Genetic study of Camelina sativa oilseed crop and selection of a new variety by the bulk method.

Camelina sativa, commonly referred to as camelina or false flax, has emerged as a promising cover crop with the potential to mitigate climate change-a pressing global challenge that demands urgent and sustainable solutions. Belonging to the Brassicaceae family and native to Europe and Central Asia, camelina is an oilseed crop known for its resilience in diverse climates, including arid and semi-arid regions, making it adaptable to various environments. A breeding program started from a study of six winter varieties and five spring varieties of camelina is described: these genetic materials were characterized by SSRs molecular markers and by GBS technique. Molecular data clearly showed all spring varieties were genetically similar and distinguishable from the winter varieties, which, in turn, clustered together. Using molecular data, parental varieties belonging to the two different clusters were selected to generate new genetic variability. The new variety obtained, selected through the bulk method based on three parameters: yield, earliness, and weight of 1000 seeds, has allowed the generation of the new genetic material provisionally named C1244. Chemical characterization was performed (bromatological and glucosinolates analysis) to better describe C1244 in comparison with benchmark varieties. The new variety exhibited early maturity, similar to spring varieties, making this genetic material promising for use in intercropping systems, a high weight of 1000 seeds (1.46 g) which improves and facilitates seeding/harvesting operations and a high oil content (33.62%) akin to winter varieties making it valuable for human and animal food purposes.

Priming Treatments with Biostimulants to Cope the Short-Term Heat Stress Response: A Transcriptomic Profile Evaluation.

Plant stress induced by high temperature is a problem in wide areas of different regions in the world. The trend of global warming is going to enhance the effects of heat stress on crops in many cultivation areas. Heat stress impairs the stability of cell membranes and many biological processes involving both primary and secondary metabolism. Biostimulants are innovative agronomical tools that can be used as a strategy to counteract the detrimental effect of abiotic stresses, including heat stress. In this work, two biostimulants based on Ascophyllum nodosum extracts (named Phylgreen) and based on animal L-α amino acids (named Delfan Plus) were applied as priming treatments to Arabidopsis thaliana plants subjected to heat stress exposure. Plants at the vegetative stage were treated with biostimulants 12 h before high temperature exposure, which consisted of maintaining the plants at 37 ± 1 °C for 4 h. Transcriptional profiles, physiological, and biochemical analyses were performed to understand the mode of action of the biostimulants in protecting the plants exposed to short-term heat stress. At a physiological level, chlorophyll, chlorophyll a fluorescence, phenolic index, total anthocyanins, reactive oxygen species (ROS) were measured, and significant variations were observed immediately after stress. Both biostimulants were able to reduce the oxidative damage in leaves and cell membrane. Transcriptomic data revealed that upregulated genes were 626 in Phylgreen and 365 in Delfan Plus, while downregulated genes were 295 in Phylgreen and 312 in Delfan Plus. Bioinformatic analysis showed that the biostimulants protected the plants from heat stress by activating specific heat shock proteins (HPS), antioxidant systems, and ROS scavengers. The results revealed that the biostimulants effectively induced the activation of heat stress-associated genes belonging to different transcription factors and HSP families. Among the heat shock proteins, the most important was the AtHSP17 family and in particular, those influenced by treatments were AtHPS17.4 and AtHPS17.6A, B, showing the most relevant changes.

Brachytic2 mutation is able to counteract the main pleiotropic effects of brown midrib3 mutant in maize.

Maize is the basis of nutrition of domesticated herbivores and one of the most promising energy crops. The presence of lignin in the cell wall, tightly associated to carbohydrates, prevents the physical access of enzymes such as cellulase, limiting the carbohydrate degradability and consequently the energy value. To increase the utilization of the biomass cellulose content, the challenge of breeding programs is to lower or modify the lignin components. In maize several mutations are able to modify the lignin content and in particular the mutation in brown midrib3 (bm3) gene appeared as one of the most promising in breeding programs. Unfortunately this mutation has several negative pleiotropic effects on various important agronomic traits such as stay green, lodging and susceptibility to several infections.The maize Brachyitic 2 (br2) gene encodes for a putative protein involved in polar movement of auxins. br2 mutant plants are characterized by shortening of lower stalk internodes, unusual stalk strength and tolerance to wind lodging, darker leaves persisting longer in the active green state in comparison to wild type plants, suggesting a possible utilization of br2 plants to counteract the negative effects of the bm3 mutation. In this work, we report the generation and a preliminary characterization of the double mutant bm3 br2, suggesting the potential use of this new genetic material to increase biomass cellulose utilization.

Food Containing Bioactive Flavonoids and Other Phenolic or Sulfur Phytochemicals With Antiviral Effect: Can We Design a Promising Diet Against COVID-19?

Since in late 2019, when the coronavirus 2 (SARS-CoV-2) pathogen of coronavirus disease 2019 (COVID-19) started to spread all over the world, causing the awful global pandemic we are still experiencing, an impressive number of biologists, infectious disease scientists, virologists, pharmacologists, molecular biologists, immunologists, and other researchers working in laboratories of all the advanced countries focused their research on the setting up of biotechnological tools, namely vaccines and monoclonal antibodies, as well as of rational design of drugs for therapeutic approaches. While vaccines have been quickly obtained, no satisfactory anti-Covid-19 preventive, or therapeutic approach has so far been discovered and approved. However, among the possible ways to achieve the goal of COVID-19 prevention or mitigation, there is one route, i.e., the diet, which until now has had little consideration. In fact, in the edible parts of plants supplying our food, there are a fair number of secondary metabolites mainly belonging to the large class of the flavonoids, endowed with antiviral or other health beneficial activities such as immunostimulating or anti-inflammatory action that could play a role in contributing to some extent to prevent or alleviate the viral infection and/or counteract the development of SARS induced by the novel coronavirus. In this review, a number of bioactive phytochemicals, in particular flavonoids, proven to be capable of providing some degree of protection against COVID-19, are browsed, illustrating their beneficial properties and mechanisms of action as well as their distribution in cultivated plant species which supply food for the human diet. Furthermore, room is also given to information regarding the amount in food, the resistance to cooking processes and, as a very important feature, the degree of bioavailability of these compounds. Concluding, remarks and perspectives for future studies aimed at increasing and improving knowledge and the possibility of using this natural complementary therapy to counteract COVID-19 and other viral pathologies are discussed.

Genetic Improvement of Arundo donax L.: Opportunities and Challenges.

Arundo donax L., the giant reed-being a long-duration, low-cost, non-food energy crop able to grow in marginal lands-has emerged as a potential alternative to produce biomass for both energy production, with low carbon emissions, and industrial bioproducts. In recent years, pioneering efforts have been made to genetically improve this very promising energy crop. This review analyses the recent advances and challenges encountered in using clonal selection, mutagenesis/somaclonal variation and transgenesis/genome editing. Attempts to improve crop yield, in vitro propagation efficiency, salt and heavy metal tolerance by clonal selection were carried out, although limited by the species' low genetic diversity and availability of mutants. Mutagenesis and somaclonal variation have also been attempted on this species; however, since Arundo donax is polyploid, it is very difficult to induce and select promising mutations. In more recent years, genomics and transcriptomics data are becoming available in Arundo, closing the gap to make possible the genetic manipulation of this energy crop in the near future. The challenge will regard the functional characterization of the genes/sequences generated by genomic sequencing and transcriptomic analysis in a complex polyploid genome.

Calcium redistribution contributes to the hard-to-cook phenotype and increases PHA-L lectin thermal stability in common bean low phytic acid 1 mutant seeds.

Seed phytic acid reduces mineral bioavailability by chelating minerals. Consumption of common bean seeds with the low phytic acid 1 (lpa1) mutation improved iron status in human trials but caused adverse gastrointestinal effects, presumably due to increased stability of lectin phytohemagglutinin L (PHA-L) compared to the wild type (wt). A hard-to-cook (HTC) defect observed in lpa1 seeds intensified this problem. We quantified the HTC phenotype of lpa1 common beans with three genetic backgrounds. The HTC phenotype in the lpa1 black bean line correlated with the redistribution of calcium particularly in the cell walls, providing support for the "phytase-phytate-pectin" theory of the HTC mechanism. Furthermore, the excess of free cations in the lpa1 mutation in combination with different PHA alleles affected the stability of PHA-L lectin.

Phlobaphenes modify pericarp thickness in maize and accumulation of the fumonisin mycotoxins.

Phlobaphenes are insoluble phenolic compounds which are accumulated in a limited number of tissues such as seed pericarp and cob glumes, conferring on them a typical red-brown pigmentation. These secondary metabolites, derived from 3-deoxy flavonoids, are thought to have an important role in plants' resistance against various pathogens, e.g. by reducing fungal infection, and also to have beneficial effects on human and animal health due to their high antioxidant power. The aim of this work was to determine the role of phlobaphenes in reducing mycotoxin contamination on maize kernels. We analysed the effect of the P1 (pericarp color 1) gene on phlobaphenes accumulation, pericarp thickness and fumonisins accumulation. Analysing fumonisins accumulation in different genetic backgrounds through three seasons, we found a clear decrease of these toxins through the three years (Wilcoxon test, Z = 2.2, p = 0.0277) in coloured lines compared with the isogenic non-coloured ones. The coloured lines, carrying P1 allele showed an increase of phlobaphenes (about 10-14 fold) with respect to colourless lines. Furthermore there was a correlation between phlobaphenes accumulation and pericarp thickness (R = 0.9318; p = 0.0067). Taken together, these results suggest that the P1 gene plays a central role in regulating phlobaphenes accumulation in maize kernels, and indirectly, also tackles mycotoxins accumulation. The development and cultivation of corn varieties rich in phlobaphenes could be a powerful tool to reduce the loss of both quality and yield due to mycotoxin contamination, increasing the safety and the quality of the maize product.

lpa1-5525: A New lpa1 Mutant Isolated in a Mutagenized Population by a Novel Non-Disrupting Screening Method.

Phytic acid, or myo-inositol 1,2,3,4,5,6-hexakisphosphate, is the main storage form of phosphorus in plants. It is localized in seeds, deposited as mixed salts of mineral cations in protein storage vacuoles; during germination, it is hydrolyzed by phytases to make available P together with all the other cations needed for seed germination. When seeds are used as food or feed, phytic acid and the bound cations are poorly bioavailable for human and monogastric livestock due to their lack of phytase activity. Therefore, reducing the amount of phytic acid is one strategy in breeding programs aimed to improve the nutritional properties of major crops. In this work, we present data on the isolation of a new maize (Zea mays L.) low phytic acid 1 (lpa1) mutant allele obtained by transposon tagging mutagenesis with the Ac element. We describe the generation of the mutagenized population and the screening to isolate new lpa1 mutants. In particular, we developed a fast, cheap and non-disrupting screening method based on the different density of lpa1 seed compared to the wild type. This assay allowed the isolation of the lpa1-5525 mutant characterized by a new mutation in the lpa1 locus associated with a lower amount of phytic phosphorus in the seeds in comparison with the wild type.

Study and characterization of an ancient European flint white maize rich in anthocyanins: Millo Corvo from Galicia.

In the second half of the last century, the American dent hybrids began to be widely grown, leading to the disappearance or marginalization of the less productive traditional varieties. Nowadays the characterization of traditional landraces can help breeders to discover precious alleles that could be useful for modern genetic improvement and allow a correct conservation of these open pollinated varieties (opvs). In this work we characterized the ancient coloured cultivar "Millo Corvo" typical of the Spanish region of Galicia. We showed that this cultivar accumulates high amounts of anthocyanins (83.4 mg/100g flour), and by TLC (Thin Layer Chromatography) and HPLC (High Pressure Liquid Chromatography) analysis, we demonstrated that they mainly consisted of cyanidin. Mapping and sequencing data demonstrate that anthocyanin pigmentation is due to the presence of the red color1 gene(r1), a transcription factor driving the accumulation of this pigment in the aleurone layer. Further chemical analysis showed that the kernels are lacking in carotenoids, as confirmed by genetic study. Finally a DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging ability test showed that Millo Corvo, even though lacking carotenoids, has a high antioxidant ability, and could be considered as a functional food due to the presence of anthocyanins.

A mutation in the FZL gene of Arabidopsis causing alteration in chloroplast morphology results in a lesion mimic phenotype.

Lesion mimic mutants (LMMs) are a class of mutants in which hypersensitive cell death and defence responses are constitutively activated in the absence of pathogen attack. Various signalling molecules, such as salicylic acid (SA), reactive oxygen species (ROS), nitric oxide (NO), Ca(2+), ethylene, and jasmonate, are involved in the regulation of multiple pathways controlling hypersensitive response (HR) activation, and LMMs are considered useful tools to understand the role played by the key elements of the HR cell death signalling cascade. Here the characterization of an Arabidopsis LMM lacking the function of the FZL gene is reported. This gene encodes a membrane-remodelling GTPase playing an essential role in the determination of thylakoid and chloroplast morphology. The mutant displayed alteration in chloroplast number, size, and shape, and the typical characteristics of an LMM, namely development of chlorotic lesions on rosette leaves and constitutive expression of genetic and biochemical markers associated with defence responses. The chloroplasts are a major source of ROS, and the characterization of this mutant suggests that their accumulation, triggered by damage to the chloroplast membranes, is a signal sufficient to start the HR signalling cascade, thus confirming the central role of the chloroplast in HR activation.

Low phytic acid 1 mutation in maize modifies density, starch properties, cations, and fiber contents in the seed.

Monogastric animals are unable to digest phytic acid, so it represents an antinutritional factor and also an environmental problem. One strategy to solve this problem is the utilization of low phytic acid (lpa) mutants that accumulate low levels of phytic P and high levels of free phosphate in the seeds; among the lpa maize mutants lpa1 exhibited the highest reduction of phytic acid in the seed. This study indicated that the low phytic acid mutations exerted pleiotropic effects not directly connected to the phytic acid pathway, such as on seed density, content of ions, and the antioxidant compounds present in the kernels. Furthermore some nutritional properties of the flour were altered by the lpa1 mutations, in particular lignin and protein content, while the starch does not seem to be modified as to the total amount and in the amylose/amylopectin ratio, but alterations were noticed in the structure and size of granules.

A loss-of-function mutation in Calmodulin2 gene affects pollen germination in Arabidopsis thaliana.

Calmodulin (CAM) is an ubiquitous calcium binding protein whose function is to translate the signals, perceived as calcium concentration variations, into the appropriate cellular responses. In Arabidopsis thaliana there are 4 CAM isoforms which are highly similar, encoded by 7 genes, and one possible explanation proposed for the evolutionary conservation of the CAM gene family is that the different genes have acquired different functions so that they play possibly overlapping but non-identical roles. Here we report the characterization of the Arabidopsis mutant cam2-2, identified among the lines of the gene-trapping collection EXOTIC because of a distorted segregation of kanamycin resistance. Phenotypic analysis showed that in normal growth conditions cam2-2 plants were indistinguishable from the wild type while genetic analysis showed a reduced transmission of the cam2-2 allele through the male gametophyte and in vitro pollen germination revealed a reduced level of germination in comparison with the wild type. These results provide genetic evidence of the involvement of a CAM gene in pollen germination and support the theory of functional diversification of the CAM gene family.

The low phytic acid1-241 (lpa1-241) maize mutation alters the accumulation of anthocyanin pigment in the kernel.

The lpa1 mutations in maize are caused by lesions in the ZmMRP4 (multidrug resistance-associated proteins 4) gene. In previous studies (Raboy et al. in Plant Physiol 124:355-368, 2000; Pilu et al. in Theor Appl Genet 107:980-987, 2003a; Shi et al. Nat Biotechnol 25:930-937, 2007), several mutations have been isolated in this locus causing a reduction of phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate, or InsP(6)) content and an equivalent increasing of free phosphate. In particular, the lpa1-241 mutation causes a reduction of up to 90% of phytic acid, associated with strong pleiotropic effects on the whole plant. In this work, we show, for the first time to our knowledge, an interaction between the accumulation of anthocyanin pigments in the kernel and the lpa mutations. In fact the lpa1-241 mutant accumulates a higher level of anthocyanins as compared to wild type either in the embryo (about 3.8-fold) or in the aleurone layer (about 0.3-fold) in a genotype able to accumulate anthocyanin. Furthermore, we demonstrate that these pigments are mislocalised in the cytoplasm, conferring a blue pigmentation of the scutellum, because of the neutral/basic pH of this cellular compartment. As a matter of fact, the propionate treatment, causing a specific acidification of the cytoplasm, restored the red pigmentation of the scutellum in the mutant and expression analysis showed a reduction of ZmMRP3 anthocyanins' transporter gene expression. On the whole, these data strongly suggest a possible interaction between the lpa mutation and anthocyanin accumulation and compartmentalisation in the kernel.