Canopy Architectural Characteristics of Ten New Olive (Olea europaea L.) Genotypes and Their Potential for Cultivation in Super-High-Density Orchards.

In recent years, there has been growing interest in olive genotypes (Olea europaea L.) suitable for super-high-density (SHD > 1200 trees/hectare) orchards. To date, only a few cultivars are considered fitting for such cultivation system. In this study, the first results on the architectural characteristics of the canopy of ten new olive genotypes are presented. Their suitability for SHD orchards was evaluated and compared with the cultivar 'Arbequina', which is considered suitable for SHD olive orchards and, for this reason, was used as the control. Several canopy measurements were taken, and some architectural parameters, such as branching frequency, branching density, and branch diameter/stem diameter ratio were calculated. The branching frequency value was greater than 0.20 in 'Arbequina' and in only four of the genotypes. The branching density in five genotypes was similar to 'Arbequina'. 'Arbequina' had the lowest value for the branch diameter/stem diameter ratio, and only three genotypes had similar values. These initial results showed that only one genotype has all canopy architectural characteristics comparable to those of the cv. 'Arbequina'. Further studies are needed to evaluate the production traits of these new genotypes and complete their characterization.

The Ancient Olive Trees (Olea europaea L.) of the Maltese Islands: A Rich and Unexplored Patrimony to Enhance Oliviculture.

A prospecting campaign in the Maltese Islands has ensured the survival of several ancient olive trees (Olea europaea L.), genetically distant from known cultivars. Most of these plants were abandoned or partially cultivated. A two-year evaluation of fruit characteristics and compositions was performed on samples collected from the main representatives of these indigenous genotypes. Analyses were carried out using Gas Chromatography, High-Performance Liquid Chromatography and Near Infrared Spectrometry. Among the fruit samples, a wide range of variations was observed. Some of the genotypes showed fruit traits suitable for table olive production. This is the case of samples with a pulp/pit ratio higher than four, such as 1Wardija, 1Caritas, 1Plattini, 1Bingemma Malta and 3Loretu, whilst 1Bidni, 1Mellieha, 2Qnotta, 3Loretu, 1Bingemma Malta and 1Caritas were suitable for dual purpose. The total phenol content ranged from 6.3 (1Wardija) to 117.9 (2Mtarfa) g/kg of fresh pulp. The average percentage of MUFA was quite low for most of the varieties. These genotypes, which presumably originated in the Maltese Islands and are well adapted to the local pedo-climatic conditions, are being propagated for the following evaluation of their bio-agronomical performance (production, suitability to intensive cultivation, environmental sustainability, product quality, etc.). The purpose is to select, among these local genotypes, the most outstanding varieties, in terms of phenolic and FA profile and agronomical potential, to spread into cultivation, thereby contributing to an increase in the quality of the local table and olive oil production, strongly linked to the territory.

Functional Characterization of MtrGSTF7, a Glutathione S-Transferase Essential for Anthocyanin Accumulation in Medicago truncatula.

Flavonoids are essential compounds widespread in plants and exert many functions such as defence, definition of organ colour and protection against stresses. In Medicago truncatula, flavonoid biosynthesis and accumulation is finely regulated in terms of tissue specificity and induction by external factors, such as cold and other stresses. Among flavonoids, anthocyanin precursors are synthesised in the cytoplasm, transported to the tonoplast, then imported into the vacuole for further modifications and storage. In the present work, we functionally characterised MtrGSTF7, a phi-class glutathione S-transferase involved in anthocyanin transport to the tonoplast. The mtrgstf7 mutant completely lost the ability to accumulate anthocyanins in leaves both under control and anthocyanin inductive conditions. On the contrary, this mutant showed an increase in the levels of soluble proanthocyanidins (Pas) in their seeds with respect to the wild type. By complementation and expression data analysis, we showed that, differently from A. thaliana and similarly to V. vinifera, transport of anthocyanin and proanthocyanidins is likely carried out by different GSTs belonging to the phi-class. Such functional diversification likely results from the plant need to finely tune the accumulation of diverse classes of flavonoids according to the target organs and developmental stages.

Bioactive Compound Profiling of Olive Fruit: The Contribution of Genotype.

The health, therapeutic, and organoleptic characteristics of olive oil depend on functional bioactive compounds, such as phenols, tocopherols, squalene, and sterols. Genotype plays a key role in the diversity and concentration of secondary compounds peculiar to olive. In this study, the most important bioactive compounds of olive fruit were studied in numerous international olive cultivars during two consecutive seasons. A large variability was measured for each studied metabolite in all 61 olive cultivars. Total phenol content varied on a scale of 1-10 (3831-39,252 mg kg-1) in the studied cultivars. Squalene values fluctuated over an even wider range (1-15), with values of 274 to 4351 mg kg-1. Total sterols ranged from 119 to 969 mg kg-1, and total tocopherols varied from 135 to 579 mg kg-1 in fruit pulp. In the present study, the linkage among the most important quality traits highlighted the scarcity of cultivars with high content of at least three traits together. This work provided sound information on the fruit metabolite profile of a wide range of cultivars, which will facilitate the studies on the genomic regulation of plant metabolites and development of new olive genotypes through genomics-assisted breeding.

Two bi-functional cytochrome P450 CYP72 enzymes from olive (Olea europaea) catalyze the oxidative C-C bond cleavage in the biosynthesis of secoxy-iridoids - flavor and quality determinants in olive oil.

Olive (Olea europaea) is an important crop in Europe, with high cultural, economic and nutritional significance. Olive oil flavor and quality depend on phenolic secoiridoids, but the biosynthetic pathway of these iridoids remains largely uncharacterized. We discovered two bifunctional cytochrome P450 enzymes, catalyzing the rare oxidative C-C bond cleavage of 7-epi-loganin to produce oleoside methyl ester (OeOMES) and secoxyloganin (OeSXS), both through a ketologanin intermediary. Although these enzymes are homologous to the previously reported Catharanthus roseus secologanin synthase (CrSLS), the substrate and product profiles differ. Biochemical assays provided mechanistic insights into the two-step OeOMES and CrSLS reactions. Model-guided mutations of OeOMES changed the product profile in a predictable manner, revealing insights into the molecular basis for this change in product specificity. Our results suggest that, in contrast to published hypotheses, in planta production of secoxy-iridoids is secologanin-independent. Notably, sequence data of cultivated and wild olives point to a relation between domestication and OeOMES expression. Thus, the discovery of this key biosynthetic gene suggests a link between domestication and secondary metabolism, and could potentially be used as a genetic marker to guide next-generation breeding programs.

Identification and functional analysis of three new anthocyanin R2R3-MYB genes in Petunia.

We identified three novel members of the R2R3-MYB clade of anthocyanin regulators in the genome of the purple flowering Petunia inflata S6 wild accession, and we called them ANTHOCYANIN SYNTHESIS REGULATOR (ASR). Two of these genes, ASR1 and ASR2, are inactivated by two different single base mutations in their coding sequence. All three of these genes are absent in the white flowering species P. axillaris N and P. parodii, in the red flowering P. exserta, and in several Petunia hybrida lines, including R27 and W115. P. violacea and other P. hybrida lines (M1, V30, and W59) instead harbor functional copies of the ASR genes. Comparative, functional and phylogenic analysis of anthocyanin R2R3-MYB genes strongly suggest that the ASR genes cluster is a duplication of the genomic fragment containing the other three R2R3-MYB genes with roles in pigmentation that were previously defined, the ANTHOCYANIN4-DEEP PURPLE-PURPLE HAZE (AN4-DPL-PHZ) cluster. An investigation of the genomic fragments containing anthocyanin MYBs in different Petunia accessions reveals that massive rearrangements have taken place, resulting in large differences in the regions surrounding these genes, even in closely related species. Yeast two-hybrid assays showed that the ASR proteins can participate in the WMBW (WRKY, MYB, B-HLH, and WDR) anthocyanin regulatory complex by interacting with the transcription factors AN1 and AN11. All three ASRs can induce anthocyanin synthesis when ectopically expressed in P. hybrida lines, but ASR1 appeared to be the most effective. The expression patterns of ASR1 and ASR2 cover several different petunia tissues with higher expression at early stages of bud development. In contrast, ASR3 is only weakly expressed in the stigma, ovary, and anther filaments. The characterization of these novel ASR MYB genes completes the picture of the MYB members of the petunia anthocyanin regulatory MBW complex and suggests possible mechanisms of the diversification of pigmentation patterns during plant evolution.

Genetic Mapping of the Incompatibility Locus in Olive and Development of a Linked Sequence-Tagged Site Marker.

The genetic control of self-incompatibility (SI) has been recently disclosed in olive. Inter-varietal crossing confirmed the presence of only two incompatibility groups (G1 and G2), suggesting a simple Mendelian inheritance of the trait. A double digest restriction associated DNA (ddRAD) sequencing of a biparental population segregating for incompatibility groups has been performed and high-density linkage maps were constructed in order to map the SI locus and identify gene candidates and linked markers. The progeny consisted of a full-sib family of 229 individuals derived from the cross 'Leccino' (G1) × 'Dolce Agogia' (G2) varieties, segregating 1:1 (G1:G2), in accordance with a diallelic self-incompatibility (DSI) model. A total of 16,743 single nucleotide polymorphisms was identified, 7,006 in the female parent 'Leccino' and 9,737 in the male parent 'Dolce Agogia.' Each parental map consisted of 23 linkage groups and showed an unusual large size (5,680 cM in 'Leccino' and 3,538 cM in 'Dolce Agogia'). Recombination was decreased across all linkage groups in pollen mother cells of 'Dolce Agogia,' the parent with higher heterozygosity, compared to megaspore mother cells of 'Leccino,' in a context of a species that showed exceptionally high recombination rates. A subset of 109 adult plants was assigned to either incompatibility group by a stigma test and the diallelic self-incompatibility (DSI) locus was mapped to an interval of 5.4 cM on linkage group 18. This region spanned a size of approximately 300 Kb in the olive genome assembly. We developed a sequence-tagged site marker in the DSI locus and identified five haplotypes in 57 cultivars with known incompatibility group assignment. A combination of two single-nucleotide polymorphisms (SNPs) was sufficient to predict G1 or G2 phenotypes in olive cultivars, enabling early marker-assisted selection of compatible genotypes and allowing for a rapid screening of inter-compatibility among cultivars in order to guarantee effective fertilization and increase olive production. The construction of high-density linkage maps has led to the development of the first functional marker in olive and provided positional candidate genes in the SI locus.

Isolation and Characterization of the Flavonol Regulator CcMYB12 From the Globe Artichoke [Cynara cardunculus var. scolymus (L.) Fiori].

Flavonoids are a well-studied group of secondary metabolites, belonging to the phenylpropanoid pathway. Flavonoids are known to exhibit health promoting effects such as antioxidant capacities, anti-cancer and anti-inflammatory activity. Globe artichoke is an important source of bioactive phenolic compounds, including flavonoids. To study the regulation of their biosynthesis, a R2R3-MYB transcription factor, CcMYB12, was isolated from artichoke leaves. Phylogenetic analysis showed that this protein belongs to the MYB subgroup 7 (flavonol-specific MYB), which includes Arabidopsis AtMYB12, grapevine VvMYBF1, and tomato SlMYB12. CcMYB12 transcripts were detected specifically in artichoke immature inflorescence and young leaves and overlapped with the profiles of flavonol biosynthetic genes. Electrophoretic mobility shift assays (EMSAs) revealed that recombinant CcMYB12 protein is able to bind to ACII element, a DNA binding site ubiquitously present in the promoters of genes encoding flavonol biosynthetic enzymes. In transgenic Arabidopsis plants, the overexpression of CcMYB12 activated the expression of endogenous flavonol biosynthesis genes, leading to an increase of flavonol accumulation and a decrease of anthocyanins in leaves. Likewise, in transgenic tobacco petals and leaves, the overexpression of CcMYB12 decreased anthocyanin levels and increased flavonols.

The R2R3-MYB TT2b and the bHLH TT8 genes are the major regulators of proanthocyanidin biosynthesis in the leaves of Lotus species.

MAIN CONCLUSION: By exploiting interspecific hybrids and their progeny, we identified key regulatory and transporter genes intimately related to proanthocyanidin biosynthesis in leaves of Lotus spp. Proanthocyanidins (PAs), known as condensed tannins, are polymeric flavonoids enriching forage legumes of key nutritional value to prevent bloating in ruminant animals. Unfortunately, major forage legumes such as alfalfa and clovers lack PAs in edible tissues. Therefore, engineering the PA trait in herbage of forage legumes is paramount to improve both ecological and economical sustainability of cattle production system. Progresses on the understanding of genetic determinants controlling PA biosynthesis and accumulation have been mainly made studying mutants of Arabidopsis, Medicago truncatula and Lotus japonicus, model species unable to synthesize PAs in the leaves. Here, we exploited interspecific hybrids between Lotus corniculatus, with high levels of PAs in the leaves, and Lotus tenuis, with no PAs in these organs, and relative F2 progeny, to identify among candidate PA regulators and transporters the genes mainly affecting this trait. We found that the levels of leaf PAs significantly correlate with the expression of MATE1, the putative transporter of glycosylated PA monomers, and, among the candidate regulatory genes, with the expression of the MYB genes TT2a, TT2b and MYB14 and the bHLH gene TT8. The expression levels of TT2b and TT8 also correlated with those of all key structural genes of the PA pathways investigated, MATE1 included. Our study unveils a different involvement of the three Lotus TT2 paralogs to the PA trait and highlights differences in the regulation of this trait in our Lotus genotypes with respect to model species. This information opens new avenues for breeding bloat safe forage legumes.

The R2R3MYB VvMYBPA1 from grape reprograms the phenylpropanoid pathway in tobacco flowers.

MAIN CONCLUSION: This work shows that, in tobacco, the ectopic expression of VvMYBPA1 , a grape regulator of proanthocyanidin biosynthesis, up- or down-regulates different branches of the phenylproanoid pathway, in a structure-specific fashion. Proanthocyanidins are flavonoids of paramount importance for animal and human diet. Research interest increasingly tilts towards generating crops enriched with these health-promoting compounds. Flavonoids synthesis is regulated by the MBW transcriptional complex, made of R2R3MYB, bHLH and WD40 proteins, with the MYB components liable for channeling the complex towards specific branches of the pathway. Hence, using tobacco as a model, here, we tested if the ectopic expression of the proanthocyanidin regulator VvMYBPA1 from grape induces the biosynthesis of these compounds in not-naturally committed cells. Here, we show, via targeted transcriptomic and metabolic analyses of primary transgenic lines and their progeny, that VvMYBPA1 alters the phenylpropanoid pathway in tobacco floral organs, in a structure-specific fashion. We also report that a modest VvMYBPA1 expression is sufficient to induce the expression of both proanthocyanidin-specific and early genes of the phenylpropanoid pathway. Consequently, proanthocyanidins and chlorogenic acids are induced or de novo synthetised in floral limbs, tubes and stamens. Other phenylpropanoid branches are conversely induced or depleted according to the floral structure. Our study documents a novel and distinct function of VvMYBPA1 with respect to other MYBs regulating proanthocyanidins. Present findings may have major implications in designing strategies for enriching crops with health-promoting compounds.

Insight into the evolution of the Solanaceae from the parental genomes of Petunia hybrida.

Petunia hybrida is a popular bedding plant that has a long history as a genetic model system. We report the whole-genome sequencing and assembly of inbred derivatives of its two wild parents, P. axillaris N and P. inflata S6. The assemblies include 91.3% and 90.2% coverage of their diploid genomes (1.4 Gb; 2n = 14) containing 32,928 and 36,697 protein-coding genes, respectively. The genomes reveal that the Petunia lineage has experienced at least two rounds of hexaploidization: the older gamma event, which is shared with most Eudicots, and a more recent Solanaceae event that is shared with tomato and other solanaceous species. Transcription factors involved in the shift from bee to moth pollination reside in particularly dynamic regions of the genome, which may have been key to the remarkable diversity of floral colour patterns and pollination systems. The high-quality genome sequences will enhance the value of Petunia as a model system for research on unique biological phenomena such as small RNAs, symbiosis, self-incompatibility and circadian rhythms.

New Challenges for the Design of High Value Plant Products: Stabilization of Anthocyanins in Plant Vacuoles.

In the last decade plant biotechnologists and breeders have made several attempt to improve the antioxidant content of plant-derived food. Most efforts concentrated on increasing the synthesis of antioxidants, in particular anthocyanins, by inducing the transcription of genes encoding the synthesizing enzymes. We present here an overview of economically interesting plant species, both food crops and ornamentals, in which anthocyanin content was improved by traditional breeding or transgenesis. Old genetic studies in petunia and more recent biochemical work in brunfelsia, have shown that after synthesis and compartmentalization in the vacuole, anthocyanins need to be stabilized to preserve the color of the plant tissue over time. The final yield of antioxidant molecules is the result of the balance between synthesis and degradation. Therefore the understanding of the mechanism that determine molecule stabilization in the vacuolar lumen is the next step that needs to be taken to further improve the anthocyanin content in food. In several species a phenomenon known as fading is responsible for the disappearance of pigmentation which in some case can be nearly complete. We discuss the present knowledge about the genetic and biochemical factors involved in pigment preservation/destabilization in plant cells. The improvement of our understanding of the fading process will supply new tools for both biotechnological approaches and marker-assisted breeding.

Lotus tenuis x L. corniculatus interspecific hybridization as a means to breed bloat-safe pastures and gain insight into the genetic control of proanthocyanidin biosynthesis in legumes.

BACKGROUND: Proanthocyanidins (PAs) are secondary metabolites that strongly affect plant quality traits. The concentration and the structure of these metabolites influence the palatability and nutritional value of forage legumes. Hence, modulating PAs in the leaves of forage legumes is of paramount relevance for forage breeders worldwide. The lack of genetic variation in the leaf PA trait within the most important forage species and the difficulties in engineering this pathway via the ectopic expression of regulatory genes, prompted us to pursue alternative strategies to enhance this trait in forage legumes of agronomic interest. The Lotus genus includes forage species which accumulate PAs in edible organs and can thus be used as potential donor parents in breeding programs. RESULTS: We recovered a wild, diploid and PA-rich population of L. corniculatus and crossed with L. tenuis. The former grows in an alkaline-salty area in Spain while the latter is a diploid species, grown extensively in South American pastures, which does not accumulate PAs in the herbage. The resulting interspecific hybrids displayed several traits of outstanding agronomic relevance such as rhizome production, PA levels in edible tissues sufficient to prevent ruminal bloating (around 5 mg of PAs/g DW), biomass production similar to the cultivated parent and potential for adaptability to marginal lands. We show that PA levels correlate with expression levels of the R2R3MYB transcription factor TT2 and, in turn, with those of the key structural genes of the epicatechin and catechin biosynthetic pathways leading to PA biosynthesis. CONCLUSIONS: The L. tenuis x L. corniculatus hybrids, reported herein, represent the first example of the introgression of the PA trait in forage legumes to levels known to provide nutritional and health benefits to ruminants. Apart from PAs, the hybrids have additional traits which may prove useful to breed forage legumes with increased persistence and adaptability to marginal conditions. Finally, our study suggests the hybrids and their progeny are an invaluable tool to gain a leap forward in our understanding of the genetic control of PA biosynthesis and tolerance to stresses in legumes.

The AD-type ectomycorrhizas, one of the most common morphotypes present in truffle fields, result from fungi belonging to the Trichophaea woolhopeia species complex.

Belowground ectomycorrhizal communities are often species rich. Characterization of the ectomycorrhizas (ECMs) underneath native truffle areas and/or cultivation sites is particularly relevant to identifying fungal species that might interfere with or promote truffle propagation and fruiting. Fungal identification at the genus/species level can now be achieved by combining detailed morphological and anatomical descriptions with molecular approaches. In a survey of the mycorrhizal biodiversity of Tuber melanosporum orchards and inoculated host plants in nurseries, we repeatedly sampled ECMs with morphological features resembling those of the ECMs widely known as the AD type. Despite the fact that the AD type is regarded as one of the most competitive fungal species towards Tuber spp., its taxonomical rank has yet to be resolved. By analyzing the 28S and internal transcribed spacer (ITS) rDNA regions, here, we show that AD-type ECMs result from host plant colonization by the pyronemataceous species Trichophaea woolhopeia. Further to this, the 28S and ITS phylogenetic trees built from the AD-type ECMs analyzed sustain the hypothesis that T. woolhopeia is a species complex.

The strawberry transcription factor FaMYB1 inhibits the biosynthesis of proanthocyanidins in Lotus corniculatus leaves.

Proanthocyanidins (PAs) are agronomically important biopolymers in higher plants composed primarily of catechin and epicatechin units. The biosynthesis of these natural products is regulated by transcription factors including proteins of the R2R3MYB class. To gain insight into the genetic control of the catechin and epicatechin branches of the PA pathway in forage legumes, here the effects of the expression of FaMYB1, a flavonoid R2R3MYB repressor from strawberry, in Lotus corniculatus (birdsfoot trefoil), were tested. It was found that in leaves of T(0) transgenic lines the degree of PA inhibition correlated with the level of FaMYB1 expression. These effects were heritable in the transgene-positive plant T(1) generation and were tissue specific as the suppression of proanthocyanidin biosynthesis was most pronounced in mesophyll cells within the leaf, whereas other flavonoid and phenolic compounds were substantially unaltered. The data suggest that FaMYB1 may counter-balance the activity of the endogenous transcriptional MYB-bHLH-WD40 (MBW) complex promoting proanthocyanidin biosynthesis via the catechin and epicatechin branches and that FaMYB1 does not interfere with the expression levels of a resident R2R3MYB activator of PAs. It is proposed that in forage legumes leaf cell commitment to synthesize proanthocyanidins relies on the balance between the activity of activator and repressor MYBs operating within the MBW complex.

Tuber melanosporum outcrosses: analysis of the genetic diversity within and among its natural populations under this new scenario.

Tuber melanosporum is an ectomycorrhizal ascomycete producing edible ascocarps. The prevalent view is that this species strictly selfs, since genetic analyses have never detected heterozygotic profiles in its putatively diploid/dikaryotic gleba. The selfing model has also forged the experimental approaches to assess the population genetic variability. Here, the hypothesis that T. melanosporum outcrosses was tested. To this end, SSR (simple sequence repeats) and ITS (internal transcribed spacer) markers were employed to fingerprint asci and the surrounding gleba within single ascocarps. The distribution of genetic variability was also investigated at different geographical levels using single (SSR and ITS) and multilocus (AFLP, amplified fragment length polymorphism) markers. It is shown that T. melanosporum outcrosses since asci display additional alleles besides those present in the surrounding, uniparental, gleba. Furthermore, SSR and AFLP data reveal a high rate of intrapopulation diversity within samples from the same ground and root apparatus and the highest rate of genetic variability within the southernmost populations of the distributional range. These data call for a profound re-examination of T. melanosporum mating system, life cycle and strategies for managing man-made plantations. They also strongly support the idea that the last glaciation restricted the species distribution to the Italian and Spanish peninsulas.

Tmt1: the first LTR-retrotransposon from a Tuber spp.

Retrotransposons are suitable targets for developing molecular markers for population genetics studies. Transposable elements have not yet been isolated from ectomycorrhizal fungi of the genus Tuber. In this paper, we report on the isolation and characterization of Tmt1, an LTR-retrotransposon from Tuber melanosporum. The Tmt1 sequence shows relatedness to Ty3/gypsy retrotransposons from which it differentiates for the presence of a dUTPase extra-domain between protease and reverse transcriptase. Phylogenetic analyses suggest a horizontal transfer of the dUTPase gene (dut) from a fungal host genome. The presence of non-identical LTRs and degenerate ORFs substantiate an ancient integration of Tmt1 in T. melanosporum genome. Furthermore, transcripts analyses proved an inactive status of Tmt1, whereas Southern analysis showed that Tmt1 is a repetitive T. melanosporum species-specific element. Tmt1-based markers will help us to gain more insights into population biology in this fungal species.