of salicyl alcohol glycosylation. Protein modelling and substrate docking analysis were used to assess the basis for the substrate specificity of SlUGT5. The analysis correctly predicted the interactions with SlUGT5 substrates, and also indicated that increased hydrogen bonding, due to the presence of a second hydrophilic group in methyl salicylate, guaiacol and hydroquinone, appeared to more favourably anchor these acceptors within the glycosylation site, leading to increased stability, higher activities and higher substrate affinities.

Much of the diversity of anthocyanins is due to the action of glycosyltransferases, which add sugar moieties to anthocyanidins. We identified two glycosyltransferases, F3GT1 and F3GGT1, from red-fleshed kiwifruit (Actinidia chinensis) that perform sequential glycosylation steps. Red-fleshed genotypes of kiwifruit accumulate anthocyanins mainly in the form of cyanidin 3-O-xylo-galactoside. Genes in the anthocyanin and flavonoid biosynthetic pathway were identified and shown to be expressed in fruit tissue. However, only the expression of the glycosyltransferase F3GT1 was correlated with anthocyanin accumulation in red tissues. Recombinant enzyme assays in vitro and in vivo RNA interference (RNAi) demonstrated the role of F3GT1 in the production of cyanidin 3-O-galactoside. F3GGT1 was shown to further glycosylate the sugar moiety of the anthocyanins. This second glycosylation can affect the solubility and stability of the pigments and modify their colour. We show that recombinant F3GGT1 can catalyse the addition of UDP-xylose to cyanidin 3-galactoside. While F3GGT1 is responsible for the end-product of the pathway, F3GT1 is likely to be the key enzyme regulating the accumulation of anthocyanin in red-fleshed kiwifruit varieties.

Fruit softening is associated with cell wall modifications produced by a set of hydrolytic enzymes and proteins. Expansins are proteins with no catalytic activity which have been associated with several processes during plant growth and development. A role for expansins has been proposed during fruit softening, and many fruit-specific expansins have been identified in a variety of species. A fruit-specific expansin gene from mountain papaya (Vasconcellea pubescens) was isolated and characterised: VpEXPA2, a 851 bp cDNA encoding a polypeptide of 258 amino acids, which contained all the characteristics of α-expansins. DNA gel blot analysis showed that VpEXPA2 is member of a small multigene family in the mountain papaya's genome. Real-time polymerase chain reaction (qPCR) analyses revealed transcript accumulation during the softening of control fruit, with maximum level after 5 d of storage at 20 °C. A reduction in transcript expression level was observed in fruit treated with an ethylene perception blocker (1-methylcyclopropene), while an earlier and higher transcript level was observed in ethylene-treated fruit, suggesting that VpEXPA2 expression is regulated by this plant hormone.

Background Mannans are hemicellulosic polysaccharides in the plant primary cell wall with two major physiological roles: as storage polysaccharides that provide energy for the growing seedling; and as structural components of the hemicellulose-cellulose network with a similar function to xyloglucans. Endo-beta-mannanases are hydrolytic enzymes that cleave the mannan backbone. They are active during seed germination and during processes of growth or senescence. The recent discovery that endo-beta-mannanase LeMAN4a from ripe tomato fruit also has mannan transglycosylase activity requires the role of endo-beta-mannanases to be reinterpreted. Aims In this review, the role of endo-beta-mannanases as mannan endotransglycosylase/hydrolases (MTHs) in remodelling the plant cell wall is considered by analogy to the role of xyloglucan endotransglucosylase/hydrolases (XTHs). The current understanding of the reaction mechanism of these enzymes, their three-dimensional protein structure, their substrates and their genes are reported. Future outlook There are likely to be more endohydrolases within the plant cell wall that can carry out hydrolysis and transglycosylation reactions. The challenge will be to demonstrate that the transglycosylation activities shown in vitro also exist in vivo and to validate a role for transglycosylation reactions during the growth and development of the plant cell wall.

Xyloglucan endotransglucosylase/hydrolase (XTH) enzymes are thought to play a key role in fruit ripening by loosening the cell wall in preparation for further modification by other cell wall-associated enzymes and through disassembly of xyloglucan. Twenty-five XTH genes were isolated and characterised from kiwifruit and apple databases containing >270,000 expressed sequence tags (ESTs). The XTH genes (14 from kiwifruit, 11 from apple) encoded putative proteins with similar molecular weights, but with isoelectric points ranging from acidic to basic. All 25 XTH amino acid sequences contained the two conserved glutamic acid residues at the active site of the XTH enzyme. Phylogenetic analysis produced trees with branches each containing kiwifruit, apple, tomato and Arabidopsis XTH sequences, suggesting the potential for functional orthology. EST expression profiling and quantitative PCR analysis were used to identify kiwifruit and apple XTH transcripts expressed in root tip, flower, young leaf and ripe fruit. In ripe apple fruit, the transcripts for two XTH genes (Md-XTH2 and Md-XTH10) were the most abundant, whilst in ripe kiwifruit three transcripts predominated (Ad-XTH4, 5 and 7). Ad-XTH7 was highly expressed in the outer pericarp of firm kiwifruit at commercial harvest and expression of this mRNA decreased during the rapid softening phase. Three kiwifruit XTH genes (Ad-XTH5, Ad-XTH7 and Ac-XTH14) were expressed in Escherichia coli and the recombinant proteins were shown to have xyloglucan endotransglucosylase activity. This research lays the groundwork for understanding the role of XTHs during fruit ripening and storage in kiwifruit and apple.

The dihydrochalcone phlorizin (phloretin 2'-glucoside) contributes to the flavor, color and health benefits of apple fruit and processed products. A genomics approach was used to identify the gene MdPGT1 in apple (Malus x domestica) with homology to the UDP-glycosyltransferase 88 family of uridine diphosphate glycosyltransferases that show specificity towards flavonoid substrates. Expressed sequence tags for MdPGT1 were found in all tissues known to produce phlorizin including leaf, flower and fruit. However, the highest expression was measured by quantitative PCR in apple root tissue. The recombinant MdPGT1 enzyme expressed in Escherichia coli glycosylated phloretin in the presence of [(3)H]-UDP-glucose, but not other apple antioxidants, including quercetin, naringenin and cyanidin. The product of phloretin and UDP-glucose co-migrated with an authentic phlorizin standard. LC/MS indicated that MdPGT1 could glycosylate phloretin in the presence of three sugar donors: UDP-glucose, UDP-xylose and UDP-galactose. This is the first report of functional characterization of a UDP-glycosyltransferase that utilizes a dihydrochalcone as its primary substrate.

Sequence analysis of left and right border integration sites of independent, single-copy T-DNA inserts in Arabidopsis thaliana revealed three previously unrecognized concomitants of T-DNA integration. First, genomic pre-insertion sites shared sequence similarity not only with the T-DNA left and right border regions, as was previously reported, but also at high frequency with the inverted complement of the T-DNA right border region. Second, palindromic sequences were frequently found to overlap or lie adjacent to genomic target sites, suggesting a high recombinogenic potential for palindromic elements during T-DNA integration and a possible role during the primary contact between the T-DNA and the target DNA. Third, "filler" DNA sequences between genomic pre-insertion site DNA and T-DNA often derive from sequences in the T-DNA left and right border regions that are clustered around palindromic sequences in these T-DNA regions, suggesting that these palindromic elements are "hot spots" for filler DNA formation. The discovery of inverted sequence similarities at the right border suggests a previously unrecognized mode of T-DNA integration that involves heteroduplex formation at both T-DNA borders and with opposite strands of the target DNA. Scanning for sequence similarities in both direct and inverted orientation may increase the probability and/or effectiveness of anchoring the T-DNA to the target DNA. Variations on this scheme may also account for inversion events at the target site of T-DNA integration and inverted T-DNA repeat formation, common sequence organization patterns associated with T-DNA integration.

To evaluate, in a randomized, open-label study, the non-inferiority of a bioequivalent fixed-dose combination of glimepiride and atorvastatin vs. separately co-administered tablets in people with Type 2 diabetes mellitus.Participants with HbA1c ≥ 53 to 80 mmol/mol (≥ 7.0 to 9.5%), average fasting blood glucose 7.0 mmol/l, who were on metformin for ≥ 3 months, were randomized to combination (n = 215) or co-administered glimepiride and atorvastatin (n = 212) once daily for 20 weeks. Up-titration of glimepiride (1-4 mg) and atorvastatin (10-20 mg) were based on average fasting blood glucose and LDL cholesterol, respectively. Co-primary endpoints were change from baseline to week 20 in HbA1c and LDL cholesterol.Non-inferiority was demonstrated for both co-primary endpoints: the upper limits of 95% CIs for differences (combination-reference) were less than the prespecified margins of 3.3 mmol/mol (0.3%) for change from baseline in HbA1c [difference 0.1 mmol/mol (95% CI -1.6, 1.9); 0.01% (95% CI -0.15, 0.17)] and 6% for percentage change from baseline in LDL cholesterol [difference 0.87% (95% CI -2.47, 4.21)]. Similar proportions of participants on combination and reference had treatment-emergent adverse events (64 vs. 61%). More participants on combination had hypoglycaemia (21 vs. 13%); most events were considered by the treating physician to be unrelated to study drug.The combination was non-inferior to separately co-administered tablets and the safety profile was consistent with the known profiles of glimepiride and atorvastatin. The observed increase in hypoglycaemia on the combination cannot be explained, but may be attributable to non-systematic collectiof glucose readings and may have been influenced by reporting bias in this open-label trial.

Background Ripening in tomato is predominantly controlled by ethylene, whilst in fruit such as grape, it is predominantly controlled by other hormones. The ripening response of many kiwifruit (Actinidia) species is atypical. The majority of ripening-associated fruit starch hydrolysis, colour change and softening occurs in the apparent absence of ethylene production (Phase 1 ripening) whilst Phase 2 ripening requires autocatalytic ethylene production and is associated with further softening and an increase in aroma volatiles. Results To dissect the ripening response in the yellow-fleshed kiwifruit A. chinensis (‘Hort16A’), a two dimensional developmental stage X ethylene response time study was undertaken. As fruit progressed through maturation and Phase 1 ripening, fruit were treated with different concentrations of propylene and ethylene. At the start of Phase 1 ripening, treated fruit responded to ethylene, and were capable of producing endogenous ethylene. As the fruit progressed through Phase 1 ripening, the fruit became less responsive to ethylene and endogeneous ethylene production was partially repressed. Towards the end of Phase 1 ripening the fruit were again able to produce high levels of ethylene. Progression through Phase 1 ripening coincided with a developmental increase in the expression of the ethylene-unresponsive MADS-box FRUITFUL-like gene (FUL1). The ability to respond to ethylene however coincided with a change in expression of another MADS-box gene SEPALLATA4/RIPENING INHIBITOR-like (SEP4/RIN). The promoter of SEP4/RIN was shown to be transactivated by EIN3-like transcription factors, but unlike tomato, not by SEP4/RIN itself. Transient over-expression of SEP4/RIN in kiwifruit caused an increase in ethylene production. Conclusions These results suggest that the non-ethylene/ethylene ripening response observed in kiwifruit is a hybrid of both the tomato and grape ripening progression, with Phase 1 being akin to the RIN/ethylene inhibitory response observed in grape and Phase 2 akin to the RIN-associated autocatalytic ethylene response observed in tomato. Electronic supplementary material The online version of this article (doi:10.1186/s12870-015-0697-9) contains supplementary material, which is available to authorized users.

Kiwellin is a cysteine-rich, cell wall-associated protein with no known structural homologues. It is one of the most abundant proteins in kiwifruit (Actinidia spp.), and has been shown to be recognised by IgE of some patients allergic to kiwifruit. Cleavage of kiwellin into an N-terminal 4 kDa peptide called kissper and a core domain called KiTH is mediated by actinidin in vitro, and isolation of the kissper peptide from green-fleshed kiwifruit extracts suggested it may result from in vivo processing of kiwellin. In solution, kissper is highly flexible and displays pore-forming activity in synthetic lipid-bilayers. We present here the 2.05 A resolution crystal structure of full-length kiwellin, purified from its native source, Actinidia chinensis (gold-fleshed kiwifruit). The structure confirms the modularity of the protein and the intrinsic flexibility of kissper and reveals that KiTH harbours a double-psi β-barrel fold hooked to an N-terminal β hairpin. Comparisons with structurally-related proteins suggest that a deep gorge located at the protein surface forms a binding site for endogenous ligands.

Glycosides are an important potential source of aroma and flavour compounds for release as volatiles in flowers and fruit. The production of glycosides is catalysed by UDP-glycosyltransferases (UGTs) that mediate the transfer of an activated nucleotide sugar to acceptor aglycones. A screen of UGTs expressed in kiwifruit (Actinidia deliciosa) identified the gene AdGT4 which was highly expressed in floral tissues and whose expression increased during fruit ripening. Recombinant AdGT4 enzyme glycosylated a range of terpenes and primary alcohols found as glycosides in ripe kiwifruit. Two of the enzyme's preferred alcohol aglycones, hexanol and (Z)-hex-3-enol, contribute strongly to the 'grassy-green' aroma notes of ripe kiwifruit and other fruit including tomato and olive. Transient over-expression of AdGT4 in tobacco leaves showed that enzyme was able to glycosylate geraniol and octan-3-ol in planta whilst transient expression of an RNAi construct in Actinidia eriantha fruit reduced accumulation of a range of terpene glycosides. Stable over-expression of AdGT4 in transgenic petunia resulted in increased sequestration of hexanol and other alcohols in the flowers. Transgenic tomato fruit stably over-expressing AdGT4 showed changes in both the sequestration and release of a range of alcohols including 3-methylbutanol, hexanol and geraniol. Sequestration occurred at all stages of fruit ripening. Ripe fruit sequestering high levels of glycosides were identified as having a less intense, earthier aroma in a sensory trial. These results demonstrate the importance of UGTs in sequestering key volatile compounds in planta and suggest a future approach to enhancing aromas and flavours in flowers and during fruit ripening. Yauk YK1, Ged C, Wang MY, Matich AJ, Tessarotto L, Cooney JM, Chervin C, Atkinson RG.

Xyloglucan endotransglycosylase (XET) from the core tissue of ripe kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson var. deliciosa cv. Hayward) was purified 3000-fold to homogeneity. The enzyme has a molecular weight of 34 kDa, is N-glycosylated, and is active between pH 5.0 and 8.0, with an optimum between 5.5 and 5.8. The Km was 0.6 mg.mL-1 for kiwifruit xyloglucan and 100 microM for [3H]XXXG-ol, a reduced heptasaccharide derived from kiwifruit xyloglucan. Kiwifruit core XET was capable of depolymerising xyloglucan in the absence of [3H]XXXG-ol by hydrolysis, and in the presence of [3H]XXXG-ol by hydrolysis and endotransglycosylation. Six cDNA clones (AdXET1-6) with homology to other reported XETs were isolated from ripe kiwifruit mRNA. The six cDNA clones share 93-99% nucleotide identity and appear to belong to a family of closely related genes. Peptide sequencing indicated that ripe kiwifruit XET was encoded by AdXET6. Northern analysis indicated that expression of the AdXET1-6 gene family was induced in ripening kiwifruit when endogenous ethylene production could first be detected, and peaked in climacteric samples when fruit were soft. A full-length cDNA clone (AdXET5) was overexpressed in E. coli to produce a recombinant protein that showed endotransglycosylase activity when refolded.

High fat diets decrease glucose tolerance and increase insulin resistance at peripheral tissues such as adipose and muscle. Fish oils, containing a high concentration of ω3 fatty acids, have been reported to counter the development of insulin resistance in rats in response to high fat diets. Because fish oils contain a number of long chain fatty acids that may be mediating this response, we investigated the specific effects of docosahexaenoic acid (DHA), which comprises approximately 10% of the fatty acids in fish oils, on the hormonal regulation of glucose uptake in isolated adipocytes. Weanling rats were fed diets containing 12% of calories as corn oil (LF-CO), or 26% of calories as safflower oil (ω6 rich, MF-SO) or DHASCO ™ (44.6% DHA, MF-DHA). Feed consumption and growth did not differ between the dietary treatments. After 8 weeks of feeding, fasting serum glucose levels were higher in both the high fat diet groups compared to LF-CO. Basal uptake of 2.5 mM [ 3 H]-2-deoxyglucose (2DG) was reduced in MF-DHA compared to LF-CO. Insulin stimulated 2-DG uptake in all three diet groups. However, despite this stimulus, uptake was lower in MF-SO rats and further reduced in MF-DHA rats. Decreased insulin-stimulated uptake was associated with a reduction in total quantity of GLUT4 in MF-SO rats, but was independent of any change in GLUT4 in MF-DHA fed rats. The β-adrenergic agonist, isoproterenol, decreased 2DG uptake in insulin-stimulated adipocytes by 51% and 36%, respectively, in the LF-CO and MF-SO groups, but had no effect after MF-DHA feeding. This loss of β-adrenergic responsiveness was associated with a decrease in quantity of immunoreactive Gα s protein. These data indicate that long-term feeding of MF-DHA diets impaired basal glucose disposal and disrupted normal hormonal regulation of glucose uptake by the adipocyte.

Polyubiquitin transcripts accumulate in plant and animal cells following a heat shock. Most species have a few to several polyubiquitin genes; within a species, the genes may differ in nucleotide (nt) sequence and (or) the number of 228-nt repeats encoding the ubiquitin monomer. This study examines three maize (inbred Oh43) polyubiquitin genes. Two of the genes, MubG9 and MubG5, possess five repeats; the third, MubG1 possesses seven repeats. Sequence analyses of the genomic clones, MubG9 and MubG1 and a cDNA clone, MubG5, reveal that they differ primarily from each other in their nt sequences 5′ and 3′ to their open reading frames. MubG1 contains a 1004-base pair (bp) intron in its 5′ untranslated region. Using gene-specific probes, we show that the amount of polyribosome-associated mRNA transcripts from MubG9 isolated from 2- and 5-day old plumules and radicles is unchanged by heat shock. While the amount of transcript from MubG1 and MubG5 on the polyribosomes in plumules and radicles of 2-day-old seedlings is also unchanged by heat shock, the levels of these transcripts are elevated considerably in similar tissues from heat-shocked 5-day-old seedlings. Similar or identical gene-specific probes were employed to map the genes using the recombinant inbred method. MubG9 maps to chromosome 4L position 186; MubG1 maps to 5L104 and MubG5 to 4L188. The opportunity to use gene-specific probes extends the evidence for distinct modulation (time and tissue) of polyubiquitin gene expression in maize and provides the basis for locus assignment within the genome.Key words: ubiquitin, maize, heat shock, heat-shock proteins, gene expression, chromosome map.

Bound volatiles are recognised as a potential source of aroma compounds in fruits. In this study, the bound volatiles of Actinidia deliciosa ‘Hayward’ and A. chinensis ‘Hort16A’ were studied at three different ripening stages. The bound volatile content tended to increase as the fruit ripened from under-ripe to ripe, and then decreased in over-ripe fruit. Glycosides of (Z)-3-hexen-1-ol and hexanol (green-note volatiles) were present in considerable amounts. β-Glucosidase activity in ‘Hayward’ and ‘Hort16A’ remained fairly constant throughout ripening. GC-olfactometry analysis of the hydrolysates of ripe ‘Hayward’ and ‘Hort16A’ revealed the presence of 2-phenylethanol, β-damascenone, vanillin and 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF). This is the first report of DMHF in ‘Hayward’ kiwifruit. For both ‘Hayward’ and ‘Hort16A’, the odour-active compounds found in the bound volatile extracts were different from those reported as contributors to the aroma of the ripe fruit, suggesting that bound volatiles are probably not significant contributors to the aroma of ripe kiwifruit.

One of many changes occurring during spontaneous and 3,5,3'-triiodothyronine (T3)-induced metamorphosis of the Rana catesbeiana tadpole is the permanent transition from an ammonotelic, aquatic larva to a ureotelic, terrestrial adult. T3-induced urea production is preceded by T3-induced elevation in the synthesis and level of liver-specific urea cycle enzymes essential for detoxication of ammonia in a terrestrial environment. This report focuses on establishing the effects heat shock (hs) has on the T3-induced expression of genes encoding three essential urea cycle enzymes. We demonstrate that hs stabilizes the intracellular existing levels of carbamyl-phosphate synthetase I (CPS I), the first enzyme in the urea cycle, while concurrently depressing its new synthesis. To establish the effects of hs on CPS I mRNA levels, we characterized cDNAs encoding an amphibian CPS I and demonstrate that it may represent an evolutionary link between microbial CPS and mammalian CPS I. Using this CPS I cDNA and other R. catesbeiana gene-specific probes, we demonstrate that hs depresses the level of T3-induced thyroid hormone receptor beta mRNAs but does not affect the level of T3-induced CPS I, ornithine transcarbamylase, and arginase mRNAs. These results support the contention that the hs response may involve the selective protection of some pre-existing mRNAs and proteins essential for an organism's survival.

In trifluoroacetic acid, in the presence of thiosalicylic acid as a trapping agent, 3-indolyl sulfides bearing a wide variety of substituents are smoothly and rapidly desulfenylated to the corresponding 3-unsubstituted indoles.

Desulfenylation of indol-3-yl sulfides liberates the most reactive position of the ring for further transformations. The usual procedure, utilizing Raney Nickel (P. G. Gassman, et. al., J. Am. Chem. SOC. 1974,96, 5495) offers a limited scope due to incompatibility of a number of functional groups towards the reducing agent. Based on our recent mechanistic studies of the acid-catalysed rearrangement of indol-3-yl sulfides to indol-2-yl sulfides (P. Hamel, et. al., Chem. Commun. 1989, 63; J. Org. Chem. 1992, 57, 2694), we have developed a novel, non-reductive desulfenylation method which permits easy access to 3-unsubstituted indoles bearing a wide array of substituents. Thus, 3-indolyl sulfides, readily obtained from appropriate phenylhydrazines (via Fischer indolization) or anilines (Gassman method, vide infra) are smoothly desulfenylated in good yields in trifluoroacetic acid in the presence of an appropriate nucleophilic trapping agent. Thiols proved to be very effective trapping agents and thi...

Fruit softening in apple (Malus × domestica) is associated with an increase in the ripening hormone ethylene. Here, we show that in cv Royal Gala apples that have the ethylene biosynthetic gene ACC OXIDASE1 suppressed, a cold treatment preconditions the apples to soften independently of added ethylene. When a cold treatment is followed by an ethylene treatment, a more rapid softening occurs than in apples that have not had a cold treatment. Apple fruit softening has been associated with the increase in the expression of cell wall hydrolase genes. One such gene, POLYGALACTURONASE1 (PG1), increases in expression both with ethylene and following a cold treatment. Transcriptional regulation of PG1 through the ethylene pathway is likely to be through an ETHYLENE-INSENSITIVE3-like transcription factor, which increases in expression during apple fruit development and transactivates the PG1 promoter in transient assays in the presence of ethylene. A cold-related gene that resembles a COLD BINDING FACTOR (CBF) class of gene also transactivates the PG1 promoter. The transactivation by the CBF-like gene is greatly enhanced by the addition of exogenous ethylene. These observations give a possible molecular mechanism for the cold- and ethylene-regulated control of fruit softening and suggest that either these two pathways act independently and synergistically with each other or cold enhances the ethylene response such that background levels of ethylene in the ethylene-suppressed apples is sufficient to induce fruit softening in apples.

The acid-catalyzed isomerization of 3-indolyl sulfides 1 to the corresponding 2-indolyl sulfides first synthesis of 3-unsubstituted 2-(arylthio)indoles, a hitherto unattainable class of compounds. When by trifluoroacetic acid, the isomerization proceeds mainly via an intermolecular mechanism involving initial disproportionation to a 2,3-indolyl bis-sulfide 5 and an substituted counterpart 6 followed by further interact of these species to yield the rearranged isomer 4

Kiwifruit vines rely on bees for pollen transfer between spatially separated male and female individuals and require synchronized flowering to ensure pollination. Volatile terpene compounds, which are important cues for insect pollinator attraction, were studied by dynamic headspace sampling in the major green-fleshed kiwifruit (Actinidia deliciosa) cultivar 'Hayward' and its male pollinator 'Chieftain'. Terpene volatile levels showed a profile dominated by the sesquiterpenes alpha-farnesene and germacrene D. These two compounds were emitted by all floral tissues and could be observed throughout the day, with lower levels at night. The monoterpene (E)-beta-ocimene was also detected in flowers but was emitted predominantly during the day and only from petal tissue. Using a functional genomics approach, two terpene synthase (TPS) genes were isolated from a 'Hayward' petal EST library. Bacterial expression and transient in planta data combined with analysis by enantioselective gas chromatography revealed that one TPS produced primarily (E,E)-alpha-farnesene and small amounts of (E)-beta-ocimene, whereas the second TPS produced primarily (+)-germacrene D. Subcellular localization using GFP fusions showed that both enzymes were localized in the cytoplasm, the site for sesquiterpene production. Real-time PCR analysis revealed that both TPS genes were expressed in the same tissues and at the same times as the corresponding floral volatiles. The results indicate that two genes can account for the major floral sesquiterpene volatiles observed in both male and female A. deliciosa flowers.

In order to investigate the selective breakdown of the blood-retinal barrier in posterior uveitis angiograms were performed using fluorescein-conjugated dextrans (FITC-dextrans) of different molecular weights (150-kDa, 20-kDa, and 4-kDa) in two healthy controls and six patients with posterior uveitis, and the results compared with those of conventional fluorescein angiography. The smallest FITC-dextran could not penetrate the healthy blood-retinal barrier. Leakage of FITC-dextrans of all sizes was seen from swollen optic discs; dextrans of 4-kDa and 20-kDa leaked from areas of macula oedema; and retinal new vessels allowed the passage of 4-kDa molecules only. These results indicate that there is a differential breakdown of the blood-retinal barrier according to molecular size in various clinical situations associated with posterior uveitis.

BACKGROUND: Actinidin has previously been reported as the major allergen in kiwifruit. Objectives To investigate the relevance of actinidin in a well-characterized population of UK patients with kiwifruit allergy. METHODS: To identify the allergens in kiwifruit, using Western blots, we examined the IgE-binding patterns of 76 patients with a history of kiwifruit allergy, 23 of who had had a positive double-blind, placebo-controlled food challenge. In addition, IgE binding to purified native actinidin was studied in 30 patients, and to acidic and basic isoforms of recombinant actinidin in five patients. Inhibition of IgE binding to kiwifruit protein extract by purified native actinidin was investigated by both inhibition immunoblots and inhibition ELISAs using pooled sera. RESULTS: Twelve protein bands in kiwifruit protein extract were bound by IgE. A protein band with a molecular weight of 38 kDa was the major allergen recognized by 59% of the population. IgE did not bind to actinidin in the kiwifruit protein extract, or to purified native or recombinant forms of actinidin during Western blotting. Pooled sera bound to kiwifruit protein extract but not purified actinidin on ELISA, and pre-incubating sera with actinidin did not inhibit IgE binding to kiwifruit protein extract on immunoblot or ELISA. CONCLUSION: A novel 38 kDa protein, not actinidin, is the major allergen in this large study population. Identification of major allergens in one patient group is therefore not necessarily reproducible in another; therefore, major allergens should not be defined until there is a sufficient body of data from diverse geographical and cultural populations.