Profiling Volatile Compounds in Blackcurrant Fruit using Headspace Solid-Phase Microextraction Coupled to Gas Chromatography-Mass Spectrometry

There is an increasing interest in measuring volatile organic compounds (VOCs) emitted by ripe fruits for the purpose of breeding varieties or cultivars ...

Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”, Universidad de Málaga-Consejo Superior de Investigaciones Científicas

The aim of this work was to investigate the effect of decreased cytosolic phosphoenolpyruvate carboxykinase (PEPCK) and plastidic NADP-dependent malic enzyme (ME) on tomato (Solanum lycopersicum) ripening. Transgenic tomato plants with strongly reduced levels of PEPCK and plastidic NADP-ME were generated by RNA interference gene silencing under the control of a ripening-specific E8 promoter. While these genetic modifications had relatively little effect on the total fruit yield and size, they had strong effects on fruit metabolism. Both transformants were characterized by lower levels of starch at breaker stage. Analysis of the activation state of ADP-glucose pyrophosphorylase correlated with the decrease of starch in both transformants, which suggests that it is due to an altered cellular redox status. Moreover, metabolic profiling and feeding experiments involving positionally labeled glucoses of fruits lacking in plastidic NADP-ME and cytosolic PEPCK activities revealed differential changes in overall respiration rates and tricarboxylic acid (TCA) cycle flux. Inactivation of cytosolic PEPCK affected the respiration rate, which suggests that an excess of oxaloacetate is converted to aspartate and reintroduced in the TCA cycle via 2-oxoglutarate/glutamate. On the other hand, the plastidic NADP-ME antisense lines were characterized by no changes in respiration rates and TCA cycle flux, which together with increases of pyruvate kinase and phosphoenolpyruvate carboxylase activities indicate that pyruvate is supplied through these enzymes to the TCA cycle. These results are discussed in the context of current models of the importance of malate during tomato fruit ripening.

Alteration of the interconversion of pyruvate and malate in the plastid or cytosol of ripening tomato fruit invokes diverse consequences on sugar but similar effects on cellular organic acid, metabolism, and transitory starch accumulation.

Phytohormones are key signaling molecules that coordinate plant growth and development through a range of complex interactions. Since the vast majority of plant responses to given stimuli result, amongst other factors, from a crosstalk between hormones, simultaneous analysis of multiple hormones is vital to improve our understanding of these interactions. This chapter describes a sensitive, reliable, and inexpensive method for quantification of multiple phytohormones by gas chromatography-mass spectrometry (GC-MS). 

Simultaneous Determination of Plant Hormones by GC-TOF-MS.

The cell wall invertase gene (LIN5) was reported to be a key enzyme influencing sugar uptake of tomato (Solanum lycopersicum) fruit. It was additionally revealed to be a key regulator of total soluble solids content in fruit as well as for reproductive development, being mainly involved in flower development, early fruit and seed development but also in ripening. Here, we demonstrate that silencing of the LIN5 gene promotes changes affecting fruit cuticle development which has a direct effect on postharvest properties. Transformants were characterized by reduced transpirational water loss in mature fruits accompanied by several other changes in the cuticle. Quantitative chemical composition, coupled with microscopy of isolated cuticle fruits revealed that the cuticle of the transformants were characterized by an increase of the thickness as well as significant increase in the content of cuticle components (cutin, phenolic compounds, and waxes). Furthermore, detailed analysis of the waxes revealed that the transformants displayed changes in waxes composition, showing higher levels of n-alkanes and triterpenoids which can shift the proportion of crystalline and amorphous waxes and change the water flux through the cuticle. Expression of the genes involved in cuticle biosynthesis indicated that LIN5 influences the biosynthesis of components of the cuticle, indicating that this process is coupled to sugar uploading via a mechanism which links carbon supply with the capacity for fruit expansion.

Postharvest changes in LIN5-down-regulated plants suggest a role for sugar deficiency in cuticle metabolism during ripening.

Plants synthesize and emit a large range of volatile organic compounds (VOCs) that play important roles in their interactions with the environment, from attracting pollinators and seed dispersers to protectants such as repellants and pathogen inhibitors. As such, the development of techniques for headspace collection of volatiles in combination with gas chromatography-mass spectrometry (GC-MS) has an important impact on our understanding of the biosynthesis of plant VOCs. Furthermore, knowledge of the plant VOCs can be valuable in relation to plant breeding for improving fruit flavor or enhancing resistance to insects or pathogens. This chapter describes a reliable method for extracting volatile compounds by headspace solid-phase microextraction (HS-SPME), and separate and detect them by GC-MS.

Acquisition of Volatile Compounds by Gas Chromatography-Mass Spectrometry (GC-MS).

High-throughput metabolomics technologies can provide the quantification of metabolites levels across various biological processes in different tissues, organs and species, allowing the identification of genes underpinning these complex traits. Information about changes of metabolites during strawberry development and ripening processes is key to aiding the development of new approaches to improve fruit attributes. We used network-based methods and multivariate statistical approaches to characterize and investigate variation in the primary and secondary metabolism of seven domesticated and seven wild strawberry fruit accessions at three different fruit development and ripening stages. Our results demonstrated that Fragaria sub-species can be identified solely based on the gathered metabolic profiles. We also showed that domesticated accessions displayed highly similar metabolic changes due to shared domestication history. Differences between domesticated and wild accessions were detected at the level of metabolite associations which served to rank metabolites whose regulation was mostly altered in the process of domestication. The discovery of comprehensive metabolic variation among strawberry accessions offers opportunities to probe into the genetic basis of variation, providing insights into the pathways to relate metabolic variation with important traits.

Genetic diversity of strawberry germplasm using metabolomic biomarkers.

Fruit flavor and nutritional characteristics are key quality traits and ones of the main factors influencing consumer preference. Central carbon metabolism, also known as primary metabolism, contributes to the synthesis of intermediate compounds that act as precursors for plant secondary metabolism. Specific and specialized metabolic pathways that evolved from primary metabolism play a key role in the plant's interaction with its environment. In particular, secondary metabolites present in the fruit serve to increase its attractiveness to seed dispersers and to protect it against biotic and abiotic stresses. As a consequence, several important organoleptic characteristics, such as aroma, color, and fruit nutritional value, rely upon secondary metabolite content. Phenolic and terpenoid compounds are large and diverse classes of secondary metabolites that contribute to fruit quality and have their origin in primary metabolic pathways, while the delicate aroma of ripe fruits is formed by a unique combination of hundreds of volatiles that are derived from primary metabolites. In this review, we show that the manipulation of primary metabolism is a powerful tool to engineer quality traits in fruits, such as the phenolic, terpenoid, and volatile content. The enzymatic reactions responsible for the accumulation of primary precursors are bottlenecks in the transfer of metabolic flux from central to specialized metabolism and should be taken into account to increase the yield of the final products of the biosynthetic pathways. In addition, understanding the connection and regulation of the carbon flow between primary and secondary metabolism is a key factor for the development of fruit cultivars with enhanced organoleptic and nutritional traits.

From Central to Specialized Metabolism: An Overview of Some Secondary Compounds Derived From the Primary Metabolism for Their Role in Conferring Nutritional and Organoleptic Characteristics to Fruit.

The strawberry fruit is perishable due to its high water content and soft texture, yet exhibits pleasant organoleptic and nutritional profile. Here we conducted a metabolomics-driven analysis followed by linear modelling to dissect the molecular processes in strawberry postharvest. Fruits from five cultivars were harvested and refrigerated during a ten-day period under three different atmospheres: ambient, CO-enriched and O-enriched. These analyses revealed that metabolites involved in, (i) organoleptic and nutritional properties; (ii) stress tolerance displayed duration and postharvest treatment-dependent levels. Ozone-enriched atmosphere appears to counteract postharvest negative effects, with fruits exhibiting lower levels of fermentative metabolites when compared to fruits kept in an ambient atmosphere. Furthermore, metabolic reconfiguration towards the synthesis of protective metabolites of those fruits can possibly confer enhanced tolerance to postharvest abiotic stresses. Finally, results from the linear modelling identified metabolites which could be used as biomarkers to assess strawberry quality during its postharvest shelf life.

Metabolic reconfiguration of strawberry physiology in response to postharvest practices.

Metabolic changes occurring in ripe or senescent fruits during postharvest storage lead to a general deterioration in quality attributes, including decreased flavor and 'off-aroma' compound generation. As a consequence, measures to reduce economic losses have to be taken by the fruit industry and have mostly consisted of storage at cold temperatures and the use of controlled atmospheres or ripening inhibitors. However, the biochemical pathways and molecular mechanisms underlying fruit senescence in commercial storage conditions are still poorly understood. In this sense, metabolomic platforms, enabling the profiling of key metabolites responsible for organoleptic and health-promoting traits, such as volatiles, sugars, acids, polyphenols and carotenoids, can be a powerful tool for further understanding the biochemical basis of postharvest physiology and have the potential to play a critical role in the identification of the pathways affected by fruit senescence. Here, we provide an overview of the metabolic changes during postharvest storage, with special attention to key metabolites related to fruit quality. The potential use of metabolomic approaches to yield metabolic markers useful for chemical phenotyping or even storage and marketing decisions is highlighted.

Metabolite Changes during Postharvest Storage: Effects on Fruit Quality Traits.

Sugars play a key role in fruit quality, as they directly influence taste, and thus consumer acceptance. Carbohydrates are the main resources needed by the plant for carbon and energy supply and have been suggested to be involved in all the important developmental processes, including embryogenesis, seed germination, stress responses, and vegetative and reproductive growth. Recently, considerable progresses have been made in understanding regulation of fruit ripening mechanisms, based on the role of ethylene, auxins, abscisic acid, gibberellins, or jasmonic acid, in both climacteric and non-climacteric fruits. However, the role of sugar and its associated molecular network with hormones in the control of fruit development and ripening is still poorly understood. In this review, we focus on sugar signaling mechanisms described up to date in fruits, describing their involvement in ripening-associated processes, such as pigments accumulation, and their association with hormone transduction pathways, as well as their role in stress-related responses.

Sugar Signaling During Fruit Ripening.

The fruits of diploid and octoploid strawberry ( spp) show substantial natural variation in color due to distinct anthocyanin accumulation and distribution patterns. Anthocyanin biosynthesis is controlled by a clade of R2R3 MYB transcription factors, among which MYB10 is the main activator in strawberry fruit. Here, we show that mutations in  cause most of the variation in anthocyanin accumulation and distribution observed in diploid woodland strawberry () and octoploid cultivated strawberry ( ×). Using a mapping-by-sequencing approach, we identified a -transposon in  that truncates the protein and knocks out anthocyanin biosynthesis in a white-fruited  ecotype. Two additional loss-of-function mutations in  were identified among geographically diverse white-fruited  ecotypes. Genetic and transcriptomic analyses of octoploid  spp revealed that , one of three  homoeologs identified, regulates anthocyanin biosynthesis in developing fruit. Furthermore, independent mutations in  are the underlying cause of natural variation in fruit skin and flesh color in octoploid strawberry. We identified a CACTA-like transposon () insertion in the  promoter of red-fleshed accessions that was associated with enhanced expression. Our findings suggest that cis-regulatory elements in  are responsible for enhanced  expression and anthocyanin biosynthesis in strawberry fruit flesh.

Allelic Variation of  Is the Major Force Controlling Natural Variation in Skin and Flesh Color in Strawberry ( spp.) Fruit.

Phenylpropanoids are a large class of plant secondary metabolites, which play essential roles in human health mainly associated with their antioxidant activity. Strawberry (Fragaria × ananassa) is a rich source of phytonutrients, including phenylpropanoids, which have been shown to have beneficial effects on human health. In this study, using the F. × ananassa '232' × '1392' F segregating population, we analyzed the genetic control of individual phenylpropanoid metabolites, total polyphenol content (TPC) and antioxidant capacity (TEAC) in strawberry fruit over two seasons. We have identified a total of 7, 9, and 309 quantitative trait loci (QTL) for TPC, TEAC and for 77 polar secondary metabolites, respectively. Hotspots of stable QTL for health-related antioxidant compounds were detected on linkage groups LG IV-3, LG V-2 and V-4, and LG VI-1 and VI-2, where associated markers represent useful targets for marker-assisted selection of new varieties with increased levels of antioxidant secondary compounds. Moreover, differential expression of candidate genes for major and stable mQTLs was studied in fruits of contrasting lines in important flavonoids. Our results indicate that higher expression of FaF3'H, which encodes the flavonoid 3'-hydroxylase, is associated with increased content of these important flavonoids.

Sonia Osorio

Departamento de Biología Molecular y Bioquímica,
Campus de Teatinos,
Departamento de Biología Molecular y Bioquímica, Campus de Teatinos

Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”, Universidad de Málaga-Consejo Superior de Investigaciones Científicas

Profiling Volatile Compounds in Blackcurrant Fruit using Headspace Solid-Phase Microextraction Coupled to Gas Chromatography-Mass Spectrometry

Sonia Osorio

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Departamento de Biología Molecular y Bioquímica,Campus de Teatinos,Departamento de Biología Molecular y Bioquímica, Campus de Teatinos

Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”, Universidad de Málaga-Consejo Superior de Investigaciones Científicas

Profiling Volatile Compounds in Blackcurrant Fruit using Headspace Solid-Phase Microextraction Coupled to Gas Chromatography-Mass Spectrometry

Departamento de Biología Molecular y Bioquímica,
Campus de Teatinos,
Departamento de Biología Molecular y Bioquímica, Campus de Teatinos