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.Fil: Escaray, Francisco José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Passeri, Valentina. Institute of Biosciences and BioResources; Italia. Consiglio Nazionale delle Ricerche; ItaliaFil: Babuin, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Marco, Francisco. Universidad de Valencia; EspañaFil: Carrasco, Pedro. Universidad de Valencia; EspañaFil: Damiani, Francesco. Consiglio Nazionale delle Ricerche; Italia. Institute of Biosciences and BioResources; ItaliaFil: Pieckenstain, Fernando Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Paolocci, Francesco. Consiglio Nazionale delle Ricerche; Italia. Institute of Biosciences and BioResources; ItaliaFil: Ruiz, Oscar Adolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentin

Multiple bHLH/MYB-based protein complexes regulate proanthocyanidin biosynthesis in the herbage of Lotus spp.

[EN] The environmental impact and health of ruminants fed with forage legumes depend on the herbage's concentration and structure of proanthocyanidins (PAs). Unfortunately, the primary forage legumes (alfalfa and clover) do not contain substantial levels of PAs. No significant progress has been made to induce PAs to agronomically valuable levels in their edible organs by biotechnological approaches thus far. Building this trait requires a profound knowledge of PA regulators and their interplay in species naturally committed to accumulating these metabolites in the target organs. Against this background, we compared the shoot transcriptomes of two inter-fertile Lotus species, namely Lotus tenuis and Lotus corniculatus, polymorphic for this trait, to search for differentially expressed MYB and bHLH genes. We then tested the expression of the above-reported regulators in L. tenuis x L. corniculatus interspecific hybrids, several Lotus spp., and different L. corniculatus organs with contrasting PA levels. We identified a novel MYB activator and MYB-bHLH-based complexes that, when expressed in Nicotiana benthamiana, trans-activated the promoters of L. corniculatus anthocyanidin reductase and leucoanthocyanidin reductase 1 genes. The last are the two critical structural genes for the biosynthesis of PAs in Lotus spp. Competition between MYB activators for the transactivation of these promoters also emerged. Overall, by employing Lotus as a model genus, we refined the transcriptional network underlying PA biosynthesis in the herbage of legumes. These findings are crucial to engineering this trait in pasture legumes.This study was partially supported by CNR (Italy)-CONICET (Argentina) 2021-2022 Bilateral Agreement, by the project Pict 2021-2023 from Agencia de Promocion Cientifica y Tecnologica (Argentina), by Margin Up, funded by the European Union's Horizon research and innovation program GA number no 101082089, and by the Italian Agritech National Research Center funded by the European Union Next-Generation EU (Piano Nazionale di Ripresa e Resilienza (PNRR)- Missione 4 Componente 2, Investitmento 1.4 - D.D. 1032 17/06/2022, CN00000022). In particular, our study represents an original paper related to the Spoke 4 "Multifunctional and resilient agriculture and forestry systems for the mitigation of climate change risks" of the PNRR project. However, views and opinions expressed are those of the authors and do not necessarily reflect those of the European Union or the European Research Agency (REA). Neither the European Union nor the granting authorities can be held responsible. Open access funding provided by Consiglio Nazionale Delle Ricerche (CNR) within the CRUI-CARE Agreement.Escaray, FJ.; Valeri, MC.; Damiani, F.; Ruiz, OA.; Carrasco, P.; Paolocci, F. (2024). Multiple bHLH/MYB-based protein complexes regulate proanthocyanidin biosynthesis in the herbage of Lotus spp. Planta. 259(1). https://doi.org/10.1007/s00425-023-04281-2259

Périgord black truffle genome uncovers evolutionary origins and mechanisms of symbiosis

LetterInternational audienceThe Périgord black truffle ($Tuber\ melanosporum$ Vittad.) and the Piedmont white truffle dominate today's truffle market. The hypogeous fruiting body of $T.\ melanosporum$ is a gastronomic delicacy produced by an ectomycorrhizal symbiont endemic to calcareous soils in southern Europe. The worldwide demand for this truffle has fuelled intense efforts at cultivation. Identification of processes that condition and trigger fruit body and symbiosis formation, ultimately leading to efficient crop production, will be facilitated by a thorough analysis of truffle genomic traits. In the ectomycorrhizal $Laccaria\ bicolor$, the expansion of gene families may have acted as a 'symbiosis toolbox'. This feature may however reflect evolution of this particular taxon and not a general trait shared by all ectomycorrhizal species. To get a better understanding of the biology and evolution of the ectomycorrhizal symbiosis, we report here the sequence of the haploid genome of $T.\ melanosporum$, which at $\sim$125 megabases is the largest and most complex fungal genome sequenced so far. This expansion results from a proliferation of transposable elements accounting for $\sim$58% of the genome. In contrast, this genome only contains $\sim$7,500 protein-coding genes with very rare multigene families. It lacks large sets of carbohydrate cleaving enzymes, but a few of them involved in degradation of plant cell walls are induced in symbiotic tissues. The latter feature and the upregulation of genes encoding for lipases and multicopper oxidases suggest that $T.\ melanosporum$ degrades its host cell walls during colonization. Symbiosis induces an increased expression of carbohydrate and amino acid transporters in both $L.\ bicolor$ and $T.\ melanosporum$, but the comparison of genomic traits in the two ectomycorrhizal fungi showed that genetic predispositions for symbiosis $-$'the symbiosis toolbox'$-$ evolved along different ways in ascomycetes and basidiomycete

Reevaluation of the Life Cycle of Tuber magnatum

Tuber spp. are ectomycorrhizal ascomycetes that produce ascocarps known as truffles. Basic aspects of Tuber biology have yet to be fully elucidated. In particular, there are conflicting hypotheses concerning the mating system and the ploidy level of the mycorrhizal and truffle hyphae. We used polymorphic microsatellites to compare the allelic configurations of asci with those from the network of the surrounding hyphae in single Tuber magnatum truffles. We then used these truffles to inoculate host plants and evaluated the microsatellite configurations of the resulting mycorrhizal root tips. These analyses provide direct evidence that T. magnatum outcrosses and that its life cycle is predominantly haploid. In addition to its scientific significance, this basic understanding of the T. magnatum life cycle may have practical importance in developing strategies to obtain and select nursery-produced mycorrhizal plants as well as in the management of artificial plantations of this and other Tuber spp

Du sexe de la truffe à ses qualités organoleptiques

National audienc

Molecular phylogenetic analyses show that Amanita ovoidea and Amanita proxima are distinct species and suggest their assignment to Roanokenses section

Amanita ovoidea is generally considered edible and is morphologically similar to Amanita proxima that contains nephrotoxins. Both species were assigned to Amanita section Amidella. To study their phylogenetic relationships, we performed nrITS- and RPB2-based analyses of these and other Amanita species. Our analyses show that (i) A. ovoidea and A. proxima are distinct species; (ii) A. ovoidea has an extensively heterogeneous ITS with up to 4.4% pairwise genetic distance between individuals; (iii) in A. ovoidea, pairwise genetic distance between ITS clones from a single basidiome was as high as 3.2%, possibly because of hybridization events and subsequent failure of the rDNA repeats to homogenize; (iv) A. aminoaliphatica and A. proxima are taxonomic synonyms; and (v) A. proxima and A. ovoidea cluster with species belonging to section Roanokenses rather than with those of section Amidella sensu stricto. This study supports the development of species-specific markers for diagnosis of the toxic and the edible species here examined. © 2019, German Mycological Society and Springer-Verlag GmbH Germany, part of Springer Nature

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

Transparent electrodes made from carbon nanotube polyelectrolytes and application to acidic environments

Carbon nanotube (CNT)-based transparent conducting films (TCFs) have been prepared by filtration of (i) surfactant-based aqueous dispersions and (ii) organic solutions obtained by reductive dissolution of an alkali metal salt of polyelectrolyte nanotubes. Starting from the same source of nanotubes, it is shown that films obtained by the reductive dissolution route present up to one order of magnitude better conductivity for the same transmittance. Light scattering experiments show that the average CNT length is much larger for the reductive dissolution-based organic solutions than for the sonication aided aqueous dispersions. Values of surface resistivity of 200 ohm per square have been obtained for 80% transmittance. Additionally, it is shown thatthe CNT-based TCFs are undistinguishable from indium tin oxide (ITO) as electrodes in regular environments, whereas they perform efficiently in acidic environments where ITO fails

Ectopic expression of a basic helix-loop-helix gene transactivates parallel pathways of proanthocyanidin biosynthesis. Structure, expression analysis, and genetic control of leucoanthocyanidin 4-reductase and anthocyanidin reductase genes in Lotus corniculatus

Proanthocyanidins (PAs) are plant secondary metabolites and are composed primarily of catechin and epicatechin units in higher plant species. Due to the ability of PAs to bind reversibly with plant proteins to improve digestion and reduce bloat, engineering this pathway in leaves is a major goal for forage breeders. Here, we report the cloning and expression analysis of anthocyanidin reductase (ANR) and leucoanthocyanidin 4-reductase (LAR), two genes encoding enzymes committed to epicatechin and catechin biosynthesis, respectively, in Lotus corniculatus. We show the presence of two LAR gene families (LAR1 and LAR2) and that the steady-state levels of ANR and LAR1 genes correlate with the levels of PAs in leaves of wild-type and transgenic plants. Interestingly, ANR and LAR1, but not LAR2, genes produced active proteins following heterologous expression in Escherichia coli and are affected by the same basic helix-loop-helix transcription factor that promotes PA accumulation in cells of palisade and spongy mesophyll. This study provides direct evidence that the same subclass of transcription factors can mediate the expression of the structural genes of both branches of PA biosynthesis