Highly Predictive Genetic Markers Distinguish Drug-Type from Fiber-Type Cannabis sativa L.

Genetic markers can be used in seeds and in plants to distinguish drug-type from fiber-type Cannabis Sativa L. varieties even at early stages, including pre-germination when cannabinoids are not accumulated yet. With this aim, this paper reports sequencing results for tetrahydrocannabinolic acid synthase (THCAS) and cannabidiolic acid synthase (CBDAS) genes from 21 C. sativa L. varieties. Taking into account that THCAS- and CBDAS-derived enzymes compete for the same substrate, the novelty of this work relies in the identification of markers based on both THCAS and CBDAS rather than THCAS alone. Notably, in our panel, we achieved an adequate degree of discrimination (AUC 100%) between drug-type and fiber-type cannabis samples. Our sequencing approach allowed identifying multiple genetic markers (single-nucleotide polymorphisms-SNPs-and a deletion/insertion) that effectively discriminate between the two subgroups of cannabis, namely fiber type vs. drug type. We identified four functional SNPs that are likely to induce decreased THCAS activity in the fiber-type cannabis plants. We also report the finding on a deletion in the CBDAS gene sequence that produces a truncated protein, possibly resulting in loss of function of the enzyme in the drug-type varieties. Chemical analyses for the actual concentration of cannabinoids confirmed the identification of drug-type rather than fiber-type genotypes. Genetic markers permit an early identification process for forensic applications while simplifying the procedures related to detection of therapeutic or industrial hemp.

Transcriptomic and metabolomic analysis of ZmYUC1 mutant reveals the role of auxin during early endosperm formation in maize.

Kernel size in cereal is an important agronomic trait controlled by the interaction of genetic and environmental factors. The endosperm occupies most of the kernel area; for this reason, the endosperm cells dimension, number and metabolic content strongly influence kernel properties. This paper presents the transcriptomic and metabolomic analysis of the maize defective endosperm 18 (de18) mutant, where auxin accumulation in the endosperm is impaired. This mutation, involving the ZmYuc1 gene, leads to a reduced kernel size compared to the wild-type line B37. Our results mainly indicate that IAA concentration controls sugar and protein metabolism during kernel differentiation and it is necessary for BETL formation. Furthermore, a fine tuning of different auxin conjugates is reported as the main mechanism to counteract the auxin deficit. Some candidates as master regulators of endosperm transcriptional regulation mediated by auxin are found between MYB and MADS-box gene families. A link between auxin and storage protein accumulation is highlighted, suggesting that IAA directly or indirectly, through CK or ABA, regulates the transcription of zein coding genes. This study represents a move forward with respect to the current knowledge about the role of auxin during maize endosperm differentiation thus revealing the genes that are modulated by auxin and that control agronomic traits as kernel size and metabolic composition.

Phenolic Profile and Susceptibility to Fusarium Infection of Pigmented Maize Cultivars.

Maize is a staple food source in the world, whose ancient varieties or landraces are receiving a growing attention. In this work, two Italian maize cultivars with pigmented kernels and one inbred line were investigated for untargeted phenolic profile, in vitro antioxidant capacity and resistance to Fusariumverticillioides infection. "Rostrato Rosso" was the richest in anthocyanins whilst phenolic acids were the second class in abundance, with comparable values detected between cultivars. Tyrosol equivalents were also the highest in "Rostrato Rosso" (822.4 mg kg-1). Coherently, "Rostrato Rosso" was highly resistant to fungal penetration and diffusion. These preliminary findings might help in breeding programs, aiming to develop maize lines more resistant to infections and with improved nutraceutical value.

Effect of dietary polyphenols on the in vitro starch digestibility of pigmented maize varieties under cooking conditions.

Interest in using polyphenols as modulators of the activity of starch digestive enzymes is increasing. The main purpose of this study was to investigate the role of phenolic compounds characterising pigmented maize flours in the modulation of in vitro starch digestibility. Flours from three different pigmented maize varieties were evaluated under cooking conditions and compared to common yellow maize (YM). The untargeted metabolomics-based approach comprehensively annotated around 300 phenolic compounds, with a high distribution of anthocyanins and phenolic acids (in free and bound fractions of maize samples) and significant differences across genotypes. Following in vitro starch digestion, the cooked pigmented maize flours showed higher resistant starch content (from 5.1 to 6.9 g /100 g dry matter), as well as lower starch hydrolysis index (HI) when compared to YM flour, with the "Rostrato Rosso" maize having the lowest HI (i.e., 61). Coherently, multivariate statistics following metabolomics showed the discrimination potential of anthocyanins' profile after cooking, characterising the "Rostrato Rosso" during in vitro digestion. These findings might be related to the modulation of enzyme activity by phenolic compounds during in vitro digestion. Therefore, the use of pigmented maize flours might help in the formulation of gluten-free foods with slowly digestible starches by exploiting the wide phenolic composition of these matrices.

Phenolic Profiling for Traceability of Vanilla ×tahitensis.

Vanilla is a flavoring recovered from the cured beans of the orchid genus Vanilla. Vanilla ×tahitensis is traditionally cultivated on the islands of French Polynesia, where vanilla vines were first introduced during the nineteenth century and, since the 1960s, have been introduced to other Pacific countries such as Papua New Guinea (PNG), cultivated and sold as "Tahitian vanilla," although both sensory properties and aspect are different. From an economic point of view, it is important to ensure V. ×tahitensis traceability and to guarantee that the marketed product is part of the future protected designation of the origin "Tahitian vanilla" (PDO), currently in progress in French Polynesia. The application of metabolomics, allowing the detection and simultaneous analysis of hundreds or thousands of metabolites from different matrices, has recently gained high interest in food traceability. Here, metabolomics analysis of phenolic compounds profiles was successfully applied for the first time to V. ×tahitensis to deepen our knowledge of vanilla metabolome, focusing on phenolics compounds, for traceability purposes. Phenolics were screened through a quadrupole-time-of-flight mass spectrometer coupled to a UHPLC liquid chromatography system, and 260 different compounds were clearly evidenced and subjected to different statistical analysis in order to enable the discrimination of the samples based on their origin. Eighty-eight and twenty three compounds, with a prevalence of flavonoids, resulted to be highly discriminant through ANOVA and Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) respectively. Volcano plot analysis and pairwise comparisons were carried out to determine those compounds, mainly responsible for the differences among samples as a consequence of either origin or cultivar. The samples from PNG were clearly different from the Tahitian samples that were further divided in two different groups based on the different phenolic patterns. Among the 260 compounds, metabolomics analysis enabled the detection of previously unreported phenolics in vanilla (such as flavonoids, lignans, stilbenes and other polyphenols).

Phenolic Compounds and Sesquiterpene Lactones Profile in Leaves of Nineteen Artichoke Cultivars.

Leaves of globe artichoke are food industry byproducts gaining interest due to their therapeutic and nutraceutical potential. The total phenolics, flavonoids, and flavonols content as well as radical scavenging capacity and reducing antioxidant power were determined in leaves of 19 artichoke cultivars. An untargeted analysis based on high-resolution mass spectrometry was then carried out to profile phenolic compounds and sesquiterpene lactones (STLs). The phenolic profile of leaf extracts from different cultivars was widely diverse and included flavonoids, hydroxycinnamic acids, tyrosols, and lignans. Grosheimin and its derivative were the most abundant STLs in all artichoke cultivars. Among the examined cultivars, "Campagnano", "Grato 1", and "Violetto di Provenza" were found to be the richest in polyphenols and presented the highest antioxidant activity, whereas "Blanca de Tudela" and "Carderas" were characterized by a high STLs content. Hence, specific artichoke cultivars can be selected as the source of natural antioxidants with a desired profile of nutraceutical compounds like phenolics and STLs.

Mild Potassium Chloride Stress Alters the Mineral Composition, Hormone Network, and Phenolic Profile in Artichoke Leaves.

There is a growing interest among consumers and researchers in the globe artichoke [Cynara cardunculus L. subsp. scolymus (L.) Hegi] leaf extract due to its nutraceutical and therapeutic properties. The application of an abiotic stress such as salinity can activate the stress-signaling pathways, thus enhancing the content of valuable phytochemicals. The aim of this study was to assess the metabolic changes in artichokes by probing the leaf metabolome of artichoke plants grown in a floating system and exposed to a relatively mild (30 mM) potassium chloride (KCl) salt stress. Potassium chloride treatment decreased the leaf dry biomass of artichoke, macro- and microelements in leaves (e.g., Ca, Mg, Mn, Zn, and B) but increased the concentrations of K and Cl. Metabolomics highlighted that the hormonal network of artichokes was strongly imbalanced by KCl. The indole-3-acetic acid conjugates, the brassinosteroids hormone 6-deoxocastasterone, and even more the cytokinin precursor N(6)-(Delta-2-isopentenyl)-adenosine-5'-triphosphate, strongly increased in leaves of KCl-treated plants. Moreover, KCl saline treatment induced accumulation of GA4, a bioactive form additional to the already known GA3. Another specific response to salinity was changes in the phenolic compounds profile, with flavones and isoflavones being decreased by KCl treatment, whereas flavonoid glycosides increased. The osmotic/oxidative stress that salinity generates also induced some expected changes at the biochemical level (e.g., ascorbate degradation, membrane lipid peroxidation, and accumulation of mannitol phosphate). These latter results help explain the molecular/physiological mechanisms that the plant uses to cope with potassium chloride stress exposure.

Current perspectives on the hormonal control of seed development in Arabidopsis and maize: a focus on auxin.

The seed represents the unit of reproduction of flowering plants, capable of developing into another plant, and to ensure the survival of the species under unfavorable environmental conditions. It is composed of three compartments: seed coat, endosperm and embryo. Proper seed development depends on the coordination of the processes that lead to seed compartments differentiation, development and maturation. The coordination of these processes is based on the constant transmission/perception of signals by the three compartments. Phytohormones constitute one of these signals; gradients of hormones are generated in the different seed compartments, and their ratios comprise the signals that induce/inhibit particular processes in seed development. Among the hormones, auxin seems to exert a central role, as it is the only one in maintaining high levels of accumulation from fertilization to seed maturation. The gradient of auxin generated by its PIN carriers affects several processes of seed development, including pattern formation, cell division and expansion. Despite the high degree of conservation in the regulatory mechanisms that lead to seed development within the Spermatophytes, remarkable differences exist during seed maturation between Monocots and Eudicots species. For instance, in Monocots the endosperm persists until maturation, and constitutes an important compartment for nutrients storage, while in Eudicots it is reduced to a single cell layer, as the expanding embryo gradually replaces it during the maturation. This review provides an overview of the current knowledge on hormonal control of seed development, by considering the data available in two model plants: Arabidopsis thaliana, for Eudicots and Zea mays L., for Monocots. We will emphasize the control exerted by auxin on the correct progress of seed development comparing, when possible, the two species.

Insight into the role of anthocyanin biosynthesis-related genes in Medicago truncatula mutants impaired in pigmentation in leaves.

Flavonoids are the most common antioxidant compounds produced in plants. In this study, two wild types and two independent mutants of Medicago truncatula with altered anthocyanin content in leaves were characterized at the phenotype, metabolite profile, gene structure and transcript levels. Flavonoid profiles showed conserved levels of dihydroflavonols, leucoanthocyanidins and flavonols, while anthocyanidin, anthocyanin and isoflavone levels were lower in the mutants (up to 90% less) compared with the wild types. Genes encoding key enzymes of the anthocyanin pathway and transcriptional factors were analyzed by RT-PCR. Genes involved in the later steps of the anthocyanin pathway (dihydrokaempferol reductase 2, UDP-glucose:anthocyanin 3-O-glucosyltransferase and glutathione S-transferase) were found under-expressed in both mutants. Dihydrokaempferol reductase 1 was downregulated two-fold in the anthocyanin-less mutant while the UDP-glucose:anthocyanin 5-O-glucosyltransferase was strongly repressed only in the mutant with low pigmentation, suggesting a different regulation in the two genotypes. The common feature was that the first enzymes of the flavonoid biosynthesis pathway were not altered in rate of expression. A very high reduction in transcript accumulation was also found for two homologous R2R3 MYB genes, namely MtMYBA and AN2, suggesting that these genes have a role in anthocyanin accumulation in leaves. More evidence was found on analyzing their nucleotide sequence: several SNPs, insertions and deletions in the coding and non-coding regions of both MYB genes were found between mutants and wild types that could influence anthocyanin biosynthesis. Moreover, a subfamily of eight MYB genes with a high homology to MtMYBA was discovered in tandem on chromosome 5 of M. truncatula.

Impaired auxin biosynthesis in the defective endosperm18 mutant is due to mutational loss of expression in the ZmYuc1 gene encoding endosperm-specific YUCCA1 protein in maize.

The phytohormone auxin (indole-3-acetic acid [IAA]) plays a fundamental role in vegetative and reproductive plant development. Here, we characterized a seed-specific viable maize (Zea mays) mutant, defective endosperm18 (de18) that is impaired in IAA biosynthesis. de18 endosperm showed large reductions of free IAA levels and is known to have approximately 40% less dry mass, compared with De18. Cellular analyses showed lower total cell number, smaller cell volume, and reduced level of endoreduplication in the mutant endosperm. Gene expression analyses of seed-specific tryptophan-dependent IAA pathway genes, maize Yucca1 (ZmYuc1), and two tryptophan-aminotransferase co-orthologs were performed to understand the molecular basis of the IAA deficiency in the mutant. Temporally, all three genes showed high expression coincident with high IAA levels; however, only ZmYuc1 correlated with the reduced IAA levels in the mutant throughout endosperm development. Furthermore, sequence analyses of ZmYuc1 complementary DNA and genomic clones revealed many changes specific to the mutant, including a 2-bp insertion that generated a premature stop codon and a truncated YUC1 protein of 212 amino acids, compared with the 400 amino acids in the De18. The putative, approximately 1.5-kb, Yuc1 promoter region also showed many rearrangements, including a 151-bp deletion in the mutant. Our concurrent high-density mapping and annotation studies of chromosome 10, contig 395, showed that the De18 locus was tightly linked to the gene ZmYuc1. Collectively, the data suggest that the molecular changes in the ZmYuc1 gene encoding the YUC1 protein are the causal basis of impairment in a critical step in IAA biosynthesis, essential for normal endosperm development in maize.

Use of a custom array to study differentially expressed genes during blood orange (Citrus sinensis L. Osbeck) ripening.

A flesh-specific oligonucleotide custom array was designed to study gene expression during blood orange ripening. The array included 301 probes derived from a subtracted SSH library, a cDNA-AFLP collection, and a set of regulatory genes from the Harvest citrus database. The custom array was hybridized using samples of Moro, a pigmented cultivar, and Cadenera, a common cultivar, at three different ripening stages: the immature phase, the halfway point of maturation (corresponding to the start of Moro pigmentation) and the full ripening. Of the 301 probes, 27 in total, corresponding to 20 different transcripts, indicated differential expression in stage-to-stage and/or cultivar-to-cultivar comparisons. Transcripts encoding for anthocyanin biosynthesis represented most of the total over-expressed probes. The remaining differentially expressed transcripts were functionally associated with primary metabolism as flavor biosynthesis, defense and signal transduction. The expressed products associated with probes indicating differential expression were confirmed by qRT-PCR. The microarray was designed considering a small collection of sequences useful for monitoring specific pathways and regulatory genes related to fruit ripening and anthocyanin pigmentation. The main novelty of this customization is the use of expressed sequences specifically derived from blood orange flesh to study different cultivars and ripening stages, and the provision of further information about processes related to anthocyanin pigmentation in citrus fruit flesh.

Transgene organisation in potato after particle bombardment-mediated (co-)transformation using plasmids and gene cassettes.

Protocols for efficient co-transformation of potato internodes with genes contained in separate plasmids or gene cassettes (i.e., linear PCR fragments comprising a promoter-gene-terminator) using particle bombardment were established. Twenty-eight out of 62 (45%) and 11 out of 65 (17%) plants transformed with a plasmid containing the selectable marker contained one and two additional non-selected genes, respectively. When gene cassettes were used in transformation, six out of eight plants were co-transformed. Expression analysis showed that 75-80% of the plants transformed with two transgenes expressed both of them, irrespective of the use of plasmids or gene cassettes. Thirty-eight plants containing the gusA reporter-gene and the nptII selectable-marker have been characterised with respect to the molecular organisation of the donor DNAs. Seventeen out of 49 (35%) gusA sites of integration contained one copy of the gene. Only 11 gusA sites (22%) were linked to the site of integration of the selectable marker. When one site of integration contained several copies of the transgene, a predominance of 3'-3' inverted re-arrangement repeats was observed.