Phytochemistry

Five undescribed C₂₁ pregnane glycosides, epigycosides D−H, together with four known analogues, two lignans, and a flavonoid have been isolated from the stems of Epigynum cochinchinensis. The structures of pregnane glycosides were elucidated using spectroscopic techniques and acid hydrolysis. The in vitro immunological activities were assessed against Con A-stimulated proliferation of mice splenocytes. The C₂₁ pregnane glycosides showed immunosuppressive activity in a concentration-dependent manner. Moreover, epigycoside E exhibited a potent immunosuppressive effect, and the IC₅₀ value on Con A-stimulated mice splenocytes was 22.1 ± 6.4 μM. Epigycoside E also caused G₀/G₁ arrest, and inhibited TNF-α and IL-2 production.

Endophytic fungi are microorganisms located in the inter- or intracellular compartments of plant tissues but with no harmful effects. They are considered a potential source of biological compounds. The present study was conducted to investigate the molecular identification of endophytic fungi isolated from the roots of Lithospermum officinale and their potential production of shikonin. Phylogenetic analysis was performed based on the Internal Transcribed Spacer (ITS) region and the isolates were classified into five genera as follows: Alternaria, Chaetosphaeronema, Fusarium, Mucor, and Trichoderma. The study on the methanol extracts of endophytic fungi indicated that total polyphenol content had a positive relationship with antioxidant activities and the highest antioxidant activity belonged to the methanol extracts of Fusarium tricinctum and Alternaria altenata. Then, to investigate the ability of the fungal isolates to produce shikonin, a naphthoquinone compound with high biological activity, the extracts were subjected to HPLC. The results obtained from HPLC-mass spectrometry showed that shikonin could be produced only by F. tricinctum. Thus, F. tricinctum isolated from the roots of L. officinale can be presented as a new source of shikonin.

Sixteen diterpenoids including nine undescribed ones, named rhodoauriculatols A−I, were isolated from the leaves of Rhododendron auriculatum Hemsl. Sixteen diterpenoids belong to seven diverse carbon skeletons, which were classified into 1,10-seco-grayanane, 1,10:2,3-diseco-grayanane, A-homo-B-nor-ent-kaurane, ent-kaurane, 4,5-seco-ent-kaurane, leucothane, and grayanane, respectively. Their structures were determined by the detailed HRESIMS, 1D and 2D NMR, UV, and IR data analysis, and their absolute configurations were established by single crystal X-ray diffraction analysis, electronic circular dichroism (ECD) data analysis, ECD calculation, as well as chemical methods. Rhodoauriculatols A−C possess a rare 1,10-seco-grayanane diterpene skeleton. Rhodoauriculatol D is the second example of the 1,10:2,3-diseco-grayanane diterpenoids, and rhodoauriculatol E is the fourth example of the A-homo-B-nor-ent-kaurane diterpenoids. Rhodomicranone E was reported as a natural product for the first time. All the isolated sixteen diterpenoids showed analgesic activities in the acetic acid-induced writhing test. Rhodoauriculatols B, E−G, rhodomicranone E, pierisformoside F, and micranthanoside A showed significant analgesic activities with the inhibition rates over 40%, and their preliminary structures-activity relationships were studied.

Strigolactone (SL) plant hormones control plant architecture and are key players in both symbiotic and parasitic interactions. GR24, a synthetic SL analog, is the worldwide reference compound used in all bioassays for investigating the role of SLs in plant development and in rhizospheric interactions. In 2012, the first characterization of the SL receptor reported the detection of an unknown compound after incubation of GR24 samples with the SL receptor. We reveal here the origin of this compound (P270), which comes from a by-product formed during GR24 chemical synthesis. We present the identification of this by-product, named contalactone. A proposed chemical pathway for its formation is provided as well as an evaluation of its bioactivity on pea, Arabidopsis, root parasitic plant seeds and AM fungi, characterizing it as a SL mimic. Quality of GR24 samples can be easily checked by carrying out microscale hydrolysis in a basic aqueous medium to easily detect P270 as indicator of the presence of the contalactone impurity. In all cases, before being used for bioassays, GR24 must be careful purified by preparative HPLC.

Eight undescribed ergostane-type steroids, (22E,24R)-ergosta-7,22-dien-3β,5α-diol- 6,5-olide, (22E,24R)-ergosta-7,9(11),22-trien-3β,5β,6β-triol, (22E,24R)-6β-methoxy ergosta-7,9(11),22-trien-3β,5α,14β-triol, (22E,24R)-9α,15α-dihydroxyergosta-4,6,8 (14),22-tetraen-3-one, (22E,24R)-ergosta-5,8,22-trien-3β,11α-dihydroxyl-7-one, (22E,24R)-ergosta-4,7,22-trien-3β,9α,14β-trihydroxyl-6-one, (22E,24R)-ergosta-7,22- dien-3β,9α,14β-trihydroxyl-6-one, and (22E,24R)-6β-methoxyergosta-7,22-dien-3β, 5α,9α,14β-tetraol, and twenty-one known analogues were isolated from the fruiting bodies of Ganoderma resinaceum Boud. Their chemical structures were determined on the basis of comprehensive spectroscopic analysis and X-ray crystal diffraction, as well as empirical pyridine-induced deshielding effects. Furthermore, selected compounds were evaluated for their inhibitory effects on macrophage activation using an inhibition of nitric oxide production assay. Finally, (22E,24R)-ergosta-5,8,22- trien-3β,11α-dihydroxyl-7-one, (22E,24R)-ergosta-4,7,22-trien-3β,9α,14β-tri hydroxyl-6-one, (22E,24R)-6β-methoxyergosta-7,22-dien-3β,5α,9α,14β-tetraol, (22E,24R)-ergosta-6,9,22-trien-3β,5α,8α-triol,ergost-6,22-dien-3β,5α,8α-triol, 5α,6α-epoxy-(22E,24R)-ergosta-8,22-diene-3β,7α-diol, 5α,6α-epoxy-(22E,24R)- ergosta-8(14),22-diene-3β,7α-diol, 5α,6α-epoxy-(22E,24R)-ergosta-8(14),22-diene-3β, 7β-diol, and 22E-7α-methoxy-5α,6α-epoxyergosta-8(14),22-dien-3β-ol showed inhibitory effects on NO production with IC₅₀ values ranging from 3.24 ± 0.02 to 35.19 ± 0.41 μM compared with L-NMMA (IC₅₀ 49.86 ± 2.13 μM), indicating that they have potential anti-inflammatory activity.

Based on the occurrence of indole alkaloids in so-called “chloroform leaf surface extracts”, it was previously deduced that these alkaloids are present in the cuticle at the leaf surface of Catharanthus roseus and Vinca minor. As no symplastic markers were found in these extracts this deduction seemed to be sound. However, since chloroform is known to destroy biomembranes very rapidly, these data have to be judged with scepticism.

We reanalyzed the alleged apoplastic localization of indole alkaloids by employing slightly acidic aqueous surface extracts and comparing the corresponding alkaloid patterns with those of aqueous total leaf extracts. Whereas in the “chloroform leaf surface extracts” all alkaloids are present in the same manner as in the total leaf extracts, no alkaloids occur in the aqueous leaf surface extracts. These results clearly show that chloroform had rapidly destroyed cell integrity, and the related extracts also contain the alkaloids genuinely accumulated within the protoplasm. The related decompartmentation was verified by the massively enhanced concentration of amino acids in aqueous surface extracts of chloroform treated leaves. Furthermore, the chloroform-induced cell disintegration was vividly visualized by confocal laser scanning microscopical analyses, which clearly displayed a strong decrease in the chlorophyll fluorescence in chloroform treated leaves. These findings unequivocally display that the indole alkaloids are not located in the apoplastic space, but exclusively are present symplastically within the cells of V. minor and C. roseus leaves. Accordingly, we have to presume that also other leaf surface extracts employing organic solvents have to be re-investigated.

Idesia polycarpa is a valuable oil-producing tree and can potentially be used for edible oil and biofuel production. The fruits of I. polycarpa are unique in that they contain both saturated and unsaturated lipids. Fatty acid desaturase 2 (FAD2), also as known as omega-6 fatty acid desaturase in endoplasmic, is a key enzyme for linoleic acid and α-linolenic acid biosynthesis. However, bioinformatics and expression of FAD2 in I. polycarpa are still absent. Here, to gain insight into the lipid and linoleic synthesis of I. polycarpa, we compared the fruits from different growth stages. Lipid accumulation rates, final lipid content, linoleic accumulation rates and final linoleic content were significantly different among the different stages. In a further step, the FAD2 gene from fruits of I. polycarpa, named IpFAD2, was cloned and characterized. A partial fragment of 169 bp of IpFAD2 was amplified by degenerate PCR. Full cDNA of IpFAD2 was obtained by the RACE technique. The open-reading frame of IpFAD2 was 1149 bp in length, encoding 382 amino acids. A comparison of the deduced amino acids sequence of IpFAD2 with FAD2 from other species showed high similarities, ranging from 78.8 to 92.6%. The IpFAD2-predicted protein has a theoretical molecular mass of 44.03 kDa and an isoelectric point (pI) of 8.04. It has five transmembrane helices located on the endoplasmic reticulum. The IpFAD2-predicted protein was classified as belonging to the Membrane-FADS-like superfamily based on its conserved domain analysis. Expression analysis based on qRT-PCR indicated that IpFAD2 was expressed in different fruit growth stages, with the highest expression level at 80 DAP and the lowest at 130 DAP. The expression of IpFAD2 was positively correlated with the linoleic accumulation rates in I. polycarpa fruits. Prokaryotic expression in Escherichia. Coli BL21(DE3) indicated that IpFAD2 gene could encode a bio-functional omega-6 fatty acid desaturase. Heterologous expression in Arabidopsis thaliana confirmed that the isolated IpFAD2 proteins could catalyse linoleic synthesis. This is the first cloning and expression analysis of FAD2 from I. polycarpa, significantly contributing to our understanding of the role of IpFAD2 in linoleic synthesis, esp. in terms of genetic engineering breeding for linoleic production.

Nine undescribed polyacetylated 18-norspirostanol saponins, trilliumosides A‒J, were obtained after a guidance based on a molecular networking strategy from the rhizomes of Trillium tschonoskii. Their structures were established by analysis of comprehensive spectroscopic data and chemical methods after their isolation in pure form. All isolated saponins were evaluated for their cytotoxicities against five selected human cancer cell lines (Huh7,A549,MCF-7,HepG2, and MOLT-4) and anti-inflammatory effects on a lipopolysaccharide (LPS)-stimulated NO production model in RAW264.7 macrophages. Trilliumoside D showed significant cytotoxicity against MOLT-4 cell lines with an IC₅₀ value of 1.0 ± 0.1 μM, whereas trilliumosides H and I displayed remarkable anti-inflammatory effects on NO production with inhibitory rates of 56.3 ± 1.5 and 56.2 ± 2.2% at the concentration of 1.0 μM, respectively.

A C₂₀-diterpenoid alkaloid with an unprecedented carbon skeleton, acoapetaludine A, together with ten undescribed aconitine-type C₁₉-diterpenoid alkaloids, acoapetaludines B–K, were isolated from the whole plants of Aconitum apetalum (Huth) B. Fedtsch. (Ranunculaceae). The structures were elucidated based on a comprehensive spectroscopic data analysis. The absolute configuration of acoapetaludine A was determined by quantum ECD calculation. Acoapetaludines D and E exhibited weak anti-Helicobacter pylori activity at a minimum inhibitory concentration (MIC) of 100 and 50 μg/mL, respectively.

In an effort to identify inhibitors of Chikungunya virus (CHIKV) replication, a systematic study of 594 extracts of plant species originating from the French Guiana plateau region was performed in a virus-cell-based assay for CHIKV assay. The extract obtained from the stem bark of Sagotia racemosa was selected for its potent antiviral activity. Using a classical bioassay-guided procedure, three undescribed degraded diterpenoids, i.e. trigohowilols C and D and trigoflavidol D, as well as trigoxyphin K, stictic acid, hyperhomosekikaic acid and five known flavonoids were isolated. The structures of these compounds were elucidated by extensive NMR spectroscopic data analysis. Although trigohowilols C and D were isolated from the most active fraction they didn't show any antiviral activity. By using the Feature-Based Molecular Networking (FBMN) and Network Annotation Propagation (NAP) workflows, it has been shown that the strong anti-CHIKV activity found for this fraction might be due to the presence of analogues of 12-O-tetradecanoylphorbol-13-acetate (TPA), one of the most potent inhibitors of CHIKV replication identified to date.

Vitamin A deficiency (VAD) is a global health problem, which despite significant financial investments and initiatives has not been eradicated. Biofortification of staple crops with β-carotene (provitamin A) in Low Medium Income Countries (LMICs) is the approach advocated and adopted by the WHO and HarvestPlus programme. The accurate determination of β-carotene is key to the assessment of outputs from these activities. In the present study, HPLC-PDA analysis displayed superior resolving power, separating and identifying 23 carotenoids in the orange sweet potato (Ipomoea batatas) variety used, including only eight carotenoids with provitamin A properties. Additionally, the results evidently displayed that the use of lyophilised material facilitated the extraction of twice the amount of pigments compared to fresh material, which impacts the precise calculation of the provitamin A content. These results highlight that yellow to orange starchy edible crops produce a wide array of carotenoids in addition to β-carotene. Biosynthetically it is clear from the intermediates and products accumulating that the β-branch of the carotenoid pathway persists in sweet potato tuber material. Collectively, the data also have implications with respect to the determination and biosynthesis of provitamin A among staple crops for developing countries.

Although Morinda umbellata L. has been used in numerous folk medicines, there is a lack of phytochemical studies on this plant. Sixteen undescribed quinones, namely, ten anthraquinones (umbellatas A–J), one naphthohydroquinone (umbellata K), one naphthohydroquinone dimer (umbellata L), and four dinaphthofuran quinones (umbellatas M–P), were isolated from the aerial parts of Morinda umbellata L. (Rubiaceae). The structures of all the isolated quinones were elucidated based on spectroscopic methods. Four of the unknown quinones (umbellatas A, H, K and M) showed potent cytotoxic effects against A431, A2780, NCI-H460, HCT116, HepG2, and MCF-7 human cancer cell lines with IC₅₀ values of 1.3–7.1 μM. These results reveal potential lead compounds for the development of new anticancer agents.

Poor pancreatic cancer (PC) prognosis has been attributed to its resistance to apoptosis and propensity for early systemic dissemination. Existing therapeutic strategies are often circumvented by the molecular crosstalk between cell-signalling pathways. p53 is mutated in more than 50% of PC and NFκB is constitutively activated in therapy-resistant residual disease; these mutations and activations account for the avoidance of cell death and metastasis. Recently, we demonstrated the anti-PC potential of fucoidan extract from marine brown alga, Turbinaria conoides (J. Agardh) Kützing (Sargassaceae). In this study, we aimed to characterize the active fractions of fucoidan extract to identify their select anti-PC efficacy, and to define the mechanism(s) involved. Five fractions of fucoidan isolated by ion exchange chromatography were tested for their potential in genetically diverse human PC cell lines. All fractions exerted significant dose-dependent and time-dependent regulation of cell survival. Fucoidans induced apoptosis, activated caspase −3, −8 and −9, and cleaved Poly ADP ribose polymerase (PARP). Pathway-specific transcriptional analysis recognized inhibition of 57 and 38 nuclear factor κB (NFκB) pathway molecules with fucoidan-F5 in MiaPaCa-2 and Panc-1 cells, respectively. In addition, fucoidan-F5 inhibited both the constitutive and Tumor necrosis factor-α (TNFα)-mediated NFκB DNA-binding activity in PC cells. Upregulation of cytoplasmic IκB levels and significant reduction of NFκB-dependent luciferase activity further substantiate the inhibitory potential of seaweed fucoidans on NFκB. Moreover, fucoidan(s) treatment increased cellular p53 in PC cells and reverted NFκB forced-expression-related p53 reduction. The results suggest that fucoidan regulates PC progression and that fucoidans may target p53–NFκB crosstalk and dictate apoptosis in PC cells.

This work used Δ⁵-sterols and their degradation products to compare the efficiency of biotic and abiotic degradation processes in senescent Mediterranean marine (Posidonia oceanica) and terrestrial (Quercus ilex and Smilax aspera) angiosperms. Type II photosensitized oxidation processes appeared to be more efficient in P. oceanica than in Q. ilex and S. aspera. The low efficiency of these processes in senescent terrestrial angiosperms was attributed to: (i) the fast degradation of the sensitizer (chlorophyll) in these organisms and (ii) the relatively high on-ground temperatures observed in Mediterranean regions favoring the diffusion of singlet oxygen outside the membranes. Senescent leaves of P. oceanica contained the highest proportions of photochemically-produced 6-hydroperoxysterols, likely due to the presence of trace amounts of metal ions in seawater catalyzing selective homolytic cleavage of 5- and 7-hydroperoxysterols. Bacterial metabolites of sitosterol and its photooxidation products could be detected in senescent leaves of P. oceanica but not Q. ilex or S. aspera. These results confirmed that biotic and abiotic degradation processes may be intimately linked in the environment.

Six undescribed phenolic derivatives along with thirty two known compounds were isolated from the twigs of Betula schmidtii. The chemical structures were characterized through extensive spectroscopic analysis and chemical methods. All known compounds were first isolated in this plant. The anti-inflammatory effect of the isolates was tested by measuring nitric oxide production in lipopolysaccharide-activated BV-2 cells. Isotachioside, 4-allyl-2-hydrophenyl 1-O-β-D-apiosyl-(1 → 6)-β-D-glucopyranoside, genistein 5-O-β-D-glucoside, and prunetinoside showed a slight potency to lower the NO production against LPS-activated microglia with IC₅₀ values of 23.9, 25.3, 28.8, and 34.0 μM, respectively.

Labdane diterpenes (LDs), and especially sclareol, are important feedstocks for the pharmaceutical and cosmetic industries, and therefore several lines of research have led to their heterologous production in non-photosynthetic microbes and higher plants. The potential of microalgae as bioreactors of natural products has been established for a variety of bioactive metabolites, including terpenes. In this work, a codon optimized sequence encoding a key plant labdane-type diterpene (LD) cyclase, copal-8-ol diphosphate synthase from Cistus creticus (CcCLS), was introduced into the chloroplast genome of Chlamydomonas reinhardtii. Of 49 transplastomic algal lines, 12 produced variable amounts of four LD compounds, namely ent-manoyl oxide, sclareol, labda-13-ene-8α,15-diol and ent-13-epi-manoyl oxide. The total LD concentrations measured in the transplastomic lines reached 1.172 ± 0.05 μg/mg cell DW for the highest overall producer, while the highest yield for sclareol was 0.038 ± 0.001 μg/mg cell DW. Thus, transplastomic expression of a key plant labdane diterpene cyclase in the C. reinhardtii chloroplast genome enabled the production of important plant-specific LD compounds.

Heisteria parvifolia Sm. is prescribed in traditional medecine against numerous diseases in Côte d'Ivoire. Due to the shortcoming in scientifical knowledge of use of this species, our investigations revealed five undescribed cyclopeptide alkaloids added to one known derivative namely anorldianine. These compounds were elucidated by 1D and 2D-NMR experiments and comparison with literature data, and confirmed by HR-ESI-MS. Cytotoxic activity evaluation of these compounds against the chronic myeloid leukemia (K565) cell line exhibited an antiproliferative activity with cell growth inhibition from 13% to 46%.

Changes in specialized metabolites were analyzed in wheat leaves inoculated with Bipolaris sorokiniana, the causal agent of spot blotch of Poaceae species. HPLC analysis detected the accumulation of six compounds in B. sorokiniana-infected leaves. Of these, we purified two compounds by silica gel and ODS column chromatography and preparative HPLC, and identified them as cinnamic acid amides, N-cinnamoyl-9-hydroxy-8-oxotryptamine and N-cinnamoyl-8-oxotryptamine, by spectroscopic analyses. The remaining four compounds were predicted to be p-coumaric acid amides of hydroxyputrescine, hydroxyagmatine, hydroxydehydroagmatine, and agmatine by mass spectrometry. The accumulation of two cinnamic acid amides was also induced by Fusarium graminearum infection, and by treatment with CuCl₂, jasmonic acid, and isopentenyladenine. Antifungal activity of these amides was shown by inhibition of conidial germination and germ tube elongation of F. graminearum and Alternaria brassicicola, indicating that they act as phytoalexins. The accumulation of these amides also detected in barley leaves treated with CuCl₂. We examined the accumulation of 25 phenylamides in B. sorokiniana-infected wheat leaves using LC-MS/MS. Hydroxycinnamic acid amides of tryptamine, serotonin, putrescine, and agmatine, were induced after infection with B. sorokiniana. Thus, the induced accumulation of two groups of phenylamides, cinnamic acid amides with indole amines, and p-coumaric acid amides with putrescine and agmatine related amines, represents a major metabolic response of wheat to pathogen infection.

White mold is a disease caused by the fungus Sclerotinia sclerotiorum, a highly destructive necrotrophic pathogen that infects soybean crops, among others. Usually, the infection triggers the plant defense system to minimize the damages. However, the effects of the infection on soybean plant metabolism are still unclear. In this work, the metabolic profiles of soybean stems and leaves were accessed using ¹H HR-MAS NMR spectroscopy to identify metabolic changes as a response to S. sclerotiorum infection. This fungus widely affects the central metabolism of soybean plants, and most of the altered metabolites are involved in carbon metabolism, as suggested by the results. Furthermore, the metabolites of central metabolism can be associated with the production of several polyphenolic metabolites. Changes in metabolic profile of leaves indicate systemic effects.

Bambusa pervariabilis × Dendrocalamopsis grandis blight, caused by Arthrinium phaeospermum, is one of the most common and serious diseases in bamboo and occurs in the newly born twigs. Bamboo has suffered large dead areas, including more than 3000 hm², which greatly threatens the process of returning farmlands to forests and the construction of ecological barriers. To identify differential metabolites and metabolic pathways associated with B. pervariabilis × D. grandis to A. phaeospermum, ultra-performance liquid chromatography (UPLC) and quadrupole-time of flight (Q-TOF) Mass Spectrometry (MS) combined with a data-dependent acquisition method was used to analyse the entire sample spectrum. In total, 13223 positive ion peaks and 10616 negative ion peaks were extracted. OPLS-DA and several other analyses were performed using the original data. The OPLS-DA models showed good quality and had strong predictive power, indicating clear trends in the analyses of the treatment and control groups. Clustering and KEGG pathway analyses were used to screen the differential metabolites in the treatment and control groups from the three B. pervariabilis × D. grandis varieties and reflected their metabolic responses induced by A. phaeospermum infection. The results showed that the three B. pervariabilis × D. grandis varieties mode showed significant changes in the following six resistance-related metabolites after A. phaeospermum invasion in positive and negative ion modes: proline, glutamine, dictamnine, apigenin 7-O-neohesperidoside, glutamate, and cis-Aconitate. The following four main metabolic pathways are involved: Arginine and proline metabolism, Glyoxylate and dicarboxylate metabolism, Biosynthesis of alkaloids derived from shikimate pathway, and Flavone and flavonol biosynthesis. This study lays a foundation for the later detection of differential metabolites and metabolic pathways for targeting, and provides a theoretical basis for disease-resistant breeding and the control of B. pervariabilis × D. grandis blight.

The medicinal plant drug “Tiliae flos” consists of the botanical flowers and bracts of Tilia sp., gathered almost exclusively during flowering. In this study, we examined the changes in the metabolome of specialized products in the bracts of Tilia platyphyllos from the appearance of the organ till the onset of senescence by LC-ESI-MS and data mining.

A set of 504 natural products were detected, 241 of which showed significant seasonal variation (p < 9.92E-5). Seven compounds were quantified and an additional 45 were putatively identified. These included flavonoid glycosides, catechins, procyanidins, quinic acid derivatives (including chlorogenic acid) and coumarins. Compared to bracts during flowering, young tissues were characterized by a relatively high diversity of polyphenolic substances. Higher amounts of flavonol glycosides (quercetin, kaempferol), catechins and derivatives have been observed. Deoxyhexosides were almost exclusive to this phenological stage. Changes of about one order of magnitude were not uncommon. For some substances, 5-fold differences were observed (calibration with authentic standards). Some compounds (e.g. the coumarin fraxin) were more prominent at the late fruit growth stage.

It was shown that bracts gathered before or after flowering could potentially be therapeutically useful. Changes are rapid during the early phase of bract development: three different groups of compounds presented their maxima during the first 32 days. Considering seasonal variation is of extreme importance during bioactivity tests and screening candidate sources for bioactive natural products. In the case of T. platyphyllos, young and old bracts can be of interest because of their high diversity of distinct specialized metabolites.

The worldwide-cultivated chicory (Cichorium intybus L.) produces food and beneficial compounds, and young pre-flowering inflorescence stems are newly marketed vegetables. These sink-organs undergo growth by metabolizing sugars of leaf origin; the carbohydrate content and sweetness are crucial aspects for consumers’ nutrition and acceptance. NMR profiling of 31 hydrosoluble phytochemicals showed that stem contents varied as influenced by genotype, environment and interaction, and that higher sucrose levels were associated with the sweeter of two landraces. Integrative analyses of metabolic and transcriptomic profile variations allowed the dissection of sucrose pathway. Overall, 427 and 23 unigenes respectively fell into the categories of sucrose metabolism and sugar carriers. Among 10 differentially expressed genes, the 11474/sucrose synthase, 53458/fructokinase, 9306 and 17035/hexokinases, and 20171/SWEET-type genes significantly associated to sugar content variation, and deduced proteins were characterised in silico. Correlation analyses encompassing sugar level variation, expressions of the former genes and of computationally assigned transcription factors (10938/NAC, 14712/bHLH, 40133/TALE and 17846/MIKC) revealed a gene network. The latter was minimally affected by the environment and accomplished with markers, representing a resource for biological studies and breeding.

Citrus fruits have been introduced to the Mediterranean area from Asia for centuries and spontaneous crosses have generated several hybrid forms, some of which have had agricultural or industrial success while others have remained niche food or ornamental products, or have disappeared. Pompia (C. medica tuberosa Risso & Poiteau) is an old endemic citrus fruit from Sardinia of unknown genetic origin. Initial phenotypic and molecular characterizations revealed a high degree of similarity with lemon (C. limon (L.) Burm.) and citron (C. medica L.). To identify the ancestors of Pompia, 70 citrus species of the Citrus genus were genotyped with 36 codominant molecular markers (SSR and InDel) of nuclear and cytoplasmic genomes. Diversity analysis and allelic comparisons between each citrus species at each locus indicated that Pompia resembles lemon and limonette of Marrakech, i.e. the result of a cross between sour orange (C. aurantium L.) and citron, where citron was the pollinator. Two Italian citron varieties were identified as potential male parents, i.e. Diamante and Common Poncire. However, we were unable to differentiate varieties of sour oranges because varietal diversification in this horticultural group resulted from DNA sequence variations that SSR or InDel markers could not reveal. Rhob el Arsa and Poncire de Collioure were found to be two synonyms of Pompia. Pompia appeared to be equally distinct from citron, lemon and sour orange based on the overall analysis of the fruit, leaf and seed phenotype, and juice chemical composition. At the leaf level, the Pompia essential oil (EO) composition is close to that of citron whereas the zest is much closer to that of sour orange.