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.

The role of chloroplast movement in C4 photosynthesis: a theoretical analysis using a three-dimensional reaction-diffusion model for maize.

Chloroplasts movement within mesophyll cells in C4 plants is hypothesized to enhance the CO2 concentrating mechanism, but this is difficult to verify experimentally. A three-dimensional (3D) leaf model can help analyse how chloroplast movement influences the operation of the CO2 concentrating mechanism. The first volumetric reaction-diffusion model of C4 photosynthesis that incorporates detailed 3D leaf anatomy, light propagation, ATP and NADPH production, and CO2, O2 and bicarbonate concentration driven by diffusional and assimilation/emission processes was developed. It was implemented for maize leaves to simulate various chloroplast movement scenarios within mesophyll cells: the movement of all mesophyll chloroplasts towards bundle sheath cells (aggregative movement) and movement of only those of interveinal mesophyll cells towards bundle sheath cells (avoidance movement). Light absorbed by bundle sheath chloroplasts relative to mesophyll chloroplasts increased in both cases. Avoidance movement decreased light absorption by mesophyll chloroplasts considerably. Consequently, total ATP and NADPH production and net photosynthetic rate increased for aggregative movement and decreased for avoidance movement compared with the default case of no chloroplast movement at high light intensities. Leakiness increased in both chloroplast movement scenarios due to the imbalance in energy production and demand in mesophyll and bundle sheath cells. These results suggest the need to design strategies for coordinated increases in electron transport and Rubisco activities for an efficient CO2 concentrating mechanism at very high light intensities.

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.

Robust dynamic experiments for the precise estimation of respiration and fermentation parameters of fruit and vegetables.

Dynamic models based on non-linear differential equations are increasingly being used in many biological applications. Highly informative dynamic experiments are valuable for the identification of these dynamic models. The storage of fresh fruit and vegetables is one such application where dynamic experimentation is gaining momentum. In this paper, we construct optimal O2 and CO2 gas input profiles to estimate the respiration and fermentation kinetics of pear fruit. The optimal input profiles, however, depend on the true values of the respiration and fermentation parameters. Locally optimal design of input profiles, which uses a single initial guess for the parameters, is the traditional method to deal with this issue. This method, however, is very sensitive to the initial values selected for the model parameters. Therefore, we present a robust experimental design approach that can handle uncertainty on the model parameters.

3-D microstructural changes in relation to the evolution of quality during ripening of mango (Mangifera indica L. cv. Carabao).

BACKGROUND: The ripening of mango involves changes in texture, flavor, and color, affecting the quality of the fruit. Previous studies have investigated the physiology on the evolution of quality during ripening but only a few have looked at microstructural changes during ripening. None of them has provided an insight into the relationhip between 3-D microstructure and the evolution of quality during ripening. As the 3-D microstructure of fruit tissue determines its mechanical and gas-transport properties, it is likely to affect fruit texture, respiratory metabolism, and other ripening processes. RESULTS: The present study focuses on the role of 3-D microstructural changes in relation to quality changes during mango ripening. Microstructural imaging using X-ray micro-computed tomography suggested the incidence of cell leakage, which was confirmed by the measurement of electrolyte leakage from the fruit peel. Due to cell leakage, porosity, pore connectivity, and pore local diameter were decreased whereas the tissue local diameter and pore specific area were increased. The decline in respiration and respiratory quotient during ripening followed the microstructural changes observed. Meanwhile, changes in aroma were observed such as a decrease in monoterpenes and an increase in esters and other fermentative metabolites. CONCLUSION: Overall, the results provide a complete, integrated picture of microstructural changes during ripening accompanying the evolution of fruit quality, suggesting functional relationships between the two. © 2020 Society of Chemical Industry.

In silico study of the role of cell growth factors in photosynthesis using a virtual leaf tissue generator coupled to a microscale photosynthesis gas exchange model.

Computational tools that allow in silico analysis of the role of cell growth and division on photosynthesis are scarce. We present a freely available tool that combines a virtual leaf tissue generator and a two-dimensional microscale model of gas transport during C3 photosynthesis. A total of 270 mesophyll geometries were generated with varying degrees of growth anisotropy, growth extent, and extent of schizogenous airspace formation in the palisade mesophyll. The anatomical properties of the virtual leaf tissue and microscopic cross-sections of actual leaf tissue of tomato (Solanum lycopersicum L.) were statistically compared. Model equations for transport of CO2 in the liquid phase of the leaf tissue were discretized over the geometries. The virtual leaf tissue generator produced a leaf anatomy of tomato that was statistically similar to real tomato leaf tissue. The response of photosynthesis to intercellular CO2 predicted by a model that used the virtual leaf tissue geometry compared well with measured values. The results indicate that the light-saturated rate of photosynthesis was influenced by interactive effects of extent and directionality of cell growth and degree of airspace formation through the exposed surface of mesophyll per leaf area. The tool could be used further in investigations of improving photosynthesis and gas exchange in relation to cell growth and leaf anatomy.

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.

Using a reaction-diffusion model to estimate day respiration and reassimilation of (photo)respired CO2 in leaves.

Methods using gas exchange measurements to estimate respiration in the light (day respiration Rd ) make implicit assumptions about reassimilation of (photo)respired CO2 ; however, this reassimilation depends on the positions of mitochondria. We used a reaction-diffusion model without making these assumptions to analyse datasets on gas exchange, chlorophyll fluorescence and anatomy for tomato leaves. We investigated how Rd values obtained by the Kok and the Yin methods are affected by these assumptions and how those by the Laisk method are affected by the positions of mitochondria. The Kok method always underestimated Rd . Estimates of Rd by the Yin method and by the reaction-diffusion model agreed only for nonphotorespiratory conditions. Both the Yin and Kok methods ignore reassimilation of (photo)respired CO2 , and thus underestimated Rd for photorespiratory conditions, but this was less so in the Yin than in the Kok method. Estimates by the Laisk method were affected by assumed positions of mitochondria. It did not work if mitochondria were in the cytosol between the plasmamembrane and the chloroplast envelope. However, mitochondria were found to be most likely between the tonoplast and chloroplasts. Our reaction-diffusion model effectively estimates Rd , enlightens the dependence of Rd estimates on reassimilation and clarifies (dis)advantages of existing methods.

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.

Modelling postmortem evolution of pH in beef M. biceps femoris under two different cooling regimes.

A model based on enzyme kinetics was developed to predict differences in postmortem pH change in beef muscles as affected by cooling rate. For the calibration and validation of the model, pH and temperature measurements were conducted at different positions in M. biceps femoris following conventional carcass cooling or faster cooling of the muscle after hot boning. The glycogen conversion, and, hence, the pH fall, was observed to significantly vary with position and cooling regime but only during the initial hours of cooling. Comparison of the cooling regimes indicated that fast cooling following hot boning avoids heat shortening induced by the combined effect of high temperature and low pH.

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.

Localization of (photo)respiration and CO2 re-assimilation in tomato leaves investigated with a reaction-diffusion model.

The rate of photosynthesis depends on the CO2 partial pressure near Rubisco, Cc, which is commonly calculated by models using the overall mesophyll resistance. Such models do not explain the difference between the CO2 level in the intercellular air space and Cc mechanistically. This problem can be overcome by reaction-diffusion models for CO2 transport, production and fixation in leaves. However, most reaction-diffusion models are complex and unattractive for procedures that require a large number of runs, like parameter optimisation. This study provides a simpler reaction-diffusion model. It is parameterized by both leaf physiological and leaf anatomical data. The anatomical data consisted of the thickness of the cell wall, cytosol and stroma, and the area ratios of mesophyll exposed to the intercellular air space to leaf surfaces and exposed chloroplast to exposed mesophyll surfaces. The model was used directly to estimate photosynthetic parameters from a subset of the measured light and CO2 response curves; the remaining data were used for validation. The model predicted light and CO2 response curves reasonably well for 15 days old tomato (cv. Admiro) leaves, if (photo)respiratory CO2 release was assumed to take place in the inner cytosol or in the gaps between the chloroplasts. The model was also used to calculate the fraction of CO2 produced by (photo)respiration that is re-assimilated in the stroma, and this fraction ranged from 56 to 76%. In future research, the model should be further validated to better understand how the re-assimilation of (photo)respired CO2 is affected by environmental conditions and physiological parameters.

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.

A mechanistic modelling approach to understand 1-MCP inhibition of ethylene action and quality changes during ripening of apples.

BACKGROUND: 1-Methylcyclopropene (1-MCP) inhibits ripening in climacteric fruit by blocking ethylene receptors, preventing ethylene from binding and eliciting its action. The objective of the current study was to use mathematical models to describe 1-MCP inhibition of apple fruit ripening, and to provide a tool for predicting ethylene production, and two important quality indicators of apple fruit, firmness and background colour. RESULTS: A model consisting of coupled differential equations describing 1-MCP inhibition of apple ripening was developed. Data on ethylene production, expression of ethylene receptors, firmness, and background colour during ripening of untreated and 1-MCP treated apples were used to calibrate the model. An overall adjusted R2 of 95% was obtained. The impact of time from harvest to treatment, and harvest maturity on 1-MCP efficacy was modelled. Different hypotheses on the partial response of 'Jonagold' apple to 1-MCP treatment were tested using the model. The model was validated using an independent dataset. CONCLUSIONS: Low 1-MCP blocking efficacy was shown to be the most likely cause of partial response for delayed 1-MCP treatment, and 1-MCP treatment of late-picked apples. Time from harvest to treatment was a more important factor than maturity for 1-MCP efficacy in 'Jonagold' apples. © 2017 Society of Chemical Industry.