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.

Fruit flesh volatile and carotenoid profile analysis within the Cucumis melo L. species reveals unexploited variability for future genetic breeding.

BACKGROUND: Aroma profile and carotenoids content of melon flesh are two important aspects influencing the quality of this fruit that have been characterized using only selected genotypes. However, the extant variability of the whole species remains unknown. RESULTS: A complete view of the volatile/carotenoid profiles of melon flesh was obtained analyzing 71 accessions, representing the whole diversity of the species. Gas chromatography-mass spectrometry and high-performance liquid chromatography were used to analyze 200 volatile compounds and five carotenoids. Genotypes were classified into two main clusters (high/low aroma), but with a large diversity of differential profiles within each cluster, consistent with the ripening behavior, flesh color and proposed evolutionary and breeding history of the different horticultural groups. CONCLUSION: Our results highlight the huge amount of untapped aroma diversity of melon germplasm, especially of non-commercial types. Also, landraces with high nutritional value with regard to carotenoids have been identified. All this knowledge will encourage melon breeding, facilitating the selection of the genetic resources more appropriate to develop cultivars with new aromatic profiles or to minimize the impact of breeding on melon quality. The newly characterized sources provide the basis for further investigations into specific genes/alleles contributing to melon flesh quality. © 2018 Society of Chemical Industry.

Fruit volatile profiles of two citrus hybrids are dramatically different from those of their parents.

Volatile compounds released from the fruit of two hybrid Citrus genotypes (FxCh90 and FxCh77) were compared to those from their parental varieties, Fortune mandarin and Chandler pummelo. A series of 113 compounds were identified, including 31 esters, 23 aldehydes, 20 alcohols, 17 monoterpenoids, and other compounds. The differences in the volatile profile among these four genotypes were essentially quantitative. The most striking result was that the volatile profile of the hybrids was not intermediate between their parents and completely differed from that of Chandler, but came closer to Fortune. This was because 56 of the 113 volatile compounds in the hybrids showed significantly higher or lower levels than in any of the parents. Such transgressive behavior in these hybrids was not observed for other fruit quality traits, such as acidity or soluble solid content. The combination of volatile profiling and chemometrics can be used to select new Citrus genotypes with a distinct volatile profile.

Proteomic study of 'Moncada' mandarin buds from on- versus off-crop trees.

A proteomic analysis of buds from mandarin trees with contrasting fruit load (on- and off-crop trees) was carried out during the onset of low-temperature induction. The aim of the study was to find out more about the molecular mechanism relating to alternate bearing in Citrus and its relationship with flowering. The 'Moncada' variety (Clementine 'Oroval'x'Kara' mandarin), displaying remarkable behaviour in alternate production, was used in this study. From 2D DIGE gel, 192 spots were isolated: 97 showed increased expression in the off-crop buds as compared to the on-crop buds, while 95 exhibited enhanced expression in the on-crop buds versus the off-crop buds. These spots were identified by MALDI-MS or LC-MS-MS. The largest groups of proteins up-expressed in the off-crop buds were the proteins involved in carbohydrate and amino acid metabolism, and the proteins expressed in response to stimuli such as reactive oxygen species. The largest groups of proteins up-expressed in the on-crop buds were related to primary metabolism, oxidative stress and defence responses. Depending on their function, some of these proteins can stimulate the flowering, such as fructose-bisphosphate aldolase or leucine-rich repeat transmembrane protein kinase, while others can inhibit it, such as cytochrome c oxidase subunit II. Twenty-two other proteins with unknown functions were up-expressed in the on- or off-crop buds.

Proteomic analysis of "Moncada" mandarin leaves with contrasting fruit load.

A proteomic approach was used to know more about the molecular mechanism related to Citrus alternate bearing. To this end, we researched protein expression differences between on-crop and off-crop "Moncada" [Clementine 'Oroval' (Citrus clementina Hort ex Tanaka) x 'Kara' mandarin (Citrus unshiu Marc. x Citrus nobilis Lou.)] mandarin leaves. This variety usually shows a remarkable behaviour in alternate production. Samples were collected in the period during which the fruit affect flowering induction. From 2D DIGE gel, 110 spots were isolated: 43 showed increased expression in the off-crop samples compared to on-crop samples, while 67 showed increased expression in the on-crop samples against off-crop samples. These spots were identified by MALDI-MS or LC-MS-MS. According to the up-expressed proteins in off-crop leaves such as proteins related to nutrient reservoir activity or to the pentose phosphate pathway, the primary metabolism was more active in off-crop trees than in on-crop trees. In contrast, the proteins up-expressed in on-crop samples such as catalase were related to the oxidoreductase activity and, therefore, the redox state seemed different for off-crop and for on-crop leaves. Other proteins with unknown functions were isolated, which could be also related to the alternate bearing and to the flowering induction.

Fruit load modulates flowering-related gene expression in buds of alternate-bearing 'Moncada' mandarin.

BACKGROUND AND AIMS: Gene determination of flowering is the result of complex interactions involving both promoters and inhibitors. In this study, the expression of flowering-related genes at the meristem level in alternate-bearing citrus trees is analysed, together with the interplay between buds and leaves in the determination of flowering. METHODS: First defruiting experiments were performed to manipulate blossoming intensity in 'Moncada' mandarin, Citrus clementina. Further defoliation was performed to elucidate the role leaves play in the flowering process. In both cases, the activity of flowering-related genes was investigated at the flower induction (November) and differentiation (February) stages. KEY RESULTS: Study of the expression pattern of flowering-genes in buds from on (fully loaded) and off (without fruits) trees revealed that homologues of FLOWERING LOCUS T (CiFT), TWIN SISTER OF FT (TSF), APETALA1 (CsAP1) and LEAFY (CsLFY) were negatively affected by fruit load. CiFT and TSF activities showed a marked increase in buds from off trees through the study period (ten-fold in November). By contrast, expression of the homologues of the flowering inhibitors of TERMINAL FLOWER 1 (CsTFL), TERMINAL FLOWER 2 (TFL2) and FLOWERING LOCUS C (FLC) was generally lower in off trees. Regarding floral identity genes, the increase in CsAP1 expression in off trees was much greater in buds than in leaves, and significant variations in CsLFY expression (approx. 20 %) were found only in February. Defoliation experiments further revealed that the absence of leaves completely abolished blossoming and severely affected the expression of most of the flowering-related genes, particularly decreasing the activity of floral promoters and of CsAP1 at the induction stage. CONCLUSIONS: These results suggest that the presence of fruit affects flowering by greatly altering gene-expression not only at the leaf but also at the meristem level. Although leaves are required for flowering to occur, their absence strongly affects the activity of floral promoters and identity genes.

Fruit regulates seasonal expression of flowering genes in alternate-bearing 'Moncada' mandarin.

BACKGROUND AND AIMS: The presence of fruit has been widely reported to act as an inhibitor of flowering in fruit trees. This study is an investigation into the effect of fruit load on flowering of 'Moncada' mandarin and on the expression of putative orthologues of genes involved in flowering pathways to provide insight into the molecular mechanisms underlying alternate bearing in citrus. METHODS: The relationship between fruit load and flowering intensity was examined first. Defruiting experiments were further conducted to demonstrate the causal effect of fruit removal upon flowering. Finally, the activity of flowering-related genes was investigated to determine the extent to which their seasonal expression is affected by fruit yield. KEY RESULTS: First observations and defruiting experiments indicated a significant inverse relationship between preceding fruit load and flowering intensity. Moreover, data indicated that when fruit remained on the tree from November onwards, a dramatic inhibition of flowering occurred the following spring. The study of the expression pattern of flowering-genes of on (fully loaded) and off (without fruits) trees revealed that homologues of FLOWERING LOCUS T (FT), SUPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), APETALA1 (AP1) and LEAFY (LFY) were negatively affected by fruit load. Thus, CiFT expression showed a progressive increase in leaves from off trees through the study period, the highest differences found from December onwards (10-fold). Whereas differences in the relative expression of SOC1 only reached significance from September to mid-December, CsAP1 expression was constantly higher in those trees through the whole study period. Significant variations in CsLFY expression only were found in late February (close to 20 %). On the other hand, the expression of the homologues of TERMINAL FLOWER 1 (TFL1) and FLOWERING LOCUS C (FLC) did not appear to be related to fruit load. CONCLUSIONS: These results suggest for the first time that fruit inhibits flowering by repressing CiFT and SOC1 expression in leaves of alternate-bearing citrus. Fruit also reduces CsAP1 expression in leaves, and the significant increase in leaf CsLFY expression from off trees in late February was associated with the onset of floral differentiation.

Comparative analysis of the volatile fraction of fruit juice from different Citrus species.

The volatile composition of fruit from four Citrus varieties (Powell Navel orange, Clemenules mandarine, and Fortune mandarine and Chandler pummelo) covering four different species has been studied. Over one hundred compounds were profiled after HS-SPME-GC-MS analysis, including 27 esters, 23 aldehydes, 21 alcohols, 13 monoterpene hydrocarbons, 10 ketones, 5 sesquiterpene hydrocarbons, 4 monoterpene cyclic ethers, 4 furans, and 2 aromatic hydrocarbons, which were all confirmed with standards. The differences in the volatile profile among juices of these varieties were essentially quantitative and only a few compounds were found exclusively in a single variety, mainly in Chandler. The volatile profile however was able to differentiate all four varieties and revealed complex interactions between them including the participation in the same biosynthetic pathway. Some compounds (6 esters, 2 ketones, 1 furan and 2 aromatic hydrocarbons) had never been reported earlier in Citrus juices. This volatile profiling platform for Citrus juice by HS-SPME-GC-MS and the interrelationship detected among the volatiles can be used as a roadmap for future breeding or biotechnological applications.

Root signalling and modulation of stomatal closure in flooded citrus seedlings.

In this work, we studied the sequence of responses induced by flooding in citrus plants, with the aim of identifying the signals that lead to stomatal closure. One-year-old seedlings of Carrizo citrange, grown in sand under greenhouse conditions, were waterlogged for 35 d and compared with normally watered well-drained plants. Significant decreases in stomatal conductance and transpiration were detected between flooded and control seedlings from a week after the beginning of the experiment. However ABA concentration in leaves only started to increase after three weeks of flooding, suggesting that stomata closed in the absence of a rise in foliar ABA. Therefore, stomatal closure in waterlogged seedlings does not appear to be induced by ABA, at least during the early stages of flood-stress. The low levels of ABA detected in roots and xylem sap from flooded seedlings indicated that it is very unlikely that the ABA increase in the leaves of these plants is due to ABA translocation from roots to shoots. We propose that ABA is produced in old leaves and transported to younger leaves. Flooding had no effect on water potential or the relative water content of leaves. Soil flooding reduced root hydraulic conductance in citrus seedlings. This effect was already evident after a week of waterlogging, and at the end of the experiment, flood-stressed seedlings reached values of root hydraulic conductance below 12% of that of control plants. This reduction was related to down-regulation of the expression of PIP aquaporins. In addition, whole plant transpiration was reduced by 56% after 35 d under flooding conditions. Flood-stress also decreased the pH of sap extracted from citrus roots. Evidence is presented suggesting that acidosis induced by anoxic stress in roots causes gating of aquaporins, thereby decreasing hydraulic conductance. Additionally, stomatal closure finely balances-out low pH-mediated losses of root hydraulic conductance therefore maintaining stable leaf hydration.

HS-SPME comparative analysis of genotypic diversity in the volatile fraction and aroma-contributing compounds of Capsicum fruits from the annuum-chinense-frutescens complex.

Volatile constituents of ripe fruits of 16 Capsicum accessions from the annuum-chinense-frutescens complex, with different aroma impressions and geographical origins, were isolated by headspace-solid phase microextraction (HS-SPME) and analyzed by gas chromatography-olfactometry-mass spectrometry (GC-sniffing port-MS). More than 300 individual compounds could be detected in the studied genotypes; most of them could be identified by comparing mass spectra and retention times with authentic reference substances or literature data. Esters and terpenoids were the main groups, although other minor compounds, such as nitrogen and sulfur compounds, phenol derivatives, norcarotenoids, lipoxygenase derivatives, carbonyls, alcohols, and other hydrocarbons, were also identified. The sniffing test revealed that the diversity of aromas found among the studied cultivars is due to qualitative and quantitative differences of, at least, 23 odor-contributing volatiles (OCVs). C. chinense, and C. frutescens accessions, with fruity/exotic aromas, were characterized by a high contribution of several esters and ionones and a low or nil contribution of green/vegetable OCVs. Different combinations of fruity/exotic and green/vegetable OCVs would explain the range of aroma impressions found among C. annuum accessions. Implications of these findings for breeding and phylogeny studies in Capsicum are also discussed.

Terretonins E and F, inhibitors of the mitochondrial respiratory chain from the marine-derived fungus Aspergillus insuetus (#).

Two new meroterpenoids, terretonins E and F (1, 2), together with three known compounds, aurantiamine (3), linoleic acid, and uridine, were isolated as fermentation products of the marine-derived fungus Aspergillus insuetus, which was associated with the sponge Petrosia ficiformis. Structures of all isolates were elucidated employing spectroscopic methods, mainly by two-dimensional NMR techniques. Compounds 1-3 showed activity as inhibitors of the mammalian mitochondrial respiratory chain.

Isolation and structural elucidation of eight new related analogues of the mycotoxin (-)-botryodiplodin from Penicillium coalescens.

Bioassay-guided fractionation of the organic extract derived from the terrestrial fungus Penicillium coalescens led to the isolation of the known mycotoxin (-)-botryodiplodin (1) and eight new structurally related analogues (2-9). The structures of the novel compounds were determined by MS and NMR studies, including 1D and 2D NMR. A likely biogenetic pathway from the aldehydic open form of 1 (C7 unit, U1) is proposed for these metabolites. Among all the isolated metabolites, only (-)-1 showed antifungal, antibacterial, and insecticidal activity. This latter activity appears to be a new property attributed to (-)-1.