Role of phosphoenolpyruvate carboxylase in organic acid accumulation during peach fruit development

The synthesis of organic acids was studied during fruit development of two peach ( Prunus persica L. Batsch) cultivars, Fantasia and Jalousia, having fruits with high and low organic acid content, respectively. The malate content was higher in cv. Fantasia than in cv. Jalousia at the end of the first rapid growth stage (50 days after bloom [DAB]). Malate and citrate contents were higher in Fantasia than in Jalousia during the second rapid growth stage (from 100 DAB to maturity). The expression of phospho enol pyruvate carboxylase (PEPC, EC 4.1.1.31), which is involved in organic acid synthesis, was studied during peach fruit development. PEPC mRNA levels, and protein levels on a total soluble protein basis, peaked at 23 and 108 DAB in Fantasia. In Jalousia, they were very low at 23 DAB and reached levels similar to Fantasia at 108 DAB. For both cultivars, in vitro PEPC activity expressed on a dry weight basis was maximal at 24 DAB, decreased from 24 to 60 DAB, and then remained constant. The activity of peach fruit PEPC appeared extremely sensitive to malate (I_0.5 of 100 μ M for Fantasia and 65 μ M for Jalousia at pH 7.3) and low pH. PEPC may participate in the control of organic acid accumulation during fruit development in the normal-acid fruit of Fantasia. However, mechanisms other than organic acid synthesis might account for the differences in acidity between normal-acid and non-acid peach fruit.     

Sucrose accumulation in developing peach fruit

Uptake of ¹⁴C-sugars and activities of sucrose metabolizing enzymes were determined in order to study the mechanism(s) of sucrose accumulation in developing peach fruit. Mesocarp of young peach fruit contained glucose and fructose but little sucrose. Starting 88 days after anthesis (DAA) the sucrose concentration increased greatly. The mechanism of sucrose accumulation was studied by measuring ¹⁴C-sucrose and ¹⁴C-glucose uptake rates at three different stages of fruit development, and by assaying weekly the activity of enzymes involved in the hydrolysis and/or synthesis of the soluble sugars. Uptake of 0.5–100 m M ¹⁴C-sucrose and ¹⁴C-glucose by mesocarp tissue slices showed a complex pattern at the first stage of fruit development (62 DAA). During the subsequent growth stages the pattern of sugar uptake changed and was approximately monophasic at the third stage of fruit development.
At 10 m M , glucose was taken up more rapidly than sucrose at the first and second stage of fruit development. Uptake was partially inhibited by the uncoupler carbonylcyanide m -chlorophenylhydrazone (CCCP) at 25 μ M. These results, together with the presence of a putative extracellular invertase, suggest an apoplastic route for sucrose uptake which is dependent, at least in part, on energy supply.
Activities of sucrose hydrolyzing enzymes (insoluble acid invertase, soluble acid invertase, neutral invertase, sucrose synthase) were high in young fruits and declined sharply with fruit development concomitantly with accumulation of sucrose. The storage of the sugar was not accompanied by a rise in synthetic activities (sucrose synthase, sucrose phosphate synthase), suggesting that sucrose could, at least in part enter the carbohydrate pool directly.     

Cytokinins in peach: Endogenous levels during early fruit development

Endogenous cytokinins have been studied in peach (Prunus persica [L.] Batsch). By means of coupled gas chromatography and mass spectrometry methods and using isotope-labelled standards, eleven cytokinin metabolites were quantified and endogenous levels followed during early fruit development. Analysis of cytokinins was performed on a separate basis for seed and pericarp, and correlated with growth and developmental events of fruit. Results show the prevalence of isopentenyladenine nucleotide, dihydrozeatin riboside and dihydrozeatin nucleotide, during the setting and exponential growth of fruit. Exceptionally high levels of zeatin riboside and dihydrozeatin nucleotide are also found in the seed, correlating with endosperm development and embryo growth during early fruit development. The putative role of these metabolites is discussed.     

Proteomic analysis of peach endocarp and mesocarp during early fruit development

The development of the stone and formation of peach (Prunus persica) fruit were explored in this work using a proteomic approach. Sixty-eight proteins with different expression patterns were identified in both the endocarp and mesocarp during early fruit development (from 28 to 59 days after flowering) and the majority were involved in primary or secondary metabolism. In contrast to most proteins associated with primary metabolism in the endocarp, whose expression is down-regulated, expression of pyruvate dehydrogenase (PDH) unexpectedly increased exponentially. Moreover, its expression pattern was linearly positively correlated with the exponentially growing lignin content (R = 0.940), which suggests that PDH may play a role in endocarp lignification. Our data also revealed different spatiotemporal expressions of enzymes involved in the lignin and flavonoid pathways that provided proteome-level evidence to support the hypothesis that these two pathways are competitive during endocarp development. In addition, we observed endocarp-specific oxidative stress and propose that it may act as a stimulating factor in activating lignification and subsequent programmed cell death in the endocarp.     

Analysis of genotypic variation in fruit flesh total sugar content via an ecophysiological model applied to peach

A simulation model of the evolution of total sugar content (C_TS) in fruit was developed in order to describe the within- and between-genotype variation of C_TS observed in a peach (Prunus persica (L.) Batsch) breeding population. The parameter k defines the ratio of carbon used for synthesizing compounds other than sugars for each genotype. Model input variables are dry flesh growth rate and fresh flesh mass of fruit. We estimated k for 137 peach and nectarine genotypes derived from a clone of a wild peach (Prunus davidiana) by three generations of crosses with commercial nectarine varieties. We tested the predictive quality of the model on independent datasets. Despite an underestimation of the observed C_TS, the correlation between observations and predictions was suitable (0.72). Spearman correlation coefficients between 2001 and 2002 for model input variables and parameter k were higher than for C_TS. None of the three components assimilation supply to the fruit, metabolism, or dilution, seemed to have a greater relative effect on C_TS variation than the others. Indeed, C_TS variation seemed to result from the balance between the three components. The interest of this approach, which consists of dissecting traits into components via an ecophysiological model, for breeding strategy and for sugar accumulation studies are discussed.Communicated by H.C. Becker     

Analysing the genetic control of peach fruit quality through an ecophysiological model combined with a QTL approach

Ecophysiological models are increasingly expected to include genetic information via genotype-dependent parameters. These parameters could be considered as quantitative traits and submitted to analysis. A pre-existing ecophysiological model of fruit quality was used and the distribution of the genotypic parameters in a second backcross population derived from a clone of a wild peach (Prunus davidiana) and commercial nectarine varieties (P. persica (L.) Batsch) was analysed. The correlations between the two years of experimentation were higher for the genotypic parameters than for the quality traits commonly studied by breeders. The correlations between the genotypic parameters and the quality traits were low. Quantitative trait loci (QTLs) for the genotypic key parameters of the ecophysiological model were detected by linear regression. Co-locations of QTLs for parameters were observed as well as co-locations of QTLs for parameters and quality traits. The ecophysiological model and the results of the QTL analysis were combined by substituting each parameter in the model by the sum of QTL effects. This combined model can simulate the behaviour of genotypes carrying diverse combinations of alleles. The quality of this combined model was moderately suitable, but had some shortcomings. Improvements are suggested and further use of this combined model as a tool for breeders is discussed.     

Biochemical mechanism on GABA accumulation during fruit development in tomato

A large amount of gamma-aminobutyric acid (GABA) was found to accumulate in tomato (Solanum lycopersicum) fruits before the breaker stage. Shortly thereafter, GABA was rapidly catabolized after the breaker stage. We screened the GABA-rich tomato cultivar 'DG03-9' which did not show rapid GABA catabolism after the breaker stage. Although GABA hyperaccumulation and rapid catabolism in fruits is well known, the mechanisms are not clearly understood. In order to clarify these mechanisms, we performed comparative studies of 'Micro-Tom' and 'DG03-9' fruits for the analysis of gene expression levels, protein levels and enzymatic activity levels of GABA biosynthesis- and catabolism-related enzymes. During GABA accumulation, we found positive correlations among GABA contents and expression levels of SlGAD2 and SlGAD3. Both of these genes encode glutamate decarboxylase (GAD) which is a key enzyme of GABA biosynthesis. During GABA catabolism, we found a strong correlation between GABA contents and enzyme activity of alpha-ketoglutarate-dependent GABA transaminase (GABA-TK). The contents of glutamate and aspartate, which are synthesized from GABA and glutamate, respectively, increased with elevation of GABA-TK enzymatic activity. GABA-TK is the major GABA transaminase form in animals and appears to be a minor form in plants. In 'DG03-9' fruits, GAD enzymatic activity was prolonged until the ripening stage, and GABA-TK activity was significantly low. Taken together, our results suggest that GAD and GABA-TK play crucial roles in GABA accumulation and catabolism, respectively, in tomato fruits.     

Indole-3-acetic acid concentration and ethylene evolution during early fruit development in peach

Ethylene evolution was measured from greenhouse-grown Jerseyglo peach fruits beginning 29 days after anthesis. Indole-3-acetic acid (IAA) levels were measured in the pericarp and seed tissues of individual fruits on a single shoot when variable ethylene evolution was noted. Despite hand-pollinating all flowers on the same day, variability within the shoot existed in fruit fresh weight, IAA levels, and ethylene evolution. Seed IAA concentration increased as fruit and seed fresh weight increased and ranged from 106 to 1572 ng. g^–1. As pericarp fresh weight increased, IAA levels in this tissue decreased. Ethylene evolution rates ranged from 0.21 to 1.07 nl. g.^–1 h^–1 and were not correlated with IAA concentration in seed, pericarp, or the whole fruit. High rates of ethylene evolution from the whole fruit occurred prior to increased IAA concentration in the seed.Fruits were excised from field-grown Redskin peach trees beginning 40 days after full bloom. Fruits from field sampled shoots appeared to be more physiologically advanced than the greenhouse-grown Jerseyglo fruits. Pericarp IAA concentration was low, ranging from 2.8 to 6.5 ng. g^–1. Seed concentrations accounted for 75% of the IAA found in the fruit and ranged from 239 to 1042 ng. g^–1. As with greenhouse-grown samples, whole fruit IAA concentration tended to decrease as fruits increased in fresh weight.     

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

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

A non-targeted approach unravels the volatile network in peach fruit

Volatile compounds represent an important part of the plant metabolome and are of particular agronomic and biological interest due to their contribution to fruit aroma and flavor and therefore to fruit quality. By using a non-targeted approach based on HS-SPME-GC-MS, the volatile-compound complement of peach fruit was described. A total of 110 volatile compounds (including alcohols, ketones, aldehydes, esters, lactones, carboxylic acids, phenolics and terpenoids) were identified and quantified in peach fruit samples from different genetic backgrounds, locations, maturity stages and physiological responses. By using a combination of hierarchical cluster analysis and metabolomic correlation network analysis we found that previously known peach fruit volatiles are clustered according to their chemical nature or known biosynthetic pathways. Moreover, novel volatiles that had not yet been described in peach were identified and assigned to co-regulated groups. In addition, our analyses showed that most of the co-regulated groups showed good intergroup correlations that are therefore consistent with the existence of a higher level of regulation orchestrating volatile production under different conditions and/or developmental stages. In addition, this volatile network of interactions provides the ground information for future biochemical studies as well as a useful route map for breeding or biotechnological purposes.     

New insights into the properties of pubescent surfaces: peach fruit as a model

The surface of peach (Prunus persica 'Calrico') is covered by a dense indumentum, which may serve various protective purposes. With the aim of relating structure to function, the chemical composition, morphology, and hydrophobicity of the peach skin was assessed as a model for a pubescent plant surface. Distinct physicochemical features were observed for trichomes versus isolated cuticles. Peach cuticles were composed of 53% cutan, 27% waxes, 23% cutin, and 1% hydroxycinnamic acid derivatives (mainly ferulic and p-coumaric acids). Trichomes were covered by a thin cuticular layer containing 15% waxes and 19% cutin and were filled by polysaccharide material (63%) containing hydroxycinnamic acid derivatives and flavonoids. The surface free energy, polarity, and work of adhesion of intact and shaved peach surfaces were calculated from contact angle measurements of water, glycerol, and diiodomethane. The removal of the trichomes from the surface increased polarity from 3.8% (intact surface) to 23.6% and decreased the total surface free energy chiefly due to a decrease on its nonpolar component. The extraction of waxes and the removal of trichomes led to higher fruit dehydration rates. However, trichomes were found to have a higher water sorption capacity as compared with isolated cuticles. The results show that the peach surface is composed of two different materials that establish a polarity gradient: the trichome network, which has a higher surface free energy and a higher dispersive component, and the cuticle underneath, which has a lower surface free energy and higher surface polarity. The significance of the data concerning water-plant surface interactions is discussed within a physiological context.     

Phenylalanine biosynthesis and its relationship to accumulation of capsaicinoids during Capsicum chinense fruit development

Activities of phenylalanine (Phe) biosynthetic enzymes chorismate mutase (CM) and arogenate dehydratase (ADT) and of phenylalanine ammonia lyase [PAL, an enzyme that directs Phe towards capsaicinoid (CAP) synthesis] were analyzed during Capsicum chinense Jacq. (habanero pepper) fruit development. A maximum CM activity coincided with a maximum CAP accumulation. However, ADT exhibited two activity peaks, one during the early phase (10 - 17 days post-anthesis, DPA) and another during the late phase (35 - 37 DPA); only the latter coincided with CAP. Interestingly, PAL activity was inversely related to CAP accumulation; lower activities coincided with a maximum CAP content. These results suggest the operation of a control mechanism that coordinated Phe synthesis and its channeling towards CAP synthesis during the course of fruit development.