Castanea sativa Mill. is a multi-functional species with a high ecological and cultural relevance. Thanks to its features, chestnut has become an essential element of subsistence for many societies in mountain and sub-mountainous areas.In this context, the principal aim of this study was to characterize the chestnut germplasm grown in pilot areas of the Lombardy Region (Municipality of Serle, province of Brescia, and the Varese Prealps, province of Varese) by integrating genetic and morphometric data analysis, in order to evaluate the regional chestnut genetic resources and promote their conservation. Twelve polymorphic microsatellite (SSRs) markers were used for genotyping 467 trees sampled between 2021 and 2022 in eight fruit tree orchards and three natural populations. Leaves and embryos were sampled for each individual to evaluate inter and intra-population gene flow. In parallel, a subset of fifty individuals from different collection sites were selected for replicated fruit morphological and nutritional analyses (specific carbohydrates, moisture, fat, protein, ash and fibers). The genetic data showed a number of clones identified as "Marrone" in Serle area. The analysis of the genetic structure, conducted with a Bayesian method and Principal Coordinates Analysis, showed different gene pools representing the two geographical areas. The combined data of mother plants and embryos from Varese showed high genetic similarity among individuals of the same area, whereas we found high genetic divergence between mother plants and embryos in Serle area. In addition, we found large inter-individual variation of several morphological traits, with the dimensional characteristics principally affected by local environmental drivers. Our results suggest that the genetic diversity and structure of the cultivated chestnut populations was shaped by different historical management systems in the two areas.

Objectives According to Charles Darwin, domestication can be thought as a great model to study convergent evolution. Among crops, common bean (Phaseolus vulgaris L.) represents a unique example of multiple parallel independent domestications: wild common bean is organized in two geographically isolated and genetically differentiated wild gene pools (Mesoamerican and Andean) that diverged from a common ancestral wild population, then independently domesticated in Mexico and in South America nearly 8,000 years ago. These processes resulted in morphological changes (e.g. seed and leaf sizes, seed coat color, growth habit, photoperiodic responses) that distinguish culturally adapted classes of beans. In addition to the interest emerging from its domestication history, common bean also carries a pivotal agronomic value: it is one of the most important grain legumes for human consumption and, as a legume, it also has a role in sustainable agriculture owing to its ability to fix atmospheric nitrogen. To gain deeper insights into the genetic basis of common bean domestication we used an available TILLING population, obtained by chemical EMS-mutagenesis of the Mesoamerican genotype BAT 93 (1), and developed a TILLING-by-sequencing pipeline starting as a method for the identification of mutants of candidate domestication genes. Materials and Methods DNA was extracted from seeds of 1728 M4 individuals of an EMS-mutagenized population developed in the Mesoamerican genotype BAT93 [1]. Samples were combined using a 3D pooling system (Figure1) and sequenced by Illumina at High Coverage (50x) after enriching target regions using custom capture probes [2, 3]. Probes were designed to cover a total of 719 genes of interest (approx. 491 Mb), based on the presence of: i) one or more signals of domestication or ii) differential expression between the Andean genotype and Mesoamerican genotype, and iii) known involvement in the phenomenon of shattering, in seed traits, in the cytokinin hormonal pathway and in phytic acid biosynthesis and metabolism. Results Sequencing data were aligned on the BAT 93 reference genome. Variant calling from 3D pools was performed using four variant callers and low frequency variant calling was performed by combining different algorithms (NGSEP v4.1.0, GATK Mutect2 v4.2.0, VarScan v2.4.2, CRISPR v0.2): 144 variants were identified on 128 candidate genes. Data validation on the same DNA samples was performed via PCR and Sanger re-sequencing: 84 variants were confirmed (65,6%) and were carried by a total of 78 lines, along 103 of the screened genes. Among these variants, 8 had a high inferred impact (gain of stop codon or frameshift), 50 had a moderate inferred impact (non-synonymous variants) while the remaining did not produce any amino acid change. Selected M4 (35) lines carrying high (8) and medium impact (27) mutations are being genotyped and reproduced to obtain M5 homozygous individuals; phenotypical analysis of selected lines will be also carried out. Conclusions The TILLING by Seq approach adopted here (three-dimensional DNA pooling strategy coupled with a High Coverage Illumina sequencing after enriching target regions using custom capture probes) allowed us to identify 53 target genes carrying variants with high/medium impact and whose phenotypic effects are currently under evaluation on 31 of them. Considered the relevance of these results and to provide the scientific community a structured platform to look for mutants in common bean, the development of a new TILLING population in the Meccearly dwarf borlotto type variety (Blumen) has recently started and 3516 M1 plants are currently under reproduction.

Proteases, including serine proteases, are involved in the entire life cycle of plants. Proteases are controlled by protease inhibitors (PI) to limit any uncontrolled or harmful protease activity. The role of PIs in biotic and abiotic stress tolerance is well documented, however their role in various other plant processes has not been fully elucidated. Seed development is one such area that lack detailed work on the function of PIs despite the fact that this is a key process in the life cycle of the plant. Serine protease inhibitors (SPI) such as the Bowman-Birk inhibitors and Kunitz-type inhibitors, are abundant in legume seeds and act as antinutrients in humans and animals. Their role in seed development is not fully understood and present an interesting research target. Whether lowering the levels and activity of PIs, in order to lower the antinutrient levels in seed will affect the development of viable seed, remains an important question. Studies on the function of SPI in seed development are therefore required. In this Perspective paper, we provide an overview on the current knowledge of seed storage proteins, their degradation as well as on the serine protease-SPI system in seeds and what is known about the consequences when this system is modified. We discuss areas that require investigation. This includes the identification of seed specific SPIs; screening of germplasms, to identify plants with low seed inhibitor content, establishing serine protease-SPI ratios and lastly a focus on molecular techniques that can be used to modify seed SPI activity.

In recent years, fermented foods have attracted increasing attention due to their important role in the human diet, since they supply beneficial health effects, providing important sources of nutrients. In this respect, a comprehensive characterization of the metabolite content in fermented foods is required to achieve a complete vision of physiological, microbiological, and functional traits. In the present preliminary study, the NMR-based metabolomic approach combined with chemometrics has been applied, for the first time, to investigate the metabolite content of Phaseolus vulgaris flour fermented by different lactic acid bacteria (LAB) and yeasts. A differentiation of microorganisms (LAB and yeasts), LAB metabolism (homo- and heterofermentative hexose fermentation), LAB genus (Lactobacillus, Leuconostoc, and Pediococcus), and novel genera (Lacticaseibacillus, Lactiplantibacillus, and Lentilactobacillus) was achieved. Moreover, our findings showed an increase of free amino acids and bioactive molecules, such as GABA, and a degradation of anti-nutritional compounds, such as raffinose and stachyose, confirming the beneficial effects of fermentation processes and the potential use of fermented flours in the production of healthy baking foods. Finally, among all microorganisms considered, the Lactiplantibacillus plantarum species was found to be the most effective in fermenting bean flour, as a larger amount of free amino acids were assessed in their analysis, denoting more intensive proteolytic activity.

I fagioli (Phaseolus vulgaris L.) sono un'importante fonte di nutrienti con effetti benefici sulla salute. Tuttavia, finora sono stati poco utilizzati per la preparazione di snack. La disponibilità di una farina di fagiolo priva di lectine attive (lec-), che non necessita di un trattamento prima dell'uso e biofortificata grazie ad un ridotto contenuto di acido fitico (lpa), che normalmente diminuisce la biodisponibilità dei cationi minerali, può aumentare l'utilizzo della farina di fagiolo per la preparazione di snack. L'obiettivo di questa ricerca è stato quello di valutare l'apprezzamento sensoriale di cracker, biscotti e una crema preparati con questa farina di fagiolo biofortificata e priva di lectine rispetto agli stessi prodotti ottenuti con farine tradizionali. I risultati evidenziano che i prodotti con farina di fagiolo sono meno apprezzati di quelli di riferimento, tuttavia la presenza di un intenso sapore umami nei prodotti con fagiolo risulta apprezzato.

Polybutylene succinate (PBS) is one of the most important and commercially available biodegradable polyesters obtained by polycondensation between succinic acid and butanediol [1]. It is endowed with high mechanical properties, comparable to polyethylene (PE) and polypropylene (PP); high thermal, chemical resistance and high heat deflection temperature. However, its applications suffer of some limitations due to softness, gas barrier properties and melt viscosity, limiting its practical use. To overcome these problems, the introduction of inorganic materials into biopolymers has been envisioned as a viable option to improve the structural properties of PBS and promote the exploitation in different application fields. The sequential infiltration synthesis (SIS), based on atomic deposition of Al2O3 on PBS, via trimethylaluminum (TMA) and H2O precursors, provides an attractive option for the production of a polymer-inorganic hybrid material. After the growth of Al2O3 in freestanding ~30 ?m thick PBS films by SIS process at 70°C, changes on PBS microstructure and mass uptake in the films were evaluated as a function of the number of SIS cycles [2]. Results evidenced that mass uptake in the PBS films was much higher than in standard polymethylmethacrylate (PMMA) films, at the same process conditions. This study evaluates changes on PBS microstructure vs mass uptake and explores the reactivity of the PBS functional groups, (ester and ether) after TMA treatment, through solution Nuclear Magnetic Resonance spectroscopy (NMR) thus suggesting a plausible reaction mechanism able to justify structural changes in PBS [3].

Common beans (Phaseolus vulgaris L.), a staple food in many regions of the world, are a major source of dietary fibers, essential amino acid-rich proteins, some vitamins and often display a high content in essential minerals such as iron, zinc and calcium. However, these minerals are scarcely bioavailable, mainly due to the presence of phytic acid (PA) and phenolic compounds. Besides, about 60% of common beans produced worldwide are grown in regions subjected to water stress, the second most important factor that contributes to yield reduction. To address this problem, BIO-BELIEF, a multidisciplinary project consisting of a trans-continentalconsortium of European (CREA-GB and CNR-IBBA in Italy and INRAE, INSERM in France); and African partners (CIAT in Kenya and the University of Pretoria with UKZN andStellenbosch University as local South African partners) with Embrapa in Brazil and the seedcompany Blumen in Italy as partners in kind, aims to select new biofortified more droughtresilient common bean lines, in order to promote a healthy diet in a general frame of food andnutrient security. The approach, to study biofortified common beans lines exposed to drought,has, to our knowledge, never been done before and is therefore innovative. Current researchactivities and results already obtained include that 20 common bean lines derived from Europeand Africa have been already planted for seed multiplication and the germination potential ofdifferent lines, including lpa mutant lines low in phytic acid, is currently tested in twocontinents (Europe and Africa at three test sites). First results already indicate that a low phyticacid content does not significantly affect seed germination. Common bean lines are currentlyalso tested in the field and greenhouse for drought tolerance in Italy (open field and greenhouse)and in South Africa (open field under rainout shelters) by applying the leaf wilting techniquefor monitoring drought tolerance. Selected lines will be further used for introgression of thebiofortification trait into a drought-resilient genetic background and the innovative technologyof Genome Editing (GE) will also be applied in a next step to modify/silence candidate genesinvolved in phytic acid production and drought tolerance. In particular, PvMYB 60, atranscriptional repressor of anthocyanin, and PvDREB 2, a transcription factor coding for adehydration-responsive element binding protein, which are both described as negativeregulators of the drought stress response will be selected to be edited in order to develop moredrought-tolerant common bean lines. Biofortified lines will be further exploited in the next twoyears by preparing bean-based recipes, which will be characterized for their nutritional profileand micronutrient bioavailability. All identified superior lines will be finally tested byconsumers to validate any new biofortified diets for European, African and South Americanpopulations. Overall, we expect that the project will ultimately release breeding lines, with ahigh nutritional value and more drought-resilient, suitable to be grown in Europe, Africa andLatin America. For more information including a video on the BIO-BELIEF project see forexample https://www linkedin com/company/biobelief/ as well as in the two recentpublications Losa et al. (2022). Drought and heat affect common bean minerals and humandiet--What we know and where to go. Food and Energy Security 11:e351. doi:10.1002/fes3.351 and Cominelli et al. (2022). Antinutritional factors, nutritional improvement,and future food use of common beans: A perspective. Frontiers Plant Sciences 13:992169. doi:10.3389/fpls.2022.992169

The shelf life of a food products is the time during which food remains safe under defined storage conditions, maintaining the desired sensory, chemical, physical and biological characteristics in compliance with the label declaration [1]. Many physical factors could influence the shelf life like temperature changes, light exposure, gases transmission, humidity changes as well as contamination with microorganism and spores. Packaging plays a critical role in extending the shelf life of food products, preventing or reducing the environmental interactions. Recent EU regulations promoted a growing interest in bio-based materials production for replacement of the traditional petro-plastics, limiting the accumulation problem and reducing the environmental pollution. NMR spectroscopy represents a valid approach to evaluate the effects of packaging on the shelf life of foods and possible chemical contamination. [2]. Moreover, NMR spectroscopy has already demonstrated its pivotal role in metabolomics [3,4] allowing to monitor in a single experiment different classes of chemical compounds, and its capabilities in microstructural characterization of packaging materials. In this study the analyses of polar and organic extracts of seasoned zucchini stored at 4°C for 35 days, in plastic and compostable trays, performed by NMR in combination with chemometrics, are reported.

L'analisi genetica pemette la caratterizzazione e tracciabilità delle produzioni locali e varietali. Le caratteristiche genetiche delle produzioni (frutti, semi e prodotti di trasformazione) vengono generalmente riferite al profilo genetico di una varietà di riferimento. Tale approccio non tiene conto del contributo pollinico esterno alla varietà, che può essere molto rilevante nelle piante auto-incompatibili o maschiosterili. Il castagno rappresenta un modello unico per studiare questo flusso genico, perché varietà coltivate, impollinatori selezionati e piante selvatiche possono coesistere. Inoltre, le varietà di "Marrone" sono spesso maschiosterili e ricevono flusso genico obbligato dagli impollinatori locali. Grazie ai progetti PSR "Multicast" e "Castadiva", aree castanicole delle regioni Umbria e Lombardia, sono state caratterizzate attraverso l'analisi genetica integrata di piante da frutto, frutti e impollinatori. I risultati hanno mostrato una gestione più convenzionale in Umbria, con castagneti da frutto geneticamente omogenei, innestati con poche varietà, diverse tra le aree castanicole. In Lombardia esiste invece una maggiore diversità genetica e gestionale con molte varietà o genotipi coesistenti all'interno di castagneti geneticamente eterogenei. In altre aree castanicole Lombarde, invece, i castagneti contengono principalmente piante innestate di "Marrone", insieme a poche altre varietà. In Umbria, l'analisi genetica integrata ha rivelato che i frutti delle varietà di "Marrone" mostrano un profilo genetico intermedio tra le piante coltivate e gli impollinatori selvatici locali. Ciò conferisce un'identità genetica e geografica alle produzioni. I risultati aprono prospettive per la caratterizzazione genetica e la tracciabilità delle produzioni. Questo approccio potrebbe integrare il concetto di "terroir", perché il territorio di coltivazione lascia una traccia genetica nelle produzioni locali, attraverso il flusso genico tra piante coltivate e selvatiche.

Over the centuries, the chestnut tree, cultivated for its fruits and timber, has become an essential element of subsistence for many societies in mountain and sub-mountainous areas, revealing its potential as a multifunctional species. Today most chestnut forests are in a state of decline and abandonment, mainly due to the depopulation of rural areas, global climate changes and recent outbreaks of exotic pests. With a view to the recovery and enhancement of chestnut genetic resources, the CASTADIVA project mainly aims to map the chestnut groves in pilot areas of the Lombardy Region (Municipality of Serle, BS and in the Varese Prealps, VA); evaluate the genetic diversity of chestnut stands, and genetically characterize local varieties. In parallel, the project includes a characterization of the fruits through morphological, pomological, qualitative and nutritional analyses. CASTADIVA will require integration of data obtained from genetic and morphometric analyses with ecological, silvicultural, phytosanitary and possibly socio-economic data, in order to identify the best practices and management criteria for the conservation and enhancement of chestnut genetic resources. Transferring information to local administrations and the agricultural stakeholder will be a final step of crucial importance. Preliminary results of genetic and nutritional analyses will be presented.

Common bean (Phaseolus vulgaris L.) is a staple food in many regions in the world. Bean seeds are a major source of dietary fibers, essential amino acid-rich proteins, some vitamins and often display a high content in essential minerals such as iron, zinc and calcium. However, these minerals are scarcely bioavailable, mainly due to the presence of phytic acid (PA) and phenolic compounds. Besides, about 60% of common beans produced worldwide are grown in regions subjected to water stress, thus after diseases, drought is the second most important factor that contributes to yield reduction. BIO-BELIEF (BIOfortification of common Bean to promote heaLthy dIEt and Food security) is a multidisciplinary project, financed in the frame of the ERA-NET FOSC call, born from the establishment of a trans-continental consortium between European, African and Brasilian partners, aims to select new biofortified and drought resilient bean lines, in order to promote a healthy diet in a general frame of food security. The project will release genetic materials with high nutritional values and develop nutritional improved and drought resilient beans suitable to be grown in Europe, Africa and Latin America (last two are regions where bean is a major staple food). BIO-BELIEF will capitalize previous works carried out by some partners that have selected biofortified lines with reduced level of PA, increased iron content and improved drought resilience. About 20 lines will be tested for seed quality in response to drought treatment in two continents. Meanwhile, the biofortification traits will be introgressed in the drought resilient genetic backgrounds. The innovative technology of Genome Editing (GE) will also be applied to explore candidate genes involved in drought resilience. The biofortified lines will be exploited by preparing bean-based recipes, which will be characterized for their nutritional profile and micronutrient bioavailability. The selected lines will be used for testing by the consumers to validate new biofortified diets for European, African and South American populations. Here we will present the preliminary results of BIO-BELIEF project.

Common bean seeds are an excellent source of protein as well as of carbohydrates, minerals, vitamins, and bioactive compounds reducing, when in the diet, the risks of diseases. The presence of bioactive compounds with antinutritional properties (e.g., phytic acid, lectins, raffinosaccharides, protease inhibitors) limits, however, the bean's nutritional value and its wider use in food preparations. In the last decades, concerted efforts have been, therefore, made to develop new common bean genotypes with reduced antinutritional compounds by exploiting the natural genetic variability of common bean and also applying induced mutagenesis. However, possible negative, or positive, pleiotropic effects due to these modifications, in terms of plant performance in response to stresses or in the resulting technological properties of the developed mutant genotypes, have yet not been thoroughly investigated. The purpose of the perspective paper is to first highlight the current advances, which have been already made in mutant bean characterization. A view will be further provided on future research directions to specifically explore further advantages and disadvantages of these bean mutants, their potential use in innovative foods and representing a valuable genetic reservoir of combinations to assess the true functional role of specific seed bioactive components directly in the food matrix.

The development of effective tools for the sustainable supply of phyto-ingredients and natural substances with reduced environmental footprints can help mitigate the dramatic scenario of climate change. Plant cell cultures-based biorefineries can be a technological advancement to face this challenge and offer a potentially unlimited availability of natural substances, in a standardized composition and devoid of the seasonal variability of cultivated plants. Monounsaturated (MUFA) fatty acids are attracting considerable attention as supplements for biodegradable plastics, bio-additives for the cosmetic industry, and bio-lubricants. Cardoon (Cynara cardunculus L. var. altilis) callus cultures accumulate fatty acids and polyphenols and are therefore suitable for large-scale production of biochemicals and valuable compounds, as well as biofuel precursors. With the aim of boosting their potential uses, we designed a biotechnological approach to increase oleic acid content through Agrobacterium tumefaciens-mediated metabolic engineering. Bioinformatic data mining in the C. cardunculus transcriptome allowed the selection and molecular characterization of SAD (stearic acid desaturase) and FAD2.2 (fatty acid desaturase) genes, coding for key enzymes in oleic and linoleic acid formation, as targets for metabolic engineering. A total of 22 and 27 fast-growing independent CcSAD overexpressing (OE) and CcFAD2.2 RNAi knocked out (KO) transgenic lines were obtained. Further characterization of five independent transgenic lines for each construct demonstrated that, successfully, SAD overexpression increased linoleic acid content, e.g., to 42.5%, of the relative fatty acid content, in the CcSADOE6 line compared with 30.4% in the wild type (WT), whereas FAD2.2 silencing reduced linoleic acid in favor of the accumulation of its precursor, oleic acid, e.g., to almost 57% of the relative fatty acid content in the CcFAD2.2KO2 line with respect to 17.7% in the WT. Moreover, CcSADOE6 and CcFAD2.2KO2 were also characterized by a significant increase in total polyphenolic content up to about 4.7 and 4.1 mg/g DW as compared with 2.7 mg/g DW in the WT, mainly due to the accumulation of dicaffeoyl quinic and feruloyl quinic acids. These results pose the basis for the effective creation of an engineered cardoon cells-based biorefinery accumulating high levels of valuable compounds from primary and specialized metabolism to meet the industrial demand for renewable and sustainable sources of innovative bioproducts.

I fagioli (Phaseolus vulgaris L.) sono una fonte importante di nutrienti con effetti benefici sulla salute umana. Tuttavia contengono lectine che limitano l'uso diretto della farina nelle preparazioni alimentari e acido fitico che riduce la biodisponibilità dei cationi minerali. Si sono valutati snack biofortificati e una crema ottenuti con una farina di fagiolo non trattata priva di lectine attive (lec-) e con ridotto contenuto di acido fitico (lpa) e si è valutato l'apprezzamento da parte dei consumatori. I prodotti con la farina lec- lpa non mostrano attività eritroagglutinante residua dovuta alle lectine; mostrano una maggiore attività residua dell'inibitore dell'?-amilasi, un ridotto indice glicemico in vitro e una maggiore biodisponibilità di ferro rispetto a quelli preparati con farina standard. I prodotti sono meno apprezzati di quelli di riferimento senza farina di fagiolo, ma la presenza di un intenso sapore umami nei prodotti con fagiolo può risultare apprezzata.