Targeted system approach to ethylene biosynthesis and signaling of a heat tolerant tomato cultivar; the impact of growing season on fruit ripening.

Growing tomato in hot weather conditions is challenging for fruit production and yield. Tomato cv. Savior is a heat-tolerant cultivar which can be grown during both the Vietnamese winter (mild condition) and summer (hot condition) season. Understanding the mechanisms of ethylene biosynthesis and signaling are important for agriculture, as manipulation of these pathways can lead to improvements in crop yield, stress tolerance, and fruit ripening. The objective of this study was to investigate an overview of ethylene biosynthesis and signaling from target genes to proteins and metabolites and the impact of growing season on a heat tolerant tomato cultivar throughout fruit ripening and postharvest storage. This work also showed the feasibility of absolute protein quantification of ethylene biosynthesis enzymes. Summer fruit showed the delayed peak of ethylene production until the red ripe stage. The difference in postharvest ethylene production between winter and summer fruit appears to be regulated by the difference in accumulation of 1-aminocyclopropane-1-carboxylic acid (ACC) which depends on the putative up-regulation of SAM levels. The lack of differences in protein concentrations between winter and summer fruit indicate that heat stress did not alter the ethylene biosynthesis-related protein abundance in heat tolerant cultivar. The analysis results of enzymatic activity and proteomics showed that in both winter and summer fruit, the majority of ACO activity could be mainly contributed to the abundance of ACO5 and ACO6 isoforms, rather than ACO1. Likewise, ethylene signal transduction was largely controlled by the abundance of ethylene receptors ETR1, ETR3, ETR6, and ETR7 together with the constitute triple response regulator CTR1 for both winter and summer grown tomatoes. Altogether our results indicate that in the heat tolerant tomato cv. Savior, growing season mainly affects the ethylene biosynthesis pathway and leaves the signaling pathway relatively unaffected.

Spatio-temporal dynamics of the metabolome of climacteric fruit during ripening and post-harvest storage.

Fruit quality traits are determined to a large extent by their metabolome. The metabolite content of climacteric fruit changes drastically during ripening and post-harvest storage, and has been investigated extensively. However, the spatial distribution of metabolites and how it changes in time has received much less attention as fruit are usually considered as homogenous plant organs. Yet, spatio-temporal changes of starch, which is hydrolyzed during ripening, has been used for a long time as a ripening index. As vascular transport of water, and hence convective transport of metabolites, slows down in mature fruit and even stalls after detachment, spatio-temporal changes in their concentration are probably affected by diffusive transport of gaseous molecules that act as substrate (O2), inhibitor (CO2), or regulator (ethylene and NO) of the metabolic pathways that are active during climacteric ripening. In this review, we discuss such spatio-temporal changes of the metabolome and how they are affected by transport of metabolic gases and gaseous hormones. As there are currently no techniques available to measure the metabolite distribution repeatedly by non-destructive means, we introduce reaction-diffusion models as an in silico tool to compute it. We show how the different components of such a model can be integrated and used to better understand the role of spatio-temporal changes of the metabolome in ripening and post-harvest storage of climacteric fruit that is detached from the plant, and discuss future research needs.

Sexual repurposing of juvenile aposematism in locusts.

Adaptive plasticity requires an integrated suite of functional responses to environmental variation, which can include social communication across life stages. Desert locusts (Schistocerca gregaria) exhibit an extreme example of phenotypic plasticity called phase polyphenism, in which a suite of behavioral and morphological traits differ according to local population density. Male and female juveniles developing at low population densities exhibit green- or sand-colored background-matching camouflage, while at high densities they show contrasting yellow and black aposematic patterning that deters predators. The predominant background colors of these phenotypes (green/sand/yellow) all depend on expression of the carotenoid-binding "Yellow Protein" (YP). Gregarious (high-density) adults of both sexes are initially pinkish, before a YP-mediated yellowing reoccurs upon sexual maturation. Yellow color is especially prominent in gregarious males, but the reason for this difference has been unknown since phase polyphenism was first described in 1921. Here, we use RNA interference to show that gregarious male yellowing acts as an intrasexual warning signal, which forms a multimodal signal with the antiaphrodisiac pheromone phenylacetonitrile (PAN) to prevent mistaken sexual harassment from other males during scramble mating in a swarm. Socially mediated reexpression of YP thus adaptively repurposes a juvenile signal that deters predators into an adult signal that deters undesirable mates. These findings reveal a previously underappreciated sexual dimension to locust phase polyphenism, and promote locusts as a model for investigating the relative contributions of natural versus sexual selection in the evolution of phenotypic plasticity.

Quality Evolution and Aroma Profile of Pointed Cabbage in Different Storage Regimes.

With its increasing popularity, the need for optimal storage conditions of pointed cabbages becomes more important to meet the year-round demand. Storage of the pointed varieties, however, is more difficult compared to the traditional, round varieties and is limited to a few weeks in normal air. Pointed cabbages are more susceptible to quality loss (shriveling, yellowing of leaves, weight loss, fungal, and bacterial infections) and tend to spoil much faster. In order to provide a year-round availability of the fresh product, storage under controlled atmosphere (CA) could offer a solution. In this study, pointed, white cabbage heads (Brassica oleracea var. capitata for. alba L. subv. Conica cv. 'Caraflex') were stored at 1°C from November 2018 to May 2019 under four different CA conditions (1 kPa O2 + 1.5 kPa CO2, 1 kPa O2 + 5 kPa CO2, 3 kPa O2 + 1.5 kPa CO2, and 3 kPa O2 + 5 kPa CO2), and compared to storage under normal air. Results showed that CA storage resulted in a prolonged storage life with a good quality retention for both texture and aroma. CA-stored cabbages showed less weight loss, shriveling, and yellowing. Internal quality parameters [color, soluble solids content (SSC)] were stable over the whole storage period for all objects. The aroma profiles of both the storage atmosphere and cabbage samples were impacted by storage duration. The aroma of cabbage juice was also affected by the storage regime. A clear separation was found for cabbage stored under CA compared to the reference group. From the CA-treatments studied, a combination of low oxygen (1 kPa O2) and elevated carbon dioxide levels (5 kPa CO2) showed the best results maintaining quality. Storage under CA resulted in a better resemblance to the aroma of freshly, harvested produce compared to cabbages stored in normal air.

Regulation of the Central Carbon Metabolism in Apple Fruit Exposed to Postharvest Low-Oxygen Stress.

After harvest, fruit remain metabolically active and continue to ripen. The main goal of postharvest storage is to slow down the metabolic activity of the detached fruit. In many cases, this is accomplished by storing fruit at low temperature in combination with low oxygen (O2) and high carbon dioxide (CO2) partial pressures. However, altering the normal atmospheric conditions is not without any risk and can induce low-O2 stress. This review focuses on the central carbon metabolism of apple fruit during postharvest storage, both under normal O2 conditions and under low-O2 stress conditions. While the current review is focused on apple fruit, most research on the central carbon metabolism, low-O2 stress, and O2 sensing has been done on a range of different model plants (e.g., Arabidopsis, potato, rice, and maize) using various plant organs (e.g., seedlings, tubers, roots, and leaves). This review pulls together this information from the various sources into a coherent overview to facilitate the research on the central carbon metabolism in apple fruit exposed to postharvest low-O2 stress.

Non-aqueous fractionation revealed changing subcellular metabolite distribution during apple fruit development.

In developing apple fruit, metabolic compartmentation is poorly understood due to the lack of experimental data. Distinguishing subcellular compartments in fruit using non-aqueous fractionation has been technically difficult due to the excess amount of sugars present in the different subcellular compartments limiting the resolution of the technique. The work described in this study represents the first attempt to apply non-aqueous fractionation to developing apple fruit, covering the major events occurring during fruit development (cell division, cell expansion, and maturation). Here we describe the non-aqueous fractionation method to study the subcellular compartmentation of metabolites during apple fruit development considering three main cellular compartments (cytosol, plastids, and vacuole). Evidence is presented that most of the sugars and organic acids were predominantly located in the vacuole, whereas some of the amino acids were distributed between the cytosol and the vacuole. The results showed a shift in the plastid marker from the lightest fractions in the early growth stage to the dense fractions in the later fruit growth stages. This implies that the accumulation of starch content with progressing fruit development substantially influenced the distribution of plastidial fragments within the non-aqueous density gradient applied. Results from this study provide substantial baseline information on assessing the subcellular compartmentation of metabolites in apple fruit in general and during fruit growth in particular.

Ethylene Receptors, CTRs and EIN2 Target Protein Identification and Quantification Through Parallel Reaction Monitoring During Tomato Fruit Ripening.

Ethylene, the plant ripening hormone of climacteric fruit, is perceived by ethylene receptors which is the first step in the complex ethylene signal transduction pathway. Much progress has been made in elucidating the mechanism of this pathway, but there is still a lot to be done in the proteomic quantification of the main proteins involved, particularly during fruit ripening. This work focuses on the mass spectrometry based identification and quantification of the ethylene receptors (ETRs) and the downstream components of the pathway, CTR-like proteins (CTRs) and ETHYLENE INSENSITIVE 2 (EIN2). We used tomato as a model fruit to study changes in protein abundance involved in the ethylene signal transduction during fruit ripening. In order to detect and quantify these low abundant proteins located in the membrane of the endoplasmic reticulum, we developed a workflow comprising sample fractionation and MS analysis using parallel reaction monitoring. This work shows the feasibility of the identification and absolute quantification of all seven ethylene receptors, three out of four CTRs and EIN2 in four ripening stages of tomato. In parallel, gene expression was analyzed through real-time qPCR. Correlation between transcriptomic and proteomic profiles during ripening was only observed for three of the studied proteins, suggesting that the other signaling proteins are likely post-transcriptionally regulated. Based on our quantification results we were able to show that the protein levels of SlETR3 and SlETR4 increased during ripening, probably to control ethylene sensitivity. The other receptors and CTRs showed either stable levels that could sustain, or decreasing levels that could promote fruit ripening.

Down-regulation of respiration in pear fruit depends on temperature.

The respiration rate of plant tissues decreases when the amount of available O2 is reduced. There is, however, a debate on whether the respiration rate is controlled either by diffusion limitation of oxygen or through regulatory processes at the level of the transcriptome. We used experimental and modelling approaches to demonstrate that both diffusion limitation and metabolic regulation affect the response of respiration of bulky plant organs such as fruit to reduced O2 levels in the surrounding atmosphere. Diffusion limitation greatly affects fruit respiration at high temperature, but at low temperature respiration is reduced through a regulatory process, presumably a response to a signal generated by a plant oxygen sensor. The response of respiration to O2 is time dependent and is highly sensitive, particularly at low O2 levels in the surrounding atmosphere. Down-regulation of the respiration at low temperatures may save internal O2 and relieve hypoxic conditions in the fruit.

Dynamic Labeling Reveals Temporal Changes in Carbon Re-Allocation within the Central Metabolism of Developing Apple Fruit.

In recent years, the application of isotopically labeled substrates has received extensive attention in plant physiology. Measuring the propagation of the label through metabolic networks may provide information on carbon allocation in sink fruit during fruit development. In this research, gas chromatography coupled to mass spectrometry based metabolite profiling was used to characterize the changing metabolic pool sizes in developing apple fruit at five growth stages (30, 58, 93, 121, and 149 days after full bloom) using 13C-isotope feeding experiments on hypanthium tissue discs. Following the feeding of [U-13C]glucose, the 13C-label was incorporated into the various metabolites to different degrees depending on incubation time, metabolic pathway activity, and growth stage. Evidence is presented that early in fruit development the utilization of the imported sugars was faster than in later developmental stages, likely to supply the energy and carbon skeletons required for cell division and fruit growth. The declined 13C-incorporation into various metabolites during growth and maturation can be associated with the reduced metabolic activity, as mirrored by the respiratory rate. Moreover, the concentration of fructose and sucrose increased during fruit development, whereas concentrations of most amino and organic acids and polyphenols declined. In general, this study showed that the imported compounds play a central role not only in carbohydrate metabolism, but also in the biosynthesis of amino acid and related protein synthesis and secondary metabolites at the early stage of fruit development.

Spectral Libraries for SWATH-MS Assays for Drosophila melanogaster and Solanum lycopersicum.

Quantitative proteomics methods have emerged as powerful tools for measuring protein expression changes at the proteome level. Using MS-based approaches, it is now possible to routinely quantify thousands of proteins. However, prefractionation of the samples at the protein or peptide level is usually necessary to go deep into the proteome, increasing both MS analysis time and technical variability. Recently, a new MS acquisition method named SWATH is introduced with the potential to provide good coverage of the proteome as well as a good measurement precision without prior sample fractionation. In contrast to shotgun-based MS however, a library containing experimental acquired spectra is necessary for the bioinformatics analysis of SWATH data. In this study, spectral libraries for two widely used models are built to study crop ripening or animal embryogenesis, Solanum lycopersicum (tomato) and Drosophila melanogaster, respectively. The spectral libraries comprise fragments for 5197 and 6040 proteins for S. lycopersicum and D. melanogaster, respectively, and allow reproducible quantification for thousands of peptides per MS analysis. The spectral libraries and all MS data are available in the MassIVE repository with the dataset identifiers MSV000081074 and MSV000081075 and the PRIDE repository with the dataset identifiers PXD006493 and PXD006495.

Population Modeling Approach to Optimize Crop Harvest Strategy. The Case of Field Tomato.

In this study, the aim is to develop a population model based approach to optimize fruit harvesting strategies with regard to fruit quality and its derived economic value. This approach was applied to the case of tomato fruit harvesting under Vietnamese conditions. Fruit growth and development of tomato (cv. "Savior") was monitored in terms of fruit size and color during both the Vietnamese winter and summer growing seasons. A kinetic tomato fruit growth model was applied to quantify biological fruit-to-fruit variation in terms of their physiological maturation. This model was successfully calibrated. Finally, the model was extended to translate the fruit-to-fruit variation at harvest into the economic value of the harvested crop. It can be concluded that a model based approach to the optimization of harvest date and harvest frequency with regard to economic value of the crop as such is feasible. This approach allows growers to optimize their harvesting strategy by harvesting the crop at more uniform maturity stages meeting the stringent retail demands for homogeneous high quality product. The total farm profit would still depend on the impact a change in harvesting strategy might have on related expenditures. This model based harvest optimisation approach can be easily transferred to other fruit and vegetable crops improving homogeneity of the postharvest product streams.

Delayed response to cold stress is characterized by successive metabolic shifts culminating in apple fruit peel necrosis.

BACKGROUND: Superficial scald is a physiological disorder of apple fruit characterized by sunken, necrotic lesions appearing after prolonged cold storage, although initial injury occurs much earlier in the storage period. To determine the degree to which the transition to cell death is an active process and specific metabolism involved, untargeted metabolic and transcriptomic profiling was used to follow metabolism of peel tissue over 180 d of cold storage. RESULTS: The metabolome and transcriptome of peel destined to develop scald began to diverge from peel where scald was controlled using antioxidant (diphenylamine; DPA) or rendered insensitive to ethylene using 1-methylcyclopropene (1-MCP) beginning between 30 and 60 days of storage. Overall metabolic and transcriptomic shifts, representing multiple pathways and processes, occurred alongside α-farnesene oxidation and, later, methanol production alongside symptom development. CONCLUSIONS: Results indicate this form of peel necrosis is a product of an active metabolic transition involving multiple pathways triggered by chilling temperatures at cold storage inception rather than physical injury. Among multiple other pathways, enhanced methanol and methyl ester levels alongside upregulated pectin methylesterases are unique to peel that is developing scald symptoms similar to injury resulting from mechanical stress and herbivory in other plants.

Hydrophilic interaction chromatography and evaporative light scattering detection for the determination of polar analytes in Belgian endive.

Belgian endive (Cichorium intybus L. var. foliosum Hegi), a popular produce in northern Europe, has been thoroughly studied in regard to its bitter sesquiterpene lactones content. Much less is known about on its sweetness and crunchiness, which are typically linked to the content of polar compounds such as sugars, organic acids and salts. Through HILIC-HPLC-MS, it was shown that simple sugars, amino acids, and potassium chloride are abundant in Belgian endive extracts. Subsequently, a HILIC-HPLC-ELSD method for the analysis of such compounds with run times below six minutes was developed. Recoveries varied between 80 and 110% and an average reproducibility was 7.5RSD%. Finally, the method was applied to the study of three difference Belgian endive varieties. Takine, a variety known for its sweet taste, was found to contain significantly higher levels of fructose, and lower levels of potassium and glutamine.

In-depth characterization of the tomato fruit pericarp proteome.

Since the genome of Solanum lycopersicum L. was published in 2012, some studies have explored its proteome although with a limited depth. In this work, we present an extended characterization of the proteome of the tomato pericarp at its ripe red stage. Fractionation of tryptic peptides generated from pericarp proteins by off-line high-pH reverse-phase phase chromatography in combination with LC-MS/MS analysis on a Fisher Scientific Q Exactive and a Sciex Triple-TOF 6600 resulted in the identification of 8588 proteins with a 1% FDR both at the peptide and protein levels. Proteins were mapped through GO and KEGG databases and a large number of the identified proteins were associated with cytoplasmic organelles and metabolic pathways categories. These results constitute one of the most extensive proteome datasets of tomato so far and provide an experimental confirmation of the existence of a high number of theoretically predicted proteins. All MS data are available in the ProteomeXchange repository with the dataset identifiers PXD004947 and PXD004932.

Gene expression and metabolism preceding soft scald, a chilling injury of 'Honeycrisp' apple fruit.

BACKGROUND: 'Honeycrisp' is an apple cultivar that is susceptible to soft scald, a chilling injury expressed as necrotic patches on the peel. Improved understanding of metabolism associated with the disorder would improve our understanding of soft scald and contribute to developing more effective management strategies for apple storage. It was expected that specific gene expression and specific metabolite levels in the peel would be linked with soft scald risk at harvest and/or specific time points during cold storage. RESULTS: Fruit from nine 'Honeycrisp' apple orchards that would eventually develop different incidences of soft scald between 4 and 8 weeks of cold air storage were used to contrast and determine differential transcriptomic and metabolomic changes during storage. Untargeted metabolic profiling revealed changes in a number of distinct pathways preceding and concurrent with soft scald symptom development, including elevated γ-aminobutryic acid (GABA), 1-hexanol, acylated steryl glycosides, and free p-coumaryl acyl esters. At harvest, levels of sesquiterpenoid and triterpenoid acyl esters were relatively higher in peel of fruit that did not later develop the disorder. RNA-seq driven gene expression profiling highlighted possible involvement of genes and associated metabolic processes with soft scald development. These included elevated expression of genes involved in lipid peroxidation and phenolic metabolism in fruit with soft scald, and isoprenoid/brassinosteroid metabolism in fruit that did not develop soft scald. Expression of other stress-related genes in fruit that developed soft scald included chlorophyll catabolism, cell wall loosening, and lipid transport while superoxide dismutases were up-regulated in fruit that did not develop the disorder. CONCLUSIONS: This study delineates the sequential transcriptomic and metabolomic changes preceding soft scald symptom development. Changes were differential depending on susceptibility of fruit to the disorder and could be attributed to key stress related and mediating pathways.

Slow softening of Kanzi apples (Malus×domestica L.) is associated with preservation of pectin integrity in middle lamella.

Kanzi is a recently developed apple cultivar that has an extremely low ethylene production, and maintains its crispiness during ripening. To identify key determinants of the slow softening behaviour of Kanzi apples, a comparative analysis of pectin biochemistry and tissue fracture pattern during different ripening stages of Kanzi apples was performed against Golden Delicious, a rapid softening cultivar. While substantial pectin depolymerisation and solubilisation was observed during softening in Golden Delicious apples, no depolymerisation or increased solubilisation was observed in Kanzi apples. Moreover, tissue failure during ripening was mainly by cell breakage in Kanzi apples and, in contrast, by cell separation in Golden Delicious apples. Kanzi apples had lower activity of beta-galactosidase, with no decline in the extent of branching of the pectin chain. A sudden decrease in firmness observed during senescence in Kanzi apples was not due to middle lamella dissolution, as tissue failure still occurred by cell breakage.

Spatial development of transport structures in apple (Malus × domestica Borkh.) fruit.

The void network and vascular system are important pathways for the transport of gases, water and solutes in apple fruit (Malus × domestica Borkh). Here we used X-ray micro-tomography at various spatial resolutions to investigate the growth of these transport structures in 3D during fruit development of "Jonagold" apple. The size of the void space and porosity in the cortex tissue increased considerably. In the core tissue, the porosity was consistently lower, and seemed to decrease toward the end of the maturation period. The voids in the core were more narrow and fragmented than the voids in the cortex. Both the void network in the core and in the cortex changed significantly in terms of void morphology. An automated segmentation protocol underestimated the total vasculature length by 9-12% in comparison to manually processed images. Vascular networks increased in length from a total of 5 m at 9 weeks after full bloom, to more than 20 m corresponding to 5 cm of vascular tissue per cubic centimeter of apple tissue. A high degree of branching in both the void network and vascular system and a complex three-dimensional pattern was observed across the whole fruit. The 3D visualizations of the transport structures may be useful for numerical modeling of organ growth and transport processes in fruit.

Metabolic profiling reveals ethylene mediated metabolic changes and a coordinated adaptive mechanism of 'Jonagold' apple to low oxygen stress.

Apples are predominantly stored in controlled atmosphere (CA) storage to delay ripening and prolong their storage life. Profiling the dynamics of metabolic changes during ripening and CA storage is vital for understanding the governing molecular mechanism. In this study, the dynamics of the primary metabolism of 'Jonagold' apples during ripening in regular air (RA) storage and initiation of CA storage was profiled. 1-Methylcyclopropene (1-MCP) was exploited to block ethylene receptors and to get insight into ethylene mediated metabolic changes during ripening of the fruit and in response to hypoxic stress. Metabolic changes were quantified in glycolysis, the tricarboxylic acid (TCA) cycle, the Yang cycle and synthesis of the main amino acids branching from these metabolic pathways. Partial least square discriminant analysis of the metabolic profiles of 1-MCP treated and control apples revealed a metabolic divergence in ethylene, organic acid, sugar and amino acid metabolism. During RA storage at 18°C, most amino acids were higher in 1-MCP treated apples, whereas 1-aminocyclopropane-1-carboxylic acid (ACC) was higher in the control apples. The initial response of the fruit to CA initiation was accompanied by an increase of alanine, succinate and glutamate, but a decline in aspartate. Furthermore, alanine and succinate accumulated to higher levels in control apples than 1-MCP treated apples. The observed metabolic changes in these interlinked metabolites may indicate a coordinated adaptive strategy to maximize energy production.

Chilling-related cell damage of apple (Malus × domestica Borkh.) fruit cortical tissue impacts antioxidant, lipid and phenolic metabolism.

'Soggy breakdown' (SB) is an internal flesh disorder of 'Honeycrisp' apple (Malus × domestica Borkh.) fruit that occurs during low temperature storage. The disorder is a chilling injury (CI) in which visible symptoms typically appear after several weeks of storage, but information about the underlying metabolism associated with its induction and development is lacking. The metabolic profile of flesh tissue from wholly healthy fruit and brown and healthy tissues from fruit with SB was characterized using gas chromatography-mass spectrometry (GC-MS) and liquid chromatograph-mass spectrometry (LC-MS). Partial least squares discriminant analysis (PLS-DA) and correlation networks revealed correlation among ester volatile compounds by composition and differences in phytosterol, phenolic and putative triacylglycerides (TAGs) metabolism among the tissues. anova-simultaneous component analysis (ASCA) was used to test the significance of metabolic changes linked with tissue health status. ASCA-significant components included antioxidant compounds, TAGs, and phytosterol conjugates. Relative to entirely healthy tissues, elevated metabolite levels in symptomatic tissue included γ-amino butyric acid, glycerol, sitosteryl (6'-O-palmitoyl) ß-d-glucoside and sitosteryl (6'-O-stearate) ß-d-glucoside, and TAGs containing combinations of 16:0, 18:3, 18:2 and 18:1 fatty acids. Reduced metabolite levels in SB tissue included 5-caffeoyl quinate, ß-carotene, catechin, epicatechin, α-tocopherol, violaxanthin and sitosteryl ß-d glucoside. Pathway analysis indicated aspects of primary metabolism differed according to tissue condition, although differences in metabolites involved were more subtle than those of some secondary metabolites. The results implicate oxidative stress and membrane disruption processes in SB development and constitute a diagnostic metabolic profile for the disorder.

Transcriptomic events associated with internal browning of apple during postharvest storage.

BACKGROUND: Postharvest ripening of apple (Malus x domestica) can be slowed down by low temperatures, and a combination of low O2 and high CO2 levels. While this maintains the quality of most fruit, occasionally storage disorders such as flesh browning can occur. This study aimed to explore changes in the apple transcriptome associated with a flesh browning disorder related to controlled atmosphere storage using RNA-sequencing techniques. Samples from a browning-susceptible cultivar ('Braeburn') were stored for four months under controlled atmosphere. Based on a visual browning index, the inner and outer cortex of the stored apples was classified as healthy or affected tissue. RESULTS: Over 600 million short single-end reads were mapped onto the Malus consensus coding sequence set, and differences in the expression profiles between healthy and affected tissues were assessed to identify candidate genes associated with internal browning in a tissue-specific manner. Genes involved in lipid metabolism, secondary metabolism, and cell wall modifications were highly modified in the affected inner cortex, while energy-related and stress-related genes were mostly altered in the outer cortex. The expression levels of several of them were confirmed using qRT-PCR. Additionally, a set of novel browning-specific differentially expressed genes, including pyruvate dehydrogenase and 1-aminocyclopropane-1-carboxylate oxidase, was validated in apples stored for various periods at different controlled atmosphere conditions, giving rise to potential biomarkers associated with high risk of browning development. CONCLUSIONS: The gene expression data presented in this study will help elucidate the molecular mechanism of browning development in apples at controlled atmosphere storage. A conceptual model, including energy-related (linked to the tricarboxylic acid cycle and the electron transport chain) and lipid-related genes (related to membrane alterations, and fatty acid oxidation), for browning development in apple is proposed, which may be relevant for future studies towards improving the postharvest life of apple.