ROS-scavenging-associated transcriptional and biochemical shifts during nectarine fruit development and ripening.

ROS are known as toxic by-products but also as important signaling molecules playing a key role in fruit development and ripening. To counteract the negative effects of ROS, plants and fruit own multiple ROS-scavenging mechanisms aiming to ensure a balanced ROS homeostasis. In the present study, changes in specific ROS (i.e. H2O2) as well as enzymatic (SOD, CAT, POX, APX) and non-enzymatic (phenylpropanoids, carotenoids and ascorbate) ROS-scavenging systems were investigated along four different stages of nectarine (cv. 'Diamond Ray') fruit development and ripening (39, 70, 94 and 121 DAFB) both at the metabolic (28 individual metabolites or enzymes) and transcriptional level (24 genes). Overall, our results demonstrate a complex ROS-related transcriptome and metabolome reprogramming during fruit development and ripening. At earlier fruit developmental stages an increase on the respiration rate is likely triggering an oxidative burst and resulting in the activation of specific ethylene response factors (ERF1). In turn, ROS-responsive genes or the biosynthesis of specific antioxidant compounds (i.e. phenylpropanoids) were highly expressed or accumulated at earlier fruit developmental stages (39-70 DAFB). Nonetheless, as the fruit develops, the decrease in the fruit respiration rate and the reduction of ERF1 genes leads to lower levels of most non-enzymatic antioxidants and higher accumulation of H2O2. Based on available literature and the observed accumulation dynamics of H2O2, it is anticipated that this compound may not only be a by-product of ROS-scavenging but also a signaling molecule accumulated during the ripening of nectarine fruit.

PbSRT1 and PbSRT2 regulate pear growth and ripening yet displaying a species-specific regulation in comparison to other Rosaceae spp.

Epigenetic regulation is crucial to ensure a coordinated control of the different events that occur during fruit development and ripening. Sirtuins are NAD+-dependent histone deacetylases involved in the regulation of gene expression of many biological processes. However, their implications in the Rosaceae family remains unexplored. Accordingly, in this work, we demonstrated the phylogenetic divergence of both sirtuins among Rosaceae species. We then characterized the expression pattern of both SRT1 and SRT2 in selected pome and stone fruit species. Both SRT1 and SRT2 significantly changed during the fruit development and ripening of apple, nectarine and pear fruit, displaying a different expression profile. Such differences could explain in part their different ripening behaviour. To further unravel the role of sirtuins on the fruit development and ripening processes, a deeper analysis was performed using pear as a fruit model. In pear, PbSRT1 gene expression levels were negatively correlated with specific hormones (i.e. abscisic acid, indole-3-acetic acid, gibberellin A1 and zeatin) during the first phases of fruit development. PbSRT2 seemed to directly mediate pear ripening in an ethylene-independent manner. This hypothesis was further reinforced by treating the fruit with the ethylene inhibitor 1-methylcyclopropene (1-MCP). Instead, enhanced PbSRT2 along pear growth/ripening positively correlated with the accumulation of major sugars (R2 > 0.94), reinforcing the idea that sugar metabolism may be a target of epigenetic modifications during fruit ripening. Overall, the results from this study point out, for the first time, the importance that sirtuins have in the regulation of fruit growth and ripening of pear fruit by likely regulating hormonal and sugar metabolism.

Ethylene biosynthesis and response factors are differentially modulated during the interaction of peach petals with Monilinia laxa or Monilinia fructicola.

Monilinia spp. may infect stone fruit at any growth stage, although susceptibility to brown rot depends on both host properties and climatological conditions. This said, no studies deciphering the host response in the interaction between peach blossoms and Monilinia spp. are yet available. This study presents an in-depth characterization of the role of ethylene in the interaction of 'Merrill O'Henry' peach petals (Prunus persica (L.) Batch) with Monilinia laxa and M. fructicola. We investigated the physiological responses of the host and the fungi to the application of ethylene and 1-methylcyclopropene (1-MCP) as well as the molecular patterns associated with the biosynthetic and ethylene-dependent responses during the interaction of both Monilinia species with the host. The incidence of both species was differentially affected by 1-MCP and ethylene; M. laxa was favoured by the enhanced host ethylene production associated with the treatments whereas M. fructicola reduced its infection capacity. Such differences were host-dependent as treatments did not affect growth or colony morphology of Monilinia spp. Besides, host ethylene production was altered in M. laxa inoculated petals, either by the fungus or the host itself. Molecular analysis revealed some important ERFs that could be involved in the different ability of both species to activate a cascade response of peach petals against these pathogens.

Elucidating the involvement of ethylene and oxidative stress during on- and off-tree ripening of two pear cultivars with different ripening patterns.

Scarce information is available about the ripening process of European pears attached and detached from the tree. Accordingly, this study aimed to investigate the physiological and biochemical processes underlying both on- and off-tree fruit ripening in a summer ('Conference') vs. a winter ('Flor d'Hivern') pear cultivar. For each cultivar, a batch of fruit was harvested at the commercial harvest date and ripened at 20 °C and another batch was left to ripen on the tree. In both cultivars the inability of the fruit to soften on-tree, was related to a very limited ethylene metabolism but also associated to high content of H2O2 and low lipid peroxidation levels. In contrast, ripening in detached fruit was cultivar-dependent. In 'Conference' pears, the sharp firmness loss and colour changes observed during off-tree ripening were not strictly associated to an enhanced ethylene production but rather triggered by an oxidative related process preceding the climacteric rise. In contrast, 'Flor d'Hivern' pears experienced limited softening and degreening during off-tree ripening not being related to the action of ethylene or oxidative stress. Collectively our results showed that pear ripening was not exclusively dependent of ethylene production and that the fruit potential to limit oxidative damage may be involved with the inability of some European pear cultivars to ripen on-tree.

Investigation of the transcriptomic and metabolic changes associated with superficial scald physiology impaired by lovastatin and 1-methylcyclopropene in pear fruit (cv. "Blanquilla").

To elucidate the physiology underlying the development of superficial scald in pears, susceptible "Blanquilla" fruit was treated with different compounds that either promoted (ethylene) or repressed (1-methylcyclopropene and lovastatin) the incidence of this disorder after 4 months of cold storage. Our data show that scald was negligible for the fruit treated with 1-methylcyclopropene or lovastatin, but highly manifested in untreated (78% incidence) or ethylene-treated fruit (97% incidence). The comparison between the fruit metabolomic profile and transcriptome evidenced a distinct reprogramming associated with each treatment. In all treated samples, cold storage led to an activation of a cold-acclimation-resistance mechanism, including the biosynthesis of very-long-chain fatty acids, which was especially evident in 1-methylcyclopropane-treated fruit. Among the treatments applied, only 1-methylcyclopropene inhibited ethylene production, hence supporting the involvement of this hormone in the development of scald. However, a common repression effect on the PPO gene combined with higher sorbitol content was found for both lovastatin and 1-methylcyclopropene-treated samples, suggesting also a non-ethylene-mediated process preventing the development of this disorder. The results presented in this work represent a step forward to better understand the physiological mechanisms governing the etiology of superficial scald in pears.

Interplay between hormones and assimilates during pear development and ripening and its relationship with the fruit postharvest behaviour.

The ability of European pears (Pyrus communis L.) to ripen immediately after harvest is cultivar-dependent and relies on a range of physiological and biochemical events occurring during fruit growth and development that remain largely unknown. To gain further knowledge on these events, changes in the content of sugars, acids, major hormones and ethylene precursors or related enzymes were studied in two pear varieties ('Blanquilla' and 'Conference') with known differences in their postharvest ripening behaviour. In both cultivars, low contents of abscisic acid (ABA) seemed to be a prerequisite to initiate on-tree fruit ripening including sugar accumulation and softening. In 'Blanquilla' pears, the enhanced potential to produce ethylene and thereby to ripen upon harvest was associated to a late increase in ABA content paralleled by an accumulation of indole 3-acetic acid (IAA). In turn, the inability of 'Conference' fruit to produce ethylene upon harvest appeared to be related to a coordinated action of gibberellins (more specifically GA1), salicylic acid (SA) and jasmonic acid (JA), which remained at high concentrations during the latest phases of fruit growth. Collectively, our results highlight that a complex hormonal cross-talk during the development and on-tree ripening of pear fruit may finally determine the ability of the fruit to ripen upon harvest.

A comprehensive study on the main physiological and biochemical changes occurring during growth and on-tree ripening of two apple varieties with different postharvest behaviour.

Apple quality and the storage potential likely depend on a range of physiological and biochemical events occurring throughout fruit development and ripening. In this study, we investigated the major physiological (ethylene production and respiration) and biochemical changes (related to sugar and malic acid content as well as antioxidant metabolism) occurring during growth and on-tree ripening of two apple varieties ('Granny Smith' (GS) and 'Early Red One' (ERO)) with known differences in their postharvest behaviour, mainly firmness loss and susceptibility to superficial scald. Our results demonstrate that the higher storability and the limited loss of firmness of 'GS' fruit was associated to a higher acid content, mainly malic acid, that seemed to be regulated already at fruit set (20 DAFB). The reduced loss of firmness during storage in 'GS' was also associated to the fruit inability to produce ethylene upon harvest resulting from very low 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) activity. Sugar accumulation, on the other hand, was similar among both varieties as was also observed for the rate of fruit growth or the fruit respiration pattern. In addition, the higher susceptibility of 'GS' if compared to 'ERO' to superficial scald was not associated to peroxidative damage (malondialdehyde accumulation) nor to higher levels of the sesquiterpene α-farnesene but rather mediated by a fruit antioxidant imbalance resulting from higher H2O2 levels and lower antioxidant (peroxidase) enzymatic capacity. The interplay between ethylene, respiration and antioxidants or sugars and organic acids during apple growth and development is further discussed.

Penicillium expansum (compatible) and Penicillium digitatum (non-host) pathogen infection differentially alter ethylene biosynthesis in apple fruit.

The role of ethylene on inducing plant resistance or susceptibility to certain fungal pathogens clearly depends on the plant pathogen interaction with little or no-information available focused on the apple-Penicillium interaction. Taken advantage that Penicillium expansum is the compatible pathogen and P. digitatum is the non-host of apples, the present study aimed at deciphering how each Penicillium spp. could interfere in the fruit ethylene biosynthesis at the biochemical and molecular level. The infection capacity and different aspects related to the ethylene biosynthesis were conducted at different times post-inoculation. The results show that the fruit ethylene biosynthesis was differently altered during the P. expansum infection than in response to other biotic (non-host pathogen P. digitatum) or abiotic stresses (wounding). The first symptoms of the disease due to P. expansum were visible before the initiation of the fruit ethylene climacteric burst. Indeed, the ethylene climacteric burst was reduced in response to P. expansum concomitant to an important induction of MdACO3 gene expression and an inhibition (ca. 3-fold) and overexpression (ca. 2-fold) of ACO (1-Aminocyclopropane-1-carboxylic acid oxidase) and ACS (1-Aminocyclopropane-1-carboxylic acid synthase) enzyme activities, indicating a putative role of MdACO3 in the P. expansum-apple interaction which may, in turn, be related to System-1 ethylene biosynthesis. System-1 is auto-inhibited by ethylene and is characteristic of non-climateric or pre-climacteric fruit. Accordingly, we hypothesise that P. expansum may 'manipulate' the endogenous ethylene biosynthesis in apples, leading to the circumvention or suppression of effective defences hence facilitating its colonization.

Biochemical markers to predict internal browning disorders in 'Rocha' pear during storage under high CO2.

BACKGROUND: The aim of this study was to identify biochemical markers to predict internal browning disorders (IBD) in 'Rocha' pear. Fruits from five orchards were stored for 45 days under cold air followed by 100 days in browning-inducing controlled atmosphere (CA) conditions (1 kPa O2 + 10 kPa CO2 ). RESULTS: Relationships between concentrations of ethanol (EtOH), acetaldehyde (AcDH) and ascorbic acid (AA), activities of peroxidase (POX) and polyphenoloxidase (PPO) and IBD incidence were established. The partial least square (PLS) model using the most promising markers, EtOH and AcDH, explained 89% of the variance in IBD incidence, whereas the univariate models based on the same markers explained between 89 and 94%. In contrast, the models based on AA levels and AA depletion rate only explained 57 and 82% of the variance in IBD incidence respectively. Model validation confirmed the robustness of EtOH for the prediction (R2 = 0.91, RMSE = 11.1) and allowed proposing a threshold level of 30 µL EtOH L-1 above which IBD may occur. Using this threshold value, the storage time limit associated with the occurrence of the first IBD symptoms was predicted with an acceptable RMSE of 9 days. CONCLUSION: This work clearly identifies biochemical IBD markers for 'Rocha' pear and shows that dynamic changes in ethanol concentration during the beginning of storage may be used to predict IBD development. Therefore the results presented herein represent a major step forward in the prediction of IBD in 'Rocha' pear. © 2017 Society of Chemical Industry.

Biochemical and physiological changes during fruit development and ripening of two sweet cherry varieties with different levels of cracking tolerance.

The aim of this study was to investigate the biochemical and metabolic changes, related to oxidative stress, ethylene and respiration, cell wall modification and primary metabolism, between a high ('Prime Giant') and a low ('Cristalina') cracking susceptible sweet cherry cultivar during growth and ripening. While cherries are referred as a non-climacteric fruit, our results show that an increase of endogenous ethylene production at earlier fruit developmental stages is parallel to colour development and softening during growth. Higher cracking susceptibility was clearly associated to a higher fruit growth rate and accompanied by an increase net CO2 and ethylene production, on a cherry basis, leading to an enhanced accumulation of oxidative stress markers (i.e. H2O2 and MDA). As observed in other fruit species (i.e. tomatoes) higher cracking susceptibility was also related to enhanced activity of cell wall-modifying enzymes which in turn occurred in parallel to the ethylene rise. Overall, these results suggest that cracking development may be a more complex phenomenon than a mere consequence of altered fruit water absorption or turgor and point out the importance of ethylene on sweet cherry ripening and cracking development.

Biochemical Basis of CO2-Related Internal Browning Disorders in Pears (Pyrus communis L. cv. Rocha) during Long-Term Storage.

This study aimed at understanding the biochemical basis of internal browning disorders (IBDs) in 'Rocha' pear. For this purpose, the effects of storage under normal controlled atmosphere (CA) (3 kPa of O2 + 0.5 kPa of CO2) and IBD-inducing CA (1 kPa of O2 + 10 kPa of CO2) on the antioxidant and fermentative metabolisms and polyphenol oxidase (PPO) activity and phenolics concentration were studied. The higher IBD incidence in high CO2-stored fruits was positively correlated with fermentative metabolites and negatively with ascorbate and H2O2 concentrations, and it was linked to PPO activation. These results indicate that both the antioxidant and fermentative metabolisms are involved in the occurrence of IBD in 'Rocha' pear. From the integration of the biochemical and enzymatic data, a schematic model illustrating the effects of high CO2 and low O2 in 'Rocha' pears during long-term storage was constructed.

Comparative transcriptional profiling analysis of developing melon (Cucumis melo L.) fruit from climacteric and non-climacteric varieties.

BACKGROUND: In climacteric fruit-bearing species, the onset of fruit ripening is marked by a transient rise in respiration rate and autocatalytic ethylene production, followed by rapid deterioration in fruit quality. In non-climacteric species, there is no increase in respiration or ethylene production at the beginning or during fruit ripening. Melon is unusual in having climacteric and non-climacteric varieties, providing an interesting model system to compare both ripening types. Transcriptomic analysis of developing melon fruits from Védrantais and Dulce (climacteric) and Piel de sapo and PI 161375 (non-climacteric) varieties was performed to understand the molecular mechanisms that differentiate the two fruit ripening types. RESULTS: Fruits were harvested at 15, 25, 35 days after pollination and at fruit maturity. Transcript profiling was performed using an oligo-based microarray with 75 K probes. Genes linked to characteristic traits of fruit ripening were differentially expressed between climacteric and non-climacteric types, as well as several transcription factor genes and genes encoding enzymes involved in sucrose catabolism. The expression patterns of some genes in PI 161375 fruits were either intermediate between. Piel de sapo and the climacteric varieties, or more similar to the latter. PI 161375 fruits also accumulated some carotenoids, a characteristic trait of climacteric varieties. CONCLUSIONS: Simultaneous changes in transcript abundance indicate that there is coordinated reprogramming of gene expression during fruit development and at the onset of ripening in both climacteric and non-climacteric fruits. The expression patterns of genes related to ethylene metabolism, carotenoid accumulation, cell wall integrity and transcriptional regulation varied between genotypes and was consistent with the differences in their fruit ripening characteristics. There were differences between climacteric and non-climacteric varieties in the expression of genes related to sugar metabolism suggesting that they may be potential determinants of sucrose content and post-harvest stability of sucrose levels in fruit. Several transcription factor genes were also identified that were differentially expressed in both types, implicating them in regulation of ripening behaviour. The intermediate nature of PI 161375 suggested that classification of melon fruit ripening behaviour into just two distinct types is an over-simplification, and that in reality there is a continuous spectrum of fruit ripening behaviour.

Investigation of ascorbate metabolism during inducement of storage disorders in pear.

In pear and apple, depletion of ascorbate has previously been associated with development of stress-related flesh browning. This disorder occurs in intact fruit and differs from browning associated with tissue maceration and processing. We investigated changes in ascorbate content, ascorbate peroxidase (APX) activities and gene expression of l-galactose pathway genes, ascorbate recycling genes and APXs from harvest to 30 days storage for three pear varieties ['Williams Bon Chretien' (WBC), 'Doyenne du Comice' and 'Beurre Bosc']. The pears were stored at 0.5°C in air or controlled atmosphere (CA, 2 kPa O(2) and 5 kPa CO(2)). Storage in CA caused significant amounts of storage disorders in WBC only. Ascorbate content generally declined after harvest, although a transient increase in ascorbate in the form of dehydroascorbate (DHA) between harvest and 3 days was observed in CA stored WBC, possibly due to low at-harvest monodehydroascorbate reductase and CA-decreased dehydroascorbate reductase expression. Quantitative polymerase chain reaction indicated that all cultivars responded to CA storage by increasing transcripts for APXs, and surprisingly the pre-l-galactose pathway gene GDP-mannose pyrophosphorylase, of which the product GDP mannose, is utilized either for cell wall polysaccharides, protein N-glycosylation or ascorbate production. Overall, the small differences in ascorbate we observed suggest how ascorbate is utilized, rather than ascorbate content, determines the potential to develop internal browning. Moreover, a transitory increase in DHA postharvest may indicate that fruits are at risk of developing the disorder.

Responsiveness of 'Conference' pears to 1-methylcyclopropene: the role of harvest date, orchard location and year.

BACKGROUND: In some pear varieties like 'Conference', 1-methylcyclopropene (1-MCP) treatment often impairs the ripening process indefinitely and the pears remain 'evergreen'. To better understand this behaviour, the influence of the harvest date, orchard location and year on the effectiveness of 1-MCP treatment was investigated. RESULTS: Pear softening was inhibited by 1-MCP treatment and the effectiveness of the treatment depended on harvest date, orchard location and year. Differences in the rate of softening in 1-MCP-treated pears depended mainly on the fruit physiological maturity at the moment of 1-MCP treatment. Accordingly, the combination of the Streif index and ethylene production at harvest appeared to be able to predict the evergreen behaviour. Treated pears with a low Streif index (<0.8) and high ethylene production at harvest (≥0.23 µL kg(-1) h(-1)) maintain significantly high firmness but did soften during shelf life, reaching acceptable eating quality. CONCLUSION: Evergreen behaviour was mainly influenced by the initial fruit maturity and especially by the ability of the fruit to produce ethylene at the moment of treatment. More mature fruit were able to overcome the inhibition by 1-MCP, and the solution to prevent evergreen behaviour therefore lies in the adequate determination of harvest maturity.

How to prevent ripening blockage in 1-MCP-treated 'Conference' pears.

BACKGROUND: Some European pear varieties treated with 1-methylcyclopropene (1-MCP) often remain 'evergreen', meaning that their ripening process is blocked and does not resume after removal from cold storage. In this work this was confirmed also to be the case in 'Conference' pears. To reverse the blockage of ripening 1-MCP treatments combined with external exogenous ethylene were tested. RESULTS: 1-MCP treatment of 'Conference' pears is very effective in delaying ripening and, more specifically, softening. The same 1-MCP concentration in different experimental years caused a different response. The higher dose of 1-MCP (600 nL L⁻¹) always resulted in irreversible blockage of ripening, whereas the behaviour of fruit receiving a lower dose (300 nL L⁻¹) depended on the year, and this did not depend on maturity at harvest or on storage conditions. Simultaneous exposure to 1-MCP and exogenous ethylene significantly affected fruit ripening, allowing significant softening to occur but at a lower rate compared with control fruit. CONCLUSION: The application of exogenous ethylene and 1-MCP simultaneously after harvest permitted restoration of the ripening process after storage in 'Conference' pears, extending the possibility of marketing and consumption.

'Diffuse skin browning' in 1-MCP-treated apples: etiology and systems of control.

BACKGROUND: 'Diffuse skin browning' (DSB) is a physiological disorder that affects Golden Delicious apples treated with 1-methylcyclopropene (1-MCP). Although a very high incidence is found, very little is known about the etiology of this disorder. This study aims to provide an understanding of the causes of this disorder and prevent it. RESULTS: A very high incidence of DSB was found in 1-MCP-treated apples independent of the location of the orchard. Similar to superficial scald, harvest maturity determines the DSB incidence, with the more mature fruit being less sensitive. The 1-MCP dose (156 nL L(-1) or 625 nL L(-1)) and the temperature at which the 1-MCP treatment was applied (0.5 or 20 °C) did not affect the incidence of DSB. Diphenylamine (DPA) treatment did not prevent DSB, contrary to superficial scald. Additionally, controlled atmosphere storage only partially reduced the incidence of DSB, whereas progressive cooling strategies completely inhibited DSB occurrence. A direct correlation was found between the sensitivity of the Golden Delicious clone to russeting and its sensitivity to develop DSB during storage. CONCLUSION: Our results indicated that DSB and superficial scald are two different disorders involving different oxidative processes. DSB can be prevented by progressive cooling and selection of russeting-resistant clones.