Specific Changes of Exocarp and Mesocarp Occurring during Softening Differently Affect Firmness in Melting (MF) and Non Melting Flesh (NMF) Fruits

Melting (MF) and non melting flesh (NMF) peaches differ in their final texture and firmness. Their specific characteristics are achieved by softening process and directly dictate fruit shelf life and quality. Softening is influenced by various mechanisms including cell wall reorganization and water loss. In this work, the biomechanical properties of MF Spring Crest’s and NMF Oro A’s exocarp and mesocarp along with the amount and localization of hydroxycinnamic acids and flavonoids were investigated during fruit ripening and post-harvest. The objective was to better understand the role played by water loss and cell wall reorganization in peach softening. Results showed that in ripe Spring Crest, where both cell turgor loss and cell wall dismantling occurred, mesocarp had a little role in the fruit reaction to compression and probe penetration response was almost exclusively ascribed to the epidermis which functioned as a mechanical support to the pulp. In ripe Oro A’s fruit, where cell wall disassembly did not occur and the loss of cell turgor was observed only in mesocarp, the contribution of exocarp to fruit firmness was consistent but relatively lower than that of mesocarp, suggesting that in addition to cell turgor, the integrity of cell wall played a key role in maintaining NMF fruit firmness. The analysis of phenols suggested that permeability and firmness of epidermis were associated with the presence of flavonoids and hydroxycinnamic acids.     

A comparative study of melting and non-melting flesh peach cultivars reveals that during fruit ripening endo-polygalacturonase (endo-PG) is mainly involved in pericarp textural changes, not in firmness reduction

Peach softening is usually attributed to the dismantling of the cell wall in which endo-polygalacturonase (endo-PG)-catalysed depolymerization of pectins plays a central role. In this study, the hypothesis that the function of endo-PG is critical for achieving a melting flesh fruit texture but not for reducing fruit firmness was tested by comparing pericarp morphology and endo-PG expression and localization in melting (MF) and non-melting flesh (NMF) fruit at successive stages of ripening. MF Bolero, Springbelle, and Springcrest, and NMF Oro-A and Jonia cultivars were analysed. Both MF and NMF fruit were left to ripen on the tree and reached a firmness of <10 Newtons (N). The image analysis of pericarp tissues revealed that during softening the loss of cell turgidity was a process common to mesocarp cells of all MF and NMF fruit and was clearly visible in peaches with a firmness of less than ～20?N. In contrast, the loss of cell adhesion was a feature exclusively observed in ripe MF fruit pericarp. In this ripe fruit, large numbers of endo-PG isoforms were highly expressed and the enzyme localization corresponded to the middle lamella. As a consequence, wide apoplastic spaces characterized the pericarp of ripe MF peaches. In contrast, no loss of cell adhesion was observed in any NMF fruit or in unripe MF peaches. Accordingly, no endo-PG was detected in unripe NMF fruit, whereas few and poorly expressed enzyme isoforms were revealed in ripe NMF and in unripe MF peaches. In this fruit, the poorly expressed endo-PG localized mainly in vesicles within the cytoplasm and inner primary cell wall. On the whole the results suggested that endo-PG function was needed to achieve melting flesh texture, which was characterized by wide apoplastic spaces and partially deflated mesocarp cells. Conversely, endo-PG activity had no critical influence on the reduction of fruit firmness given the capacity of NMF peaches to soften, reaching values of 5-10?N. As in tomato, the change of symplast/apoplast water status seems to be the main process through which peach fruit regulates its firmness.     

Effect of post-harvest heat treatment on proteome change of peach fruit during ripening

The extracted proteins from the heat-treated peach fruit (dipped in hot water at 48°C for 10min and then stored at room temperature (20°C-25°C) for up to 6 days) were used for proteomic analysis in order to understand the response of post-harvest peach fruit to heat treatment during ripening stage at proteomic level. After two dimensional gels electrophoresis (2-DE) was conducted, more than 600 protein spots were detected. Among them, 35 differently expressed spots (P<0.05) were selected to be excised and analyzed using MALDI-TOF/TOF, and finally 30 protein spots were confidently identified according to NCBI database. The results demonstrated that among the thirty protein spots expressed particularly induced by heat treatment, 43% were related to stress response, 17% to cell structure, 13% to protein fate, 7% to glycolytic pathway, 3% to ripening and senescence and 17% to unclassified. All of them are involved in the regulation of peach fruit development and ripening. All these indicated that the self-defense capability of peach fruit was improved by heat treatment. The study will enable future detailed investigation of gene expression and function linked with peach fruit ripening.     

Assessment of Prunus persica fruit softening using a proteomics approach

Fruit ripening in Prunus persica involves a number of physiological changes, being one of the most significant the mesocarp softening in melting varieties. In order to get a better understanding of the molecular processes involved in this phenomenon, the protein accumulation patterns in firm and soft fruit of three peach and two nectarine melting flesh varieties were assessed using 2D gel analysis. A General Linear Model (GLM) two-way analysis of variance determined that 164 of the 621 protein spots analyzed displayed a differential accumulation associated with the softening process. Among them, only 14 proteins changed their accumulation in all the varieties assessed, including proteins mostly involved in carbohydrates and cell wall metabolism as well as fruit senescence. The analysis among varieties showed that 195 and 189 spots changed within the firm and soft fruit conditions, respectively. Despite the changes in relative abundance in the spot proteins, the proteome is conserved among varieties and during the transition from firm to soft fruit. Only two spots proteins exhibited a qualitative change in all the conditions assessed. These results are in agreement with the notion that Prunus persica commercial varieties have a narrow genetic background.     

The involvement of auxin in the ripening of climacteric fruits comes of age: the hormone plays a role of its own and has an intense interplay with ethylene in ripening peaches

Ethylene has long been regarded as the main regulator of ripening in climacteric fruits. The characterization of a few tomato mutants, unable to produce climacteric ethylene and to ripen their fruits even following treatments with exogenous ethylene, has shown that other factors also play an important role in the control of climacteric fruit ripening. In climacteric peach and tomato fruits it has been shown that, concomitant with ethylene production, increases in the amount of auxin can also be measured. In this work a genomic approach has been used in order to understand if such an auxin increase is functional to an independent role played by the hormone during ripening of the climacteric peach fruits. Besides the already known indirect activity on ripening due to its up-regulation of climacteric ethylene synthesis, it has been possible to show that auxin plays a role of its own during ripening of peaches. In fact, the hormone has shown the ability to regulate the expression of a number of different genes. Moreover, many genes involved in biosynthesis and transport and, in particular, the signalling (receptors, Auxin Response Factors and Aux/IAA) of auxin had increased expression in the mesocarp during ripening, thus strengthening the idea that this hormone is actively involved in the ripening of peaches. This study has also demonstrated the existence of an important cross-talk between auxin and ethylene, with genes in the auxin domain regulated by ethylene and genes in the ethylene domain regulated by auxin.     

Biochemical characterisation of MdCXE1, a carboxylesterase from apple that is expressed during fruit ripening

Esters are an important component of apple (Malus × domestica) flavour. Their biosynthesis increases in response to the ripening hormone ethylene, but their metabolism by carboxylesterases (CXEs) is poorly understood. We have identified 16 members of the CXE multigene family from the commercial apple cultivar, ‘Royal Gala’, that contain all the conserved features associated with CXE members of the α/β hydrolase fold superfamily. The expression of two genes, MdCXE1 and MdCXE16 was characterised in an apple fruit development series and in a transgenic line of ‘Royal Gala’ (AO3) that is unable to synthesise ethylene in fruit. In wild-type MdCXE1 is expressed at low levels during early stages of fruit development, rising to a peak of expression in apple fruit at harvest maturity. It is not significantly up-regulated by ethylene in the skin of AO3 fruit. MdCXE16 is expressed constitutively in wild-type throughout fruit development, and is up-regulated by ethylene in skin of AO3 fruit. Semi-purified recombinant MdCXE1 was able to hydrolyse a range of 4-methyl umbelliferyl ester substrates that included those containing acyl moieties that are found in esters produced by apple fruit. Kinetic characterisation of MdCXE1 revealed that the enzyme could be inhibited by organophosphates and that its ability to hydrolyse esters showed increasing affinity (K_m) but decreasing turnover (k_cat) as substrate acyl carbon length increases from C2 to C16. Our results suggest that MdCXE1 may have an impact on apple flavour through its ability to hydrolyse relevant flavour esters in ripe apple fruit.     

Genome-wide identification and expression analysis of the lipoxygenase gene family during peach fruit ripening under different postharvest treatments

In plants, lipoxygenase (LOX), facilitated by the LOX family genes is closely related to fruit ripening and senescence, but research on LOX in peach fruit is limited. To study the roles of LOX family genes in fruit ripening during storage, a comprehensive overview of the LOX gene family in peach is presented, including their phylogenetic relationships, gene structures and subcellular localizations. Additionally, the fruit quality, including fruit firmness, ethylene production and soluble solids content under different storage conditions, were assessed. Finally, 12 peach genes that encode LOX proteins have been identified, and comparisons of the PpaLOX gene expression levels under different postharvest treatments in peach fruit suggest that PpaLOX2.1, PpaLOX7.1, PpaLOX7.2, and especially PpaLOX2.2, may be required in peach fruit ripening during storage. The results will be useful to further analyze the functions of the LOX family of genes in plants.     

Polygalacturonase activity and variation in ripening of papaya fruit with tissue depth and heat treatment

Effects of tissue position (viz. outer vs inner mesocarp) and heat treatment (48°C, 20 min) on variations in polygalacturonase (EC 3.2.1.15 and EC 3.2.1.67) activity and ripening of fruits of Carica papaya L. cv. Backcross Solo were investigated. Polygalacturonase activity increased during ripening concomitantly with an increase in tissue softness and soluble polyuronide level. Throughout ripening, inner mesocarp tissue was softer and contained higher polygalacturonase activity than outer mesocarp tissue. Titratable acidity as well as ß-galactosidase (EC 3.2.1.23) activity also increased during ripening; however, unlike polygalacturonase, their level or activity was lower in inner than in outer mesocarp. Ascorbic acid could partially account for the increase in titratable acidity during ripening but contributed very little to the differences in titratable acid levels between outer and inner mesocarp. Heat treatment had no effect on either fruit softness or titratable acidity, but it markedly reduced the increase in ascorbic acid and polygalacturonase activity during ripening. Ripening, as reflected by changes in tissue softness and polygalacturonase activity, progressed outwardly from the interior towards the exterior of the fruit. The effect of heat treatment in suppressing polygalacturonase activity was relatively greater in inner than in outer mesocarp, suggesting that sensitivity of the enzyme to heat treatment may vary with stage of ripeness of the tissue.     

Genetic variation and drought response in two Populus × euramericana genotypes through 2-DE proteomic analysis of leaves from field and glasshouse cultivated plants

Genotype and water deficit effects on leaf 2-DE protein profiles of two Populus deltoides × Populus nigra, cv. ‘Agathe_F’ and ‘Cima’, were analysed over a short-term period of 18 days in glasshouse using 4-month-old rooted cuttings and over a long-lasting period of 86 days in open field using 4-year-old rooted cuttings. Leaf proteomes were analyzed using two-dimensional gel electrophoresis, and proteins were identified after database searching from MS peptide spectra.A reliable genotype effect was observed in the leaf proteome over experiment locations, water regimes and sampling dates. Quantitative differences between genotypes were found. Most of them corresponded to proteins matching isoforms or post-translational modification variants. However, ‘Cima’ displayed the highest abundance of antioxidant enzymes.In response to water deficit, about 10% of the reproducible spots significantly varied regardless of the experiment location, among which about 25% also displayed genotype-dependent variations. As a whole, while ‘Cima’ differed from ‘Agathe_F’ by increased abundance of enzymes involved in photorespiration and in oxidative stress, ‘Agathe_F’ was mainly differentiated by increased abundance of enzymes involved in photosynthesis.     

Synthesis of polygalacturonase during tomato fruit ripening

The cell wall degrading enzyme polygalacturonase (E.C. 3.2.1.15) is not detectable in green tomatoes (Lycopersicon esculentum Mill). Activity appears at the onset of ripening and in ripe fruit it is one of the major cell-wall-bound proteins. Radioimmunoassay results, employing an antibody against purified polygalacturonase, suggest that during ripening the enzyme is synthesised de novo. Radioimmunoassay data also show that the low level of polygalacturonase in Never ripe mutants and the lack of activity in ripening inhibitor mutants can be correlated to the levels of immunologically detectable polygalacturonase protein.Abbreviations PG polygalacturonase - Nr Never ripe mutation - rin ripening inhibitor mutation     

A genetic map of Prunus based on an interspecific cross between peach and almond

A genetic linkage map of Prunus has been constructed using an interspecific F₂ population generated from self-pollinating a single F₁ plant from a cross between a dwarf peach selection (54P455) and an almond cultivar Padre. Mendelian segregations were observed for 118 markers including 1 morphological (dw), 6 isozymes, 12 plum genomic, 14 almond genomic and 75 peach mesocarp specific cDNA markers. One hundred and seven markers were mapped to 9 different linkage groups covering about 800 cM map distance, and 11 markers remained unlinked. Three loci identified by three cDNA clones, PC8, PC5 and PC68.1, were tightly linked to the dw locus in linkage group 5. Segregation distortion was observed for approximately one-third of the markers, perhaps due to the interspecific nature and the reproductive (i.e. self-incompatibility) differences between peach and almond. This map will be used for adding other markers and genes controlling important traits, identifying the genomic locations and genetic characterizing of the economically important genes in the genus Prunus, as well as for markerassisted selection in breeding populations. Of particular interest are the genes controlling tree growth and form, and fruit ripening and mesocarp development in peach and almond.     

Regulation of the expression of lipoxygenase genes in <Emphasis Type="Italic">Prunus persica</Emphasis> fruit ripening

Three genes of the lipoxygenase (LOX) family in peach (Prunus persica var. compressa cv. Ruipan 4) were cloned, and their expression patterns during fruit ripening were analyzed using real-time quantitative PCR. All of the three peach LOX genes had been expressed during fruit ripening; however, their expression patterns were significantly different. During the normal ripening of peach fruits, the expression levels of PpLox1, PpLox2 and PpLox3 increased in varying degrees accompanying upsurge of ethylene evolution. After treated by methyl jasmonic acid (MeJA), the peak of ethylene releasing occurred in advance, and the declining rate of fruit hardness was accelerated, the expression level of the three peach LOX genes in fruits markedly enhanced at the early stage of storage, but significantly decreased at the late storage stage. So, it could be suggested that all three LOXs relate to fruit ripening; however, their functions might be different. PpLox1 expression increase along with the upsurge of ethylene evolution in both control and MeJA-treated peach fruits suggested that PpLox1 probably played a major role in the peach fruit ripening. Expression peak of PpLox2 appeared at the 1 DAH (days after harvest) in both control and MeJA-treated peach fruits, while obvious changes in ethylene evolution and fruit hardness was not observed, which suggested that the rise of PpLox2 expression can be induced by certain stimulation related to ripening, such as harvesting stress and MeJA treatment. The expression of PpLox3 kept a lower level in the natural ripening fruits, whereas raced up at the early stage of storage in the fruits treated with MeJA, which indicated that PpLox3 was expressed inductively and had minor roles during the normal ripening of peach fruits, but when encountered with external stimulation, its expression level would rapidly enhance and accelerate the ripening of peach fruit.     

Two forms of exopolygalacturonase increase as peach fruits ripen

Abstract. Freestone peach cultivars are distinguished from clingstone cultivars by a more extensive softening of the mesocarp tissue, and by the separation of mesocarp and endocarp during ripening. Cultivars of both types have been reported to develop exopolygalacturonase activity during ripening, but the enzyme has not been characterized in any detail. During development of freestone peaches ( Prunus persica L. var Coronet), two exopolygalacturonase enzymes were detected 42, 65 and 85 d after full bloom and in ripe fruit. During ripening one enzyme (exoPG 1) increased 36-fold and the other (exoPG 2) 90-fold but exoPG 2 accounted for a 73% of the total activity in ripe fruit. ExoPG 1 was purified 24-fold and exoPG 2 540-fold. ExoPG 2 is a slightly acidic glycoprotein. ExoPG 1 and exoPG 2 differ slightly in their pH optima and in their responses to calcium: each produces monogalacturonic acid as a reaction product. Similar enzymes were found in Flavorerest, a semi-freestone peach.