Comparison of metabolomic reconfiguration between Columbia and Landsberg ecotypes subjected to the combination of high salinity and increased irradiance.

BACKGROUND: Plants growing in the field are subjected to combinations of abiotic stresses. These conditions pose a devastating threat to crops, decreasing their yield and causing a negative economic impact on agricultural production. Metabolic responses play a key role in plant acclimation to stress and natural variation for these metabolic changes could be key for plant adaptation to fluctuating environmental conditions. RESULTS: Here we studied the metabolomic response of two Arabidopsis ecotypes (Columbia-0 [Col] and Landsberg erecta-0 [Ler]), widely used as genetic background for Arabidopsis mutant collections, subjected to the combination of high salinity and increased irradiance. Our findings demonstrate that this stress combination results in a specific metabolic response, different than that of the individual stresses. Although both ecotypes displayed reduced growth and quantum yield of photosystem II, as well as increased foliar damage and malondialdehyde accumulation, different mechanisms to tolerate the stress combination were observed. These included a relocation of amino acids and sugars to act as potential osmoprotectants, and the accumulation of different stress-protective compounds such as polyamines or secondary metabolites. CONCLUSIONS: Our findings reflect an initial identification of metabolic pathways that differentially change under stress combination that could be considered in studies of stress combination of Arabidopsis mutants that include Col or Ler as genetic backgrounds.

γ-Aminobutyric acid plays a key role in plant acclimation to a combination of high light and heat stress.

Plants are frequently subjected to different combinations of abiotic stresses, such as high light (HL) intensity, and elevated temperatures. These environmental conditions pose a threat to agriculture production, affecting photosynthesis, and decreasing yield. Metabolic responses of plants, such as alterations in carbohydrates and amino acid fluxes, play a key role in the successful acclimation of plants to different abiotic stresses, directing resources toward stress responses, and suppressing growth. Here we show that the primary metabolic response of Arabidopsis (Arabidopsis thaliana) plants to HL or heat stress (HS) is different from that of plants subjected to a combination of HL and HS (HL+HS). We further demonstrate that the combined stress results in a unique metabolic response that includes increased accumulation of sugars and amino acids coupled with decreased levels of metabolites participating in the tricarboxylic acid cycle. Among the amino acids exclusively accumulated during HL+HS, we identified the nonproteinogenic amino acid γ-aminobutyric acid (GABA). Analysis of different mutants deficient in GABA biosynthesis (GLUTAMATE DESCARBOXYLASE 3 [gad3]) as well as mutants impaired in autophagy (autophagy-related proteins 5 and 9 [atg5 and atg9]), revealed that GABA plays a key role in the acclimation of plants to HL+HS, potentially by promoting autophagy. Taken together, our findings identify a role for GABA in regulating plant responses to combined stress.

The flavour of grape colour: anthocyanin content tunes aroma precursor composition by altering the berry microenvironment.

Anthocyaninless (white) instead of black/red (coloured) fruits develop in grapevine cultivars without functional VviMYBA1 and VviMYBA2 genes, and this conditions the colour of wines that can be produced. To evaluate whether this genetic variation has additional consequences on fruit ripening and composition, we performed comparisons of microenvironment, transcriptomics, and metabolomics of developing grapes between near-isogenic white- and black-berried somatic variants of Garnacha and Tempranillo cultivars. Berry temperature was as much as 3.5 ºC lower in white- compared to black-berried Tempranillo. An RNA-seq study combined with targeted and untargeted metabolomics revealed that ripening fruits of white-berried variants were characterized by the up-regulation of photosynthesis-related and other light-responsive genes and by their higher accumulation of specific terpene aroma precursors, fatty acid-derived aldehyde volatiles, and phenylpropanoid precursor amino acids. MYBA1-MYBA2 function proved essential for flavonol trihydroxylation in black-berried somatic variants, which were also characterized by enhanced expression of pathogen defence genes in the berry skin and increased accumulation of C6-derived alcohol and ester volatiles and γ-aminobutyric acid. Collectively, our results indicate that anthocyanin depletion has side-effects on grape composition by altering the internal microenvironment of the berry and the partitioning of the phenylpropanoid pathway. Our findings show how fruit colour can condition other fruit features, such as flavour potential and stress homeostasis.

Expression of two barley proteinase inhibitors in tomato promotes endogenous defensive response and enhances resistance to Tuta absoluta.

BACKGROUND: Plants and insects have coexisted for million years and evolved a set of interactions which affect both organisms at different levels. Plants have developed various morphological and biochemical adaptations to cope with herbivores attacks. However, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) has become the major pest threatening tomato crops worldwide and without the appropriated management it can cause production losses between 80 to 100%. RESULTS: The aim of this study was to investigate the in vivo effect of a serine proteinase inhibitor (BTI-CMe) and a cysteine proteinase inhibitor (Hv-CPI2) from barley on this insect and to examine the effect their expression has on tomato defensive responses. We found that larvae fed on tomato transgenic plants co-expressing both proteinase inhibitors showed a notable reduction in weight. Moreover, only 56% of these larvae reached the adult stage. The emerged adults showed wings deformities and reduced fertility. We also investigated the effect of proteinase inhibitors ingestion on the insect digestive enzymes. Our results showed a decrease in larval trypsin activity. Transgenes expression had no harmful effect on Nesidiocoris tenuis (Reuter) (Heteroptera: Miridae), a predator of Tuta absoluta, despite transgenic tomato plants attracted the mirid. We also found that barley cystatin expression promoted plant defense by inducing the expression of the tomato endogenous wound inducible Proteinase inhibitor 2 (Pin2) gene, increasing the production of glandular trichomes and altering the emission of volatile organic compounds. CONCLUSION: Our results demonstrate the usefulness of the co-expression of different proteinase inhibitors for the enhancement of plant resistance to Tuta absoluta.

Identification, introgression, and validation of fruit volatile QTLs from a red-fruited wild tomato species.

Volatile organic compounds (VOCs) are major determinants of fruit flavor, a primary objective in tomato breeding. A recombinant inbred line (RIL) population consisting of 169 lines derived from a cross between Solanum lycopersicum and a red-fruited wild tomato species Solanum pimpinellifolium accession (SP) was characterized for VOCs in three different seasons. Correlation and hierarchical cluster analyses were performed on the 52 VOCs identified, providing a tool for the putative assignation of individual compounds to metabolic pathways. Quantitative trait locus (QTL) analysis, based on a genetic linkage map comprising 297 single nucleotide polymorphisms (SNPs), revealed 102 QTLs (75% not described previously) corresponding to 39 different VOCs. The SP alleles exerted a positive effect on most of the underlying apocarotenoid volatile QTLs-regarded as desirable for liking tomato-indicating that alleles inherited from SP are a valuable resource for flavor breeding. An introgression line (IL) population developed from the same parental genotypes provided 12 ILs carrying a single SP introgression and covering 85 VOC QTLs, which were characterized at three locations. The results showed that almost half of the QTLs previously identified in the RILs maintained their effect in an IL form, reinforcing the value of these QTLs for flavor/aroma breeding in cultivated tomato.

Thermospermine levels are controlled by an auxin-dependent feedback loop mechanism in Populus xylem.

Polyamines are small polycationic amines that are widespread in living organisms. Thermospermine, synthesized by thermospermine synthase ACAULIS5 (ACL5), was recently shown to be an endogenous plant polyamine. Thermospermine is critical for proper vascular development and xylem cell specification, but it is not known how thermospermine homeostasis is controlled in the xylem. We present data in the Populus model system supporting the existence of a negative feedback control of thermospermine levels in stem xylem tissues, the main site of thermospermine biosynthesis. While over-expression of the ACL5 homologue in Populus, POPACAULIS5, resulted in strong up-regulation of ACL5 expression and thermospermine accumulation in leaves, the corresponding levels in the secondary xylem tissues of the stem were similar or lower than those in the wild-type. POPACAULIS5 over-expression had a negative effect on accumulation of indole-3-acetic acid, while exogenous auxin had a positive effect on POPACAULIS5 expression, thus promoting thermospermine accumulation. Further, over-expression of POPACAULIS5 negatively affected expression of the class III homeodomain leucine zipper (HD-Zip III) transcription factor gene PttHB8, a homologue of AtHB8, while up-regulation of PttHB8 positively affected POPACAULIS5 expression. These results indicate that excessive accumulation of thermospermine is prevented by a negative feedback control of POPACAULIS5 transcript levels through suppression of indole-3-acetic acid levels, and that PttHB8 is involved in the control of POPACAULIS5 expression. We propose that this negative feedback loop functions to maintain steady-state levels of thermospermine, which is required for proper xylem development, and that it is dependent on the presence of high concentrations of endogenous indole-3-acetic acid, such as those present in the secondary xylem tissues.

The expanded tomato fruit volatile landscape.

The present review aims to synthesize our present knowledge about the mechanisms implied in the biosynthesis of volatile compounds in the ripe tomato fruit, which have a key role in tomato flavour. The difficulties in identifiying not only genes or genomic regions but also individual target compounds for plant breeding are addressed. Ample variability in the levels of almost any volatile compound exists, not only in the populations derived from interspecific crosses but also in heirloom varieties and even in commercial hybrids. Quantitative trait loci (QTLs) for all tomato aroma volatiles have been identified in collections derived from both intraspecific and interspecific crosses with different wild tomato species and they (i) fail to co-localize with structural genes in the volatile biosynthetic pathways and (ii) reveal very little coincidence in the genomic regions characterized, indicating that there is ample opportunity to reinforce the levels of the volatiles of interest. Some of the identified genes may be useful as markers or as biotechnological tools to enhance tomato aroma. Current knowledge about the major volatile biosynthetic pathways in the fruit is summarized. Finally, and based on recent reports, it is stressed that conjugation to other metabolites such as sugars seems to play a key role in the modulation of volatile release, at least in some metabolic pathways.

A plant spermine oxidase/dehydrogenase regulated by the proteasome and polyamines.

Polyamine oxidases (PAOs) are flavin-dependent enzymes involved in polyamine catabolism. In Arabidopsis five PAO genes (AtPAO1-AtPAO5) have been identified which present some common characteristics, but also important differences in primary structure, substrate specificity, subcellular localization, and tissue-specific expression pattern, differences which may suggest distinct physiological roles. In the present work, AtPAO5, the only so far uncharacterized AtPAO which is specifically expressed in the vascular system, was partially purified from 35S::AtPAO5-6His Arabidopsis transgenic plants and biochemically characterized. Data presented here allow AtPAO5 to be classified as a spermine dehydrogenase. It is also shown that AtPAO5 oxidizes the polyamines spermine, thermospermine, and N(1)-acetylspermine, the latter being the best in vitro substrate of the recombinant enzyme. AtPAO5 also oxidizes these polyamines in vivo, as was evidenced by analysis of polyamine levels in the 35S::AtPAO5-6His Arabidopsis transgenic plants, as well as in a loss-of-function atpao5 mutant. Furthermore, subcellular localization studies indicate that AtPAO5 is a cytosolic protein undergoing proteasomal control. Positive regulation of AtPAO5 expression by polyamines at the transcriptional and post-transcriptional level is also shown. These data provide new insights into the catalytic properties of the PAO gene family and the complex regulatory network controlling polyamine metabolism.

Thermospermine catabolism increases Arabidopsis thaliana resistance to Pseudomonas viridiflava.

This work investigated the roles of the tetraamine thermospermine (TSpm) by analysing its contribution to Arabidopsis basal defence against the biotrophic bacterium Pseudomonas viridiflava. The participation of polyamine oxidases (PAOs) in TSpm homeostasis and TSpm-mediated defence was also investigated. Exogenous supply of TSpm, as well as ectopic expression of the TSpm biosynthetic gene ACL5, increased Arabidopsis Col-0 resistance to P. viridiflava, while null acl5 mutants were less resistant than Col-0 plants. The above-mentioned increase in resistance was blocked by the PAO inhibitor SL-11061, thus demonstrating the participation of TSpm oxidation. Analysis of PAO genes expression in transgenic 35S::ACL5 and Col-0 plants supplied with TSpm suggests that PAO 1, 3, and 5 are the main PAOs involved in TSpm catabolism. In summary, TSpm exhibited the potential to perform defensive functions previously reported for its structural isomer Spm, and the relevance of these findings is discussed in the context of ACL5 expression and TSpm concentration in planta. Moreover, this work demonstrates that manipulation of TSpm metabolism modifies plant resistance to pathogens.

A xanthophyll-derived apocarotenoid regulates carotenogenesis in tomato chromoplasts.

Carotenoids possess important biological functions that make them essential components of the human diet. ß-Carotene and some other carotenoids have vitamin A activity while lutein and zeaxanthin, typically referred to as the macular pigments, are involved in good vision and in delaying the onset of age-related eye diseases. In order to create a zeaxanthin-producing tomato fruit, two transgenic lines, one with a high ß-carotene cyclase activity and the other with a high ß-carotene hydroxylase activity, have been genetically crossed. Ripe fruits from the resulting progeny contained significant levels of violaxanthin, antheraxanthin, and xanthophyll esters. However, their zeaxanthin content was not as high as expected, and the total level of carotenoids was only 25% of the carotenoids found in ripe fruits of the comparator line. Targeted transcript analysis and apocarotenoids determinations indicated that transcriptional regulation of the pathway or degradation of synthesized carotenoids were not responsible for the low carotenoid content of hybrid fruits which instead appeared to result from a substantial reduction of carotenoid biosynthesis. Notably, the content of an unidentified hydroxylated cyclic (C13) apocarotenoid was 13 times higher in the hybrid fruits than in the control fruits. Furthermore, a GC-MS-based metabolite profiling demonstrated a perturbation of carotenogenesis in ripening hybrid fruits compatible with a block of the pathway. Moreover, carotenoid profiling on leaf, fruit, and petal samples from a set of experimental lines carrying the hp3 mutation, in combination with the two transgenes, indicated that the carotenoid biosynthesis in petal and fruit chromoplasts could be regulated. Altogether the data were consistent with the hypothesis of the regulation of the carotenoid pathway in tomato chromoplasts through a mechanism of feedback inhibition mediated by a xanthophyll-derived apocarotenoid. This chromoplast-specific post-transcriptional mechanism was disclosed in transgenic fruits of HU hybrid owing to the abnormal production of zeaxanthin and antheraxanthin, the more probable precursors of the apocarotenoid signal. A model describing the regulation of carotenoid pathway in tomato chromoplasts is presented.

Abscisic acid promotes plant acclimation to the combination of salinity and high light stress.

Plants encounter combinations of different abiotic stresses such as salinity (S) and high light (HL). These environmental conditions have a detrimental effect on plant growth and development, posing a threat to agricultural production. Metabolic changes play a crucial role in enabling plants to adapt to fluctuations in their environment. Furthermore, hormones such as abscisic acid (ABA), jasmonic acid (JA) and salicylic acid (SA) have been previously identified as regulators of plant responses to different abiotic stresses. Here we studied the response of Arabidopsis wild type (Col and Ler) plants and mutants impaired in hormone biosynthesis (aba2-11 and aba1-1 in ABA, aos in JA and sid2 in SA) to the combination of S and HL (S + HL). Our findings showed that aba2-11 plants displayed reduced growth, impaired photosystem II (PSII) function, increased leaf damage, and decreased survival compared to Col when subjected to stress combination. However, aos and sid2 mutants did not display significant changes in response to S + HL compared to Col, indicating a key role for ABA in promoting plant tolerance to S + HL and suggesting a marginal role for JA and SA in this process. In addition, we revealed differences in the metabolic response of plants to S + HL compared to S or HL. The analysis of altered metabolic pathways under S + HL suggested that the accumulation of flavonoids is ABA-dependent, whereas the accumulation of branched-chain amino acids (BCAAs) and proline is ABA-independent. Therefore, our study uncovered a key function for ABA in regulating the accumulation of different flavonoids in plants during S + HL.

A metabolome and transcriptome survey to tap the dynamics of fruit prolonged shelf-life and improved quality within Greek tomato germplasm.

Introduction: Tomato is a high economic value crop worldwide with recognized nutritional properties and diverse postharvest potential. Nowadays, there is an emerging awareness about the exploitation and utilization of underutilized traditional germplasm in modern breeding programs. In this context, the existing diversity among Greek accessions in terms of their postharvest life and nutritional value remains largely unexplored. Methods: Herein, a detailed evaluation of 130 tomato Greek accessions for postharvest and nutritional characteristics was performed, using metabolomics and transcriptomics, leading to the selection of accessions with these interesting traits. Results: The results showed remarkable differences among tomato Greek accessions for overall ripening parameters (color, firmness) and weight loss. On the basis of their postharvest performance, a balance between short shelf life (SSL) and long shelf life (LSL) accessions was revealed. Metabolome analysis performed on 14 selected accessions with contrasting shelf-life potential identified a total of 206 phytonutrients and volatile compounds. In turn, transcriptome analysis in fruits from the best SSL and the best LSL accessions revealed remarkable differences in the expression profiles of transcripts involved in key metabolic pathways related to fruit quality and postharvest potential. Discussion: The pathways towards cell wall synthesis, polyamine synthesis, ABA catabolism, and steroidal alkaloids synthesis were mostly induced in the LSL accession, whereas those related to ethylene biosynthesis, cell wall degradation, isoprenoids, phenylpropanoids, ascorbic acid and aroma (TomloxC) were stimulated in the SSL accession. Overall, these data would provide valuable insights into the molecular mechanism towards enhancing shelf-life and improving flavor and aroma of modern tomato cultivars.

Untargeted Metabolomics of Rind Essential Oils Allowed to Differentiate Two Closely Related Clementine Varieties.

Chemical characterization of clementine varieties (Citrus clementina Hort. ex Tan.) essential oils (EO) can lead to variety identification and valorization of their potential use in food and aroma industries. The goal of this study was the chemometric discrimination between two very closely related and morphologically identical clementine varieties, Clemenules (NL) and Clemenpons (PO), based on their rind EO, to identify the differential volatile organic compounds (VOCs) and to determine their antioxidant capacity. EO rind volatile profile was determined by gas chromatography coupled to mass spectrometry in Citrus fruit at different ripening stages grown two independent years in two different locations. Untargeted metabolomics and multivariate data analysis showed an evolution of EO volatile profiles markedly parallel in both varieties. Although EO qualitative composition was identical in both varieties, PLS-DA allowed the identification of characteristic VOCs, quantitatively discriminating them along all the ripening process. PO showed higher accumulation of several mono- and sesquiterpene compounds such as trans-carveol, while NL showed higher levels of aldehyde and alcohol non-terpenoids like dodecanal. Both varieties evinced identical EO antioxidant activities, indicating a similar value for food preservation. Hence, untargeted metabolomics approach based on rind EO volatiles was revealed as a powerful technique able to differentiate between morphologically undistinguishable Citrus varieties.

Quantitation of biogenic tetraamines in Arabidopsis thaliana.

Classical methods for the determination of polyamines in Arabidopsis, based on high-performance liquid chromatography or thin-layer chromatography, do not distinguish between aliphatic tetraamines or do not allow accurate quantitation, respectively. Here we describe a gas chromatography-mass spectrometry method that allows separation and simple quantitation of spermine and thermospermine. The reliability of the protocol has been confirmed using extracts of Arabidopsis mutants impaired in tetraamine biosynthesis. The levels of thermospermine are around 6-fold lower than those of spermine in Arabidopsis seedlings. Interestingly, hormonal effects on polyamine levels are not always correlated with those of transcripts of their biosynthetic enzymes.

Determination of Plant Volatile Apocarotenoids.

Volatile compounds derived from carotenoid cleavage are biologically relevant molecules in plants, as they are involved in different processes of plant communication and also have a significant effect on our perception of food flavor.Here we describe two methods for the determination of volatile apocarotenoids in fruit based on extraction by means of headspace solid-phase microextraction (HS-SPME) and separation and detection by means of gas chromatography coupled to mass spectrometry (GC-MS). The first method is optimised for the detection of the complete volatile profile in Citrus fruit, including those compounds derived from carotenoids. The second is a shorter method focused to the detection of volatile apocarotenoids in the tomato fruit.

Volatile Compounds in Citrus Essential Oils: A Comprehensive Review.

The essential oil fraction obtained from the rind of Citrus spp. is rich in chemical compounds of interest for the food and perfume industries, and therefore has been extensively studied during the last decades. In this manuscript, we provide a comprehensive review of the volatile composition of this oil fraction and rind extracts for the 10 most studied Citrus species: C. sinensis (sweet orange), C. reticulata (mandarin), C. paradisi (grapefruit), C. grandis (pummelo), C. limon (lemon), C. medica (citron), C. aurantifolia (lime), C. aurantium (bitter orange), C. bergamia (bergamot orange), and C. junos (yuzu). Forty-nine volatile organic compounds have been reported in all 10 species, most of them terpenoid (90%), although about half of the volatile compounds identified in Citrus peel are non-terpenoid. Over 400 volatiles of different chemical nature have been exclusively described in only one of these species and some of them could be useful as species biomarkers. A hierarchical cluster analysis based on volatile composition arranges these Citrus species in three clusters which essentially mirrors those obtained with genetic information. The first cluster is comprised by C. reticulata, C. grandis, C. sinensis, C. paradisi and C. aurantium, and is mainly characterized by the presence of a larger abundance of non-terpenoid ester and aldehyde compounds than in the other species reviewed. The second cluster is comprised by C. junos, C. medica, C. aurantifolia, and C. bergamia, and is characterized by the prevalence of mono- and sesquiterpene hydrocarbons. Finally, C. limon shows a particular volatile profile with some sulfur monoterpenoids and non-terpenoid esters and aldehydes as part of its main differential peculiarities. A systematic description of the rind volatile composition in each of the species is provided together with a general comparison with those in leaves and blossoms. Additionally, the most widely used techniques for the extraction and analysis of volatile Citrus compounds are also described.

Zoophytophagous mirids provide pest control by inducing direct defences, antixenosis and attraction to parasitoids in sweet pepper plants.

BACKGROUND: In addition to their services as predators, mirid predators are able to induce plant defences by phytophagy. However, whether this induction occurs in sweet pepper and whether it could be an additional benefit to their role as a biological control agent in this crop remain unknown. Here, these questions were investigated in two model insects, the mirids Nesidiocoris tenuis and Macrolophus pygmaeus. RESULTS: Plant feeding behaviour was observed in both N. tenuis and M. pygmaeus on sweet pepper and occupied 33% and 14% of total time spent on the plant, respectively. The punctures caused by mirid plant feeding induced the release of a blend of volatile organic compounds (VOCs) which repelled the herbivore pests Frankliniella occidentalis and Bemisia tabaci and attracted the whitefly parasitoid Encarsia formosa. The repellent effect on B. tabaci was observed for at least 7 days after initial exposure of the plant to N. tenuis, and attraction of E. formosa remained functional for 14 days. CONCLUSION: Plant defences induced by the feeding of mirid predators, their subsequent effects on the behaviour of both pests and natural enemies, and the persistence of these observed effects open the door to new control strategies in the sweet pepper crop. Further application of this research is discussed, such as the vaccination of plants by zoophytophagous mirids in the nursery before transplantation. © 2017 Society of Chemical Industry.

Genetic analysis of the wild strawberry (Fragaria vesca) volatile composition.

The volatile composition of wild strawberry (Fragaria vesca) fruit differs from that of the cultivated strawberry, having more intense and fruity aromas. Over the last few years, the diploid F. vesca has been recognized as a model species for genetic studies of cultivated strawberry (F. x ananassa), and here a previously developed F. vesca/F. bucharica Near Isogenic Line collection (NIL) was used to explore genetic variability of fruit quality traits. Analysis of fruit volatiles by GC-MS in our NIL collection revealed a complex and highly variable profile. One hundred compounds were unequivocally identified, including esters, aldehydes, ketones, alcohols, terpenoids, furans and lactones. Those in a subset, named key volatile compounds (KVCs), are likely contributors to the special aroma/flavour of wild strawberry. Genetic analysis revealed 50 major quantitative trait loci (QTL) including 14 QTL for KVCs, and one segregating as a dominant monogenetic trait for nerolidol. The most determinant regions affecting QTLs for KVCs, were mapped on LG5 and LG7. New candidate genes for the volatile QTL are proposed, based on differences in gene expression between NILs containing specific fragments of F. bucharica and the F. vesca recurrent genome. A high percentage of these candidate genes/alleles were colocalized within the boundaries of introgressed regions that contain QTLs, appearing to affect volatile metabolite accumulation acting in cis. A NIL collection is a good tool for the genetic dissection of volatile accumulation in wild strawberry fruit and a source of information for genes and alleles which may enhance aroma in cultivated strawberry.

Gene-Metabolite Networks of Volatile Metabolism in Airen and Tempranillo Grape Cultivars Revealed a Distinct Mechanism of Aroma Bouquet Production.

Volatile compounds are the major determinants of aroma and flavor in both grapes and wine. In this study, we investigated the emission of volatile and non-volatile compounds during berry maturation in two grape varieties (Airén and Tempranillo) throughout 2010 and 2011. HS-SPME coupled to gas chromatography and mass spectrometry was applied for the identification and relative quantitation of these compounds. Principal component analysis was performed to search for variability between the two cultivars and evolution during 10 developmental stages. Results showed that there are distinct differences in volatile compounds between cultivars throughout fruit development. Early stages were characterized in both cultivars by higher levels of some apocarotenoids such as ß-cyclocitral or ß-ionone, terpenoids (E)-linalool oxide and (Z)-linalool oxide and several furans, while the final stages were characterized by the highest amounts of ethanol, benzenoid phenylacetaldehyde and 2-phenylethanol, branched-amino acid-derived 3-methylbutanol and 2-methylbutanol, and a large number of lipid derivatives. Additionally, we measured the levels of the different classes of volatile precursors by using liquid chromatography coupled to high resolution mass spectrometry. In both varieties, higher levels of carotenoid compounds were detected in the earlier stages, zeaxanthin and α-carotene were only detected in Airén while neoxanthin was found only in Tempranillo; more variable trends were observed in the case of the other volatile precursors. Furthermore, we monitored the expression of homolog genes of a set of transcripts potentially involved in the biosynthesis of these metabolites, such as some glycosyl hydrolases family 1, lipoxygenases, alcohol dehydrogenases hydroperoxide lyases, O-methyltransferases and carotenoid cleavage dioxygenases during the defined developmental stages. Finally, based on Pearson correlation analyses, we explored the metabolite-metabolite fluctuations within VOCs/precursors during the berry development; as well as tentatively linking the formation of some metabolites detected to the expression of some of these genes. Our data showed that the two varieties displayed a very different pattern of relationships regarding the precursor/volatile metabolite-metabolite fluctuations, being the lipid and the carotenoid metabolism the most distinctive between the two varieties. Correlation analysis showed a higher degree of overall correlation in precursor/volatile metabolite-metabolite levels in Airén, confirming the enriched aroma bouquet characteristic of the white varieties.

Metabolomic profiling of plant tissues.

Metabolomics is a powerful discipline aimed at a comprehensive and global analysis of the metabolites present in a cell, tissue, or organism, and to which increasing attention has been paid in the last few years. Given the high diversity in physical and chemical properties of plant metabolites, not a single method is able to analyze them all.Here we describe two techniques for the profiling of two quite different groups of metabolites: polar and semi-polar secondary metabolites, including many of those involved in plant response to biotic and abiotic stress, and volatile compounds, which include those responsible of most of our perception of food flavor. According to these techniques, polar and semi-polar metabolites are extracted in methanol, separated by liquid chromatography (UPLC), and detected by a UV-VIS detector (PDA) and a time-of-flight (ToF) mass spectrometer. Volatile compounds, on the other hand, are extracted by headspace solid phase microextraction (HS-SPME), and separated and detected by gas chromatography coupled to mass spectrometry (GC-MS).