Environmentally driven transcriptomic and metabolic changes leading to color differences in "Golden Reinders" apples.

Apple is characterized by its high adaptation to diverse growing environments. However, little is still known about how different environments can regulate at the metabolic or molecular level specific apple quality traits such as the yellow fruit peel color. In this study, changes in carotenoids and chlorophylls, antioxidants as well as differences in the transcriptome were investigated by comparing the peel of "Golden Reinders" apples grown at different valley and mountain orchards. Mountain environment favored the development of yellow color, which was not caused by an enhanced accumulation of carotenoids but rather by a decrease in the chlorophyll content. The yellow phenotype was also associated to higher expression of genes related to chloroplast functions and oxidative stress. Time-course analysis over the last stages of apple development and ripening, in fruit from both locations, further revealed that the environment differentially modulated isoprenoids and phenylpropanoid metabolism and pointed out a key role for H2O2 in triggering apple peel degreening. Overall, the results presented herein provide new insights into how different environmental conditions regulate pigment and antioxidant metabolism in apple leading to noticeable differences in the apple peel color.

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.

Scrutinising the relationship between major physiological and compositional changes during 'Merrill O'Henry' peach growth with brown rot susceptibility.

In the present work, the major physiological and compositional changes occurring during 'Merrill O'Henry' peach growth and its relationship with susceptibility to three strains of Monilinia spp. at 49, 77, 126 and 160 days after full bloom were explored. Results of disease incidence indicated wide differences among phenological stages, being 49 and 126 days after full bloom the moment when peaches showed significantly lower susceptibility to brown rot (40 and 23% of rotten fruit, respectively, for strain ML8L). Variation in brown rot susceptibility among different growth stages was also strain-dependent. Lower fruit susceptibility to ML8L at 49 and 126 was accompanied by noticeable changes in the fruit ethylene and respiration patterns, and also in sugars and organic acids content. By employing a partial least squares regression model, a strong negative relationship between citric acid, and a positive association of ethylene with peach susceptibility to Monilinia spp. at diverse phenological stages were observed. The results obtained herein highlight that the content of certain compounds such as citrate, malate and sucrose; the respiratory activity and the fruit ethylene production may mediate in a coordinated manner the fruit resistance to Monilinia spp. at different phenological stages of peach fruit.

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.

Double-sided battle: The role of ethylene during Monilinia spp. infection in peach at different phenological stages.

Controversy exists on whether ethylene is involved in determining fruit resistance or susceptibility against biotic stress. In this work, the hypothesis that ethylene biosynthesis in peaches at different phenological stages may be modulated by Monilinia spp. was tested. To achieve this, at 49 and 126 d after full bloom (DAFB), ethylene biosynthesis of healthy and infected 'Merryl O'Henry' peaches with three strains of Monilinia spp. (M. fructicola (CPMC6) and M. laxa (CPML11 and ML8L) that differ in terms of aggressiveness) was analysed at the biochemical and molecular level along the course of infection in fruit stored at 20 °C. At 49 DAFB, results evidenced that infected fruit showed inhibition of ethylene production in comparison with non-inoculated fruit, suggesting that the three Monilinia strains were somehow suppressing ethylene biosynthesis to modify fruit defences to successfully infect the host. On the contrary, at 126 DAFB ethylene production increased concomitantly with brown rot spread, and values for non-inoculated fruit were almost undetectable throughout storage at 20 °C. The expression of several target genes involved in the ethylene biosynthetic pathway confirmed that they were differentially expressed upon Monilinia infection, pointing to a strain-dependent regulation. Notably, Prunus persica 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) (PpACS) family was the most over-expressed over time, demonstrating a positive ethylene regulation, especially at 126 DAFB. At this phenological stage it was demonstrated the ability of Monilinia spp. to alter ethylene biosynthesis through PpACS1 and benefit from the consequences of an ethylene burst likely on cell wall softening. Overall, our results put forward that infection not only among different strains but also at each stage is achieved by different mechanisms, with ethylene being a key factor in determining peach resistance or susceptibility to brown rot.

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 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.

Unravelling the resistance mechanisms to 2,4-D (2,4-dichlorophenoxyacetic acid) in corn poppy (Papaver rhoeas).

In southern Europe, the intensive use of 2,4-D (2,4-dichlorophenoxyacetic acid) and tribenuron-methyl in cereal crop systems has resulted in the evolution of resistant (R) corn poppy (Papaver rhoeas L.) biotypes. Experiments were conducted to elucidate (1) the resistance response to these two herbicides, (2) the cross-resistant pattern to other synthetic auxins and (3) the physiological basis of the auxin resistance in two R (F-R213 and D-R703) populations. R plants were resistant to both 2,4-D and tribenuron-methyl (F-R213) or just to 2,4-D (D-R703) and both R populations were also resistant to dicamba and aminopyralid. Results from absorption and translocation experiment revealed that R plants translocated less [14C]-2,4-D than S plants at all evaluation times. There was between four and eight-fold greater ethylene production in S plants treated with 2,4-D, than in R plants. Overall, these results suggest that reduced 2,4-D translocation is the resistance mechanism in synthetic auxins R corn poppy populations and this likely leads to less ethylene production and greater survival in R plants.

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.

Dietary intake of metals by the population of Tarragona County (Catalonia, Spain): results from a duplicate diet study.

The daily intakes of arsenic (As), beryllium, cadmium (Cd), cobalt, chromium, copper, mercury (Hg), manganese, nickel, lead (Pb), antimony, tin, thallium, uranium, vanadium, and zinc by an adult population living in Tarragona County (Catalonia, Spain) were determined by the duplicate diet method with a 10-day sampling period. Duplicate diet samples, prepared as per consumption, were collected during September 2010 in 20 restaurants offering a variety of daily menus (breakfast, lunch, and dinner). A total of 200 composite samples, corresponding to 600 individual samples, were prepared for trace elements analyses, which were carried out by inductively coupled plasma mass spectrometry. The results were compared with data from previous total diet studies (TDS) recently performed in the same geographical area. Notable differences in the intake of the toxic elements As, Cd, Hg, and Pb were found depending on the method used. It is concluded that both the duplicate diet method and the TDS may provide important and useful information to estimate human exposure to metals through the diet and their derived health risks. However, for comparison between surveys or for establishing temporal trends in the intake of metals, it seems to be recommendable to use always the same method. Notwithstanding, if the interest is only to know whether the intake of a certain element is below the tolerable intake, both methods are useful.

Monitoring environmental levels of trace elements near a hazardous waste incinerator: human health risks after a decade of regular operations.

In 1998, we initiated an environmental surveillance program of the only hazardous waste incinerator (HWI) in Spain. The concentrations of a number of metals (As, Be, Cd, Cr, Hg, Mn, Ni, Pb, Sn, Tl, and V) were analyzed in soil and vegetation samples collected around the facility. Since then, periodical measurements of these same elements have been performed in both matrices. In 2009 and 2010, soil and vegetation samples were again collected, and the levels of the above elements were determined. In general terms, the temporal trends in metal concentrations were not homogeneous, showing significant changes between the baseline and the latest surveys. Metal levels in soil and herbage samples analyzed in the current survey were similar to recent data reported for other urban and industrial areas, being notably lower than the reference values set by national regulatory organisms. Moreover, more than 10 years after regular operations of the HWI, exposure to metals would not mean, either now or in past surveys, any carcinogenic or non-carcinogenic health risks for the population living in the neighborhood.

A literature review on the safety assessment of genetically modified plants.

In recent years, there has been a notable concern on the safety of genetically modified (GM) foods/plants, an important and complex area of research, which demands rigorous standards. Diverse groups including consumers and environmental Non Governmental Organizations (NGO) have suggested that all GM foods/plants should be subjected to long-term animal feeding studies before approval for human consumption. In 2000 and 2006, we reviewed the information published in international scientific journals, noting that the number of references concerning human and animal toxicological/health risks studies on GM foods/plants was very limited. The main goal of the present review was to assess the current state-of-the-art regarding the potential adverse effects/safety assessment of GM plants for human consumption. The number of citations found in databases (PubMed and Scopus) has dramatically increased since 2006. However, new information on products such as potatoes, cucumber, peas or tomatoes, among others was not available. Corn/maize, rice, and soybeans were included in the present review. An equilibrium in the number research groups suggesting, on the basis of their studies, that a number of varieties of GM products (mainly maize and soybeans) are as safe and nutritious as the respective conventional non-GM plant, and those raising still serious concerns, was currently observed. Nevertheless, it should be noted that most of these studies have been conducted by biotechnology companies responsible of commercializing these GM plants. These findings suggest a notable advance in comparison with the lack of studies published in recent years in scientific journals by those companies. All this recent information is herein critically reviewed.

Development of a glucose biosensor for rapid assessment of strawberry quality: relationship between biosensor response and fruit composition.

Sugars are intimately related to the taste of strawberry fruit and therefore to quality. A disposable prototype glucose biosensor was constructed using glucose oxidase (GOx) immobilized onto Meldolas Blue mediated screen printed electrodes, to determine glucose content in diluted strawberry juices. Several experimental variables that affect biosensor performance, such as applied potential, GOx loading, and pH of the buffer/electrolyte solution were optimized by means of a 2(3) central composite design. Optimum applied potential, pH, and enzyme loading were +300 mV (versus Ag/AgCl), 7.2, and 20 U, respectively. Although the linear range (0-10 mM) was not significantly affected by the optimization process, the signal given by the biosensor was increased by as much as 3-fold as compared to preliminary experiments, and the reproducibility of the measurements was improved. Unlike total soluble solids (TSS), and as hypothesized, the constructed GOx biosensor was able to discriminate and rank eight different strawberry cultivars on the basis of their glucose content when compared to known concentrations measured by standard HPLC. A detailed study of the possible interferences (viz. total phenolics, antioxidant capacity, and organic acids) found in strawberry samples that could affect biosensor performance was also performed to further understand the relationship between biosensor response and sample composition. Under the imposed experimental conditions the constructed biosensor acted interference-free.