A draft genome of sweet cherry (Prunus avium L.) reveals genome-wide and local effects of domestication.

Sweet cherry (Prunus avium L.) trees are both economically important fruit crops but also important components of natural forest ecosystems in Europe, Asia and Africa. Wild and domesticated trees currently coexist in the same geographic areas with important questions arising on their historical relationships. Little is known about the effects of the domestication process on the evolution of the sweet cherry genome. We assembled and annotated the genome of the cultivated variety "Big Star*" and assessed the genetic diversity among 97 sweet cherry accessions representing three different stages in the domestication and breeding process (wild trees, landraces and modern varieties). The genetic diversity analysis revealed significant genome-wide losses of variation among the three stages and supports a clear distinction between wild and domesticated trees, with only limited gene flow being detected between wild trees and domesticated landraces. We identified 11 domestication sweeps and five breeding sweeps covering, respectively, 11.0 and 2.4 Mb of the P. avium genome. A considerable fraction of the domestication sweeps overlaps with those detected in the related species, Prunus persica (peach), indicating that artificial selection during domestication may have acted independently on the same regions and genes in the two species. We detected 104 candidate genes in sweep regions involved in different processes, such as the determination of fruit texture, the regulation of flowering and fruit ripening and the resistance to pathogens. The signatures of selection identified will enable future evolutionary studies and provide a valuable resource for genetic improvement and conservation programs in sweet cherry.

Overexpression of the MYB29 transcription factor affects aliphatic glucosinolate synthesis in Brassica oleracea.

KEY MESSAGE: Overexpression of BoMYB29 gene up-regulates the aliphatic glucosinolate pathway in Brassica oleracea plants increasing the production of the anti-cancer metabolite glucoraphanin, and the toxic and pungent sinigrin. Isothiocyanates, the bio-active hydrolysis products of glucosinolates, naturally produced by several Brassicaceae species, play an important role in human health and agriculture. This study aims at correlating the content of aliphatic glucosinolates to the expression of genes involved in their synthesis in Brassica oleracea, and perform functional analysis of BoMYB29 gene. To this purpose, three genotypes were used: a sprouting broccoli, a cabbage, and a wild genotype (Winspit), a high glucosinolate containing accession. Winspit showed the highest transcript level of BoMYB28, BoMYB29 and BoAOP2 genes, and BoAOP2 expression was positively correlated with that of the two MYB genes. Further analyses of the aliphatic glucosinolates also showed a positive correlation between the expression of BoAOP2 and the production of sinigrin and gluconapin in Winspit. The Winspit BoMYB29 CDS was cloned and overexpressed in Winspit and in the DH AG1012 line. Overexpressing Winspit plants produced higher quantities of alkenyl glucosinolates, such as sinigrin. Conversely, the DH AG1012 transformants showed a higher production of methylsulphinylalkyl glucosinolates, including glucoraphanin, and, despite an up-regulation of the aliphatic glucosinolate genes, no increase in alkenyl glucosinolates. The latter may be explained by the absence of a functional AOP2 gene in DH AG1012. Nevertheless, an extract of DH AG1012 lines overexpressing BoMYB29 provided a chemoprotective effect on human colon cells. This work exemplifies how the genetic diversity of B. oleracea may be used by breeders to select for higher expression of transcription factors for glucosinolate biosynthesis to improve its natural, health-promoting properties.

MicroRNAs in durum wheat seedlings under chronic and short-term nitrogen stress.

Nitrogen is an essential macronutrient for plant growth and reproduction. In durum wheat, an appropriate nitrogen soil availability is essential for an optimal seed development. miRNAs contribute to the environmental change adaptation of plants through the regulation of important genes involved in stress processes. In this work, nitrogen stress response was evaluated in durum wheat seedlings of Ciccio and Svevo cultivars. Eight small RNA libraries from leaves and roots of chronically stressed plants were sequenced to detect conserved and novel miRNAs. A total of 294 miRNAs were identified, 7 of which were described here for the first time. The expression level of selected miRNAs and target genes was analyzed by qPCR in seedlings subjected to chronic (Ciccio and Svevo, leaves and roots) or short-term (Svevo roots) stress conditions. Some miRNAs showed an immediate stress response, and their level of expression was either maintained or returned to a basal level during a long-term stress. Other miRNAs showed a gradual up- or downregulation during the short-term stress. The newly identified miRNA ttu-novel-106 showed an immediate strongly downregulation after nitrogen stress, which was negatively correlated with the expression of MYB-A, its putative target gene. PHO2 gene was significantly upregulated after 24-48-h stress, corresponding to a downregulation of miR399b. Ttu-miR399b putative binding sites in the 5' UTR region of the Svevo PHO2 gene were identified in the A and B genomes. Both MYB-A and PHO2 genes were validated for their cleavage site using 5' RACE assay.

The miRNAome of durum wheat: isolation and characterisation of conserved and novel microRNAs and their target genes.

BACKGROUND: The allotetraploid durum wheat [Triticum turgidum subsp. durum (Desf.) Husn.] is a highly economically important species especially in the Mediterranean basin. However, its genomics, transcriptomics and in particular microRNAome are still largely unknown. RESULTS: In the present work, two small RNA libraries from durum wheat Ciccio and Svevo cultivars were generated from different tissues at the late milk (Z77) developmental stage. A total of 167 conserved and 98 potential novel miRNAs were identified in the two libraries and interestingly, three novel miRNAs were found to be derived from ribosomal RNA. Putative target genes were predicted for conserved and novel miRNAs, the majority of which interact with nucleic acids, according to GO terms relative to molecular function. Quantitative qPCR analysis showed that several miRNAs identified were differentially expressed in the mature (Z77) developmental stage compared to young (Z14) tissues. Moreover, target gene expression analysis suggested that in roots, the putative genes encoding for the SQUAMOSA SPL2 and TGA1 proteins are regulated by ttu-miR156n, while MYB3 transcription factor by ttu-miR319f. Additionally, the Photosystem II P680 chlorophyll A apoprotein gene showed an expression level negatively correlated to that of ttu-novel-48 in leaves. CONCLUSION: Our results suggest that, in durum wheat, these genes may play important roles in root/leaf development and are subjected to miRNA regulation. The prediction of novel miRNAs putatively derived from ribosomal RNA opens new perspectives on the study of plant miRNAs.

Genetic Associations of Alexithymia in Predicting Interferon-Induced Depression in Chronic Hepatitis C.

BACKGROUND: Previous studies have shown that alexithymia is associated with gene polymorphisms that regulate the availability of serotonin (5-HT) in the brain. Since the 5-HT network is involved in interferon (IFN)-induced depression, this paper aimed to investigate the role of alexithymia and the functional gene variants of the 5-HT1A receptor (HTR1A) and the 5-HT transporter (5-HTTLPR) in induction of depression during antiviral treatment. METHODS: The depressive symptoms of 130 consecutive patients with chronic hepatitis C and no current psychopathology were measured during treatment with IFN and ribavirin (6-12 months) and at a 6-month follow-up. At baseline, alexithymia and 2 genotypes (5-HTTLPR and HTR1A) were also assessed. RESULTS: Patients with homozygosity for HTR1A-G and 5-HTTLPR long alleles had significantly higher levels of alexithymia. After controlling for sociodemographic and disease-related factors, alexithymia and HTR1A-G polymorphism, both separately (20-22%) and jointly (14-16%), significantly and independently predicted the development of IFN-induced depression. CONCLUSIONS: Subjects carrying HTR1A-G and 5-HTTLRP double long alleles are more vulnerable to alexithymia. Also patients with a higher level of alexithymia and the HTR1A-G gene variant are more vulnerable to experiencing IFN-induced depressive symptoms. The clinical implications of targeting alexithymia and HTR1A receptors as a possible treatment option for mood disorders should be investigated in further studies.

Population structure of Cynara cardunculus complex and the origin of the conspecific crops artichoke and cardoon.

BACKGROUND AND AIMS: Globe artichoke and leafy cardoon, two crops within the same species Cynara cardunculus, are traditionally cultivated in the Mediterranean region and play a significant role in the agricultural economy of this area. The two cultigens have different reproductive systems: artichoke is generally vegetatively propagated, while leafy cardoon is seed propagated. The domestication events underlying the origin of both artichoke and cultivated cardoon from their wild relative and the area of occurrence are not yet fully understood. The aim of this study was to investigate population structure in wild cardoon, globe artichoke and leafy cardoon material and infer domestication events. METHODS: Thirty-five microsatellite (simple sequence repeat) markers, distributed in the C. cardunculus genome, and a large geographical and numerical sampling in southern Europe and North Africa were used to assess population structure and diversity. KEY RESULTS: The results suggest the presence of two distinct domestication events for artichoke and leafy cardoon, and also suggest a new possible scenario, with western wild cardoon having originated from cultivated cardoon escaped from cultivation. Evidence was found for a demographic bottleneck in the past history of globe artichoke. CONCLUSIONS: The results shed new light on the relationships between the three taxa of C. cardunculus and highlight relevant aspects on the evolution of domestication of two crops with a different reproductive system within the same species. It is proposed that the probable centre of origin of artichoke is located in southern Italy, probably Sicily.

A Survey of the Transcriptomic Resources in Durum Wheat: Stress Responses, Data Integration and Exploitation.

Durum wheat (Triticum turgidum subsp. durum (Desf.) Husn.) is an allotetraploid cereal crop of worldwide importance, given its use for making pasta, couscous, and bulgur. Under climate change scenarios, abiotic (e.g., high and low temperatures, salinity, drought) and biotic (mainly exemplified by fungal pathogens) stresses represent a significant limit for durum cultivation because they can severely affect yield and grain quality. The advent of next-generation sequencing technologies has brought a huge development in transcriptomic resources with many relevant datasets now available for durum wheat, at various anatomical levels, also focusing on phenological phases and environmental conditions. In this review, we cover all the transcriptomic resources generated on durum wheat to date and focus on the corresponding scientific insights gained into abiotic and biotic stress responses. We describe relevant databases, tools and approaches, including connections with other "omics" that could assist data integration for candidate gene discovery for bio-agronomical traits. The biological knowledge summarized here will ultimately help in accelerating durum wheat breeding.

Genetic Mapping of Flavonoid Grain Pigments in Durum Wheat.

Pigmented cereal grains with high levels of flavonoid compounds have attracted the attention of nutritional science backing the development of functional foods with claimed health benefits. In this study, we report results on the genetic factors controlling grain pigmentation in durum wheat using a segregant population of recombinant inbred lines (RILs) derived from a cross between an Ethiopian purple grain accession and an Italian amber grain cultivar. The RIL population was genotyped by the wheat 25K SNP array and phenotyped for total anthocyanin content (TAC), grain color, and the L*, a*, and b* color index of wholemeal flour, based on four field trials. The mapping population showed a wide variation for the five traits in the different environments, a significant genotype x environment interaction, and high heritability. A total of 5942 SNP markers were used for constructing the genetic linkage map, with an SNP density ranging from 1.4 to 2.9 markers/cM. Two quantitative trait loci (QTL) were identified for TAC mapping on chromosome arms 2AL and 7BS in the same genomic regions of two detected QTL for purple grain. The interaction between the two QTL was indicative of an inheritance pattern of two loci having complementary effects. Moreover, two QTL for red grain color were detected on chromosome arms 3AL and 3BL. The projection of the four QTL genomic regions on the durum wheat Svevo reference genome disclosed the occurrence of the candidate genes Pp-A3, Pp-B1, R-A1, and R-B1 involved in flavonoid biosynthetic pathways and encoding of transcription factors bHLH (Myc-1) and MYB (Mpc1, Myb10), previously reported in common wheat. The present study provides a set of molecular markers associated with grain pigments useful for the selection of essential alleles for flavonoid synthesis in durum wheat breeding programs and enhancement of the health-promoting quality of derived foods.

In-silico and in-vivo analyses of EST databases unveil conserved miRNAs from Carthamus tinctorius and Cynara cardunculus.

BACKGROUND: MicroRNAs (miRNAs) are small RNAs (21-24 bp) providing an RNA-based system of gene regulation highly conserved in plants and animals. In plants, miRNAs control mRNA degradation or restrain translation, affecting development and responses to stresses. Plant miRNAs show imperfect but extensive complementarity to mRNA targets, making their computational prediction possible, useful when data mining is applied on different species. In this study we used a comparative approach to identify both miRNAs and their targets, in artichoke and safflower. RESULTS: Two complete expressed sequence tags (ESTs) datasets from artichoke (3.6 · 10(4) entries) and safflower (4.2 · 10(4)), were analysed with a bioinformatic pipeline and in vitro experiments, identifying 17 potential miRNAs. For each EST, using RNAhybrid program and 953 non redundant miRNA mature sequences, available in mirBase as reference, we searched matching putative targets. 8730 out of 42011 ESTs from safflower and 7145 of 36323 ESTs from artichoke showed at least one predicted miRNA target. BLAST analysis showed that 75% of all ESTs shared at least a common homologous region (E-value < 10(-4)) and about 50% of these displayed 400 bp or longer aligned sequences as conserved homologous/orthologous (COS) regions. 960 and 890 ESTs of safflower and artichoke organized in COS shared 79 different miRNA targets, considered functionally conserved, and statistically significant when compared with random sequences (signal to noise ratio > 2 and specificity ≥ 0.85). Four highly significant miRNAs selected from in silico data were experimentally validated in globe artichoke leaves. CONCLUSIONS: Mature miRNAs and targets were predicted within EST sequences of safflower and artichoke. Most of the miRNA targets appeared highly/moderately conserved, highlighting an important and conserved function. In this study we introduce a stringent parameter for the comparative sequence analysis, represented by the identification of the same target in the COS region. After statistical analysis 79 targets, found on the COS regions and belonging to 60 miRNA families, have a signal to noise ratio > 2, with ≥ 0.85 specificity. The putative miRNAs identified belong to 55 dicotyledon plants and to 24 families only in monocotyledon.

The miRNAome of globe artichoke: conserved and novel micro RNAs and target analysis.

BACKGROUND: Plant microRNAs (miRNAs) are involved in post-transcriptional regulatory mechanisms of several processes, including the response to biotic and abiotic stress, often contributing to the adaptive response of the plant to adverse conditions. In addition to conserved miRNAs, found in a wide range of plant species a number of novel species-specific miRNAs, displaying lower levels of expression can be found. Due to low abundance, non conserved miRNAs are difficult to identify and isolate using conventional approaches. Conversely, deep-sequencing of small RNA (sRNA) libraries can detect even poorly expressed miRNAs.No miRNAs from globe artichoke have been described to date. We analyzed the miRNAome from artichoke by deep sequencing four sRNA libraries obtained from NaCl stressed and control leaves and roots. RESULTS: Conserved and novel miRNAs were discovered using accepted criteria. The expression level of selected miRNAs was monitored by quantitative real-time PCR. Targets were predicted and validated for their cleavage site. A total of 122 artichoke miRNAs were identified, 98 (25 families) of which were conserved with other plant species, and 24 were novel. Some miRNAs were differentially expressed according to tissue or condition, magnitude of variation after salt stress being more pronounced in roots. Target function was predicted by comparison to Arabidopsis proteins; the 43 targets (23 for novel miRNAs) identified included transcription factors and other genes, most of which involved in the response to various stresses. An unusual cleaved transcript was detected for miR393 target, transport inhibitor response 1. CONCLUSIONS: The miRNAome from artichoke, including novel miRNAs, was unveiled, providing useful information on the expression in different organs and conditions. New target genes were identified. We suggest that the generation of secondary short-interfering RNAs from miR393 target can be a general rule in the plant kingdom.

Polyphenolic Compound Variation in Globe Artichoke Cultivars as Affected by Fertilization and Biostimulants Application.

Globe artichoke is an ancient herbaceous plant native to the Mediterranean Basin. The edible part of the plant is limited to the fleshy leaves (bracts) and receptacle of a large immature inflorescence (head) that has been shown to be a rich source of bioactive compounds. Nutritional and pharmacological properties of artichoke heads and leaves are attributed mainly to polyphenolic compounds and inulin present at high concentration. In this study, polyphenols were investigated in two artichoke cultivars (Opal and Madrigal) in response to four nitrogen rates and foliar applications of biostimulating products under drip irrigation. Field experiments were carried out over two growing seasons (2015-2016, 2016-2017) in Policoro (MT), Southern Italy, on a deep clay soil in sub-humid climate conditions. Phenolic compounds were isolated and characterized by means of high-performance liquid chromatography with photodiode array detection and electrospray ionization/mass spectrometry (HPLC-DAD-MS/MS) analysis. In both cultivars, caffeoylquinic acids were more abundant when a dose of 100 kg ha-1 of ammonium nitrate was provided, whereas apigenins were not affected by nitrogen fertilization. Luteolins increased in cv Opal and decreased in cv Madrigal following N fertilization. The application of biostimulants (3 L ha-1) favored the accumulation of polyphenols, in particular of caffeoylquinic acids and apigenin, in artichoke heads in both cultivars. The results obtained highlight some positive aspects related to the synergistic effect of nitrogen fertilization and biostimulant foliar application.

R2R3-MYBs in Durum Wheat: Genome-Wide Identification, Poaceae-Specific Clusters, Expression, and Regulatory Dynamics Under Abiotic Stresses.

MYB transcription factors (TFs) represent one of the biggest TF families in plants, being involved in various specific plant processes, such as responses to biotic and abiotic stresses. The implication of MYB TFs in the tolerance mechanisms to abiotic stress is particularly interesting for crop breeding, since environmental conditions can negatively affect growth and productivity. Wheat is a worldwide-cultivated cereal, and is a major source of plant-based proteins in human food. In particular, durum wheat plays an important role in global food security improvement, since its adaptation to hot and dry conditions constitutes the base for the success of wheat breeding programs in future. In the present study, a genome-wide identification of R2R3-MYB TFs in durum wheat was performed. MYB profile search and phylogenetic analyses based on homology with Arabidopsis and rice MYB TFs led to the identification of 233 R2R3-TdMYB (Triticum durum MYB). Three Poaceae-specific MYB clusters were detected, one of which had never been described before. The expression of eight selected genes under different abiotic stress conditions, revealed that most of them responded especially to salt and drought stress. Finally, gene regulatory network analyses led to the identification of 41 gene targets for three TdR2R3-MYBs that represent novel candidates for functional analyses. This study provides a detailed description of durum wheat R2R3-MYB genes and contributes to a deeper understanding of the molecular response of durum wheat to unfavorable climate conditions.

Whole-exome sequencing of selected bread wheat recombinant inbred lines as a useful resource for allele mining and bulked segregant analysis.

Although wheat (Triticum aestivum L.) is the main staple crop in the world and a major source of carbohydrates and proteins, functional genomics and allele mining are still big challenges. Given the advances in next-generation sequencing (NGS) technologies, the identification of causal variants associated with a target phenotype has become feasible. For these reasons, here, by combining sequence capture and target-enrichment methods with high-throughput NGS re-sequencing, we were able to scan at exome-wide level 46 randomly selected bread wheat individuals from a recombinant inbred line population and to identify and classify a large number of single nucleotide polymorphisms (SNPs). For technical validation of results, eight randomly selected SNPs were converted into Kompetitive Allele-Specific PCR (KASP) markers. This resource was established as an accessible and reusable molecular toolkit for allele data mining. The dataset we are making available could be exploited for novel studies on bread wheat genetics and as a foundation for starting breeding programs aimed at improving different key agronomic traits.

Sweet Cherry Diversity and Relationships in Modern and Local Varieties Based on SNP Markers.

The sweet cherry is an important fruit species that is widespread globally. In addition to the well-known traditional and modern varieties, a myriad of landraces is present in Europe, as well as in southern Italy. This study aims to evaluate the population structure, genetic relationships, and cases of duplicate samples in a collection of 143 accessions using GBS-derived SNP markers. The genetic material under investigation includes modern commercial varieties, ancient European and American varieties, landraces, and individuals retrieved from small orchards. Some of the known varieties were genetically analyzed here for the first time. In addition, several genotypes were collected from the Basilicata region (southern Italy), an area largely unexplored for sweet cherry genetic resources. The relationships among genotypes were assessed using four different methods: allele frequency and ancestry estimation, principal component analysis, Neighbor-Joining tree, and identity-by-state estimation. The analyses returned quite congruent results and highlighted the presence of four main genetic groups, namely: (i) American varieties, (ii) the 'Germersdorfer-Ferrovia' cluster, (iii) the 'Burlat' group, and (iv) the group of Italian landraces. The main drivers of clustering were ancestry, geographical distribution, and some important traits such as self-compatibility. The sweet cherries from Basilicata, herewith examined for the first time, were mostly distributed within the group of Italian landraces, being particularly linked to the autochthonous varieties of the Campania region. However, some genotypes were outside this group, thus suggesting the introduction of genetic material from other Italian regions or from European countries. The considerable amount of American and European modern varieties analyzed are genetically very closely related, suggesting a reduced genetic basis. In addition, we highlighted the discriminating ability of SNP markers to distinguish between an original variety and its mutant. Overall, our results may be useful in defining conservation strategies for sweet cherry germplasm and developing future breeding programs to enlarge the genetic basis of commercial varieties.

Novel hydroxycinnamoyl-coenzyme A quinate transferase genes from artichoke are involved in the synthesis of chlorogenic acid.

Artichoke (Cynara cardunculus subsp. scolymus) extracts have high antioxidant capacity, due primarily to flavonoids and phenolic acids, particularly chlorogenic acid (5-caffeoylquinic acid [CGA]), dicaffeoylquinic acids, and caffeic acid, which are abundant in flower bracts and bioavailable to humans in the diet. The synthesis of CGA can occur following different routes in plant species, and hydroxycinnamoyl-coenzyme A transferases are important enzymes in these pathways. Here, we report on the isolation and characterization of two novel genes both encoding hydroxycinnamoyl-coenzyme A quinate transferases (HQT) from artichoke. The recombinant proteins (HQT1 and HQT2) were assayed after expression in Escherichia coli, and both showed higher affinity for quinate over shikimate. Their preferences for acyl donors, caffeoyl-coenzyme A or p-coumaroyl-coenzyme A, were examined. Modeling and docking analyses were used to propose possible pockets and residues involved in determining substrate specificities in the HQT enzyme family. Quantitative real-time polymerase chain reaction analysis of gene expression indicated that HQT1 might be more directly associated with CGA content. Transient and stable expression of HQT1 in Nicotiana resulted in a higher production of CGA and cynarin (1,3-dicaffeoylquinic acid). These findings suggest that several isoforms of HQT contribute to the synthesis of CGA in artichoke according to physiological needs and possibly following various metabolic routes.

Genetic map of artichoke × wild cardoon: toward a consensus map for Cynara cardunculus.

An integrated consensus linkage map is proposed for globe artichoke. Maternal and paternal genetic maps were constructed on the basis of an F(1) progeny derived from crossing an artichoke genotype (Mola) with its progenitor, the wild cardoon (Tolfa), using EST-derived SSRs, genomic SSRs, AFLPs, ten genes, and two morphological traits. For most genes, mainly belonging to the chlorogenic acid pathway, new markers were developed. Five of these were SNP markers analyzed through high-resolution melt technology. From the maternal (Mola) and paternal (Tolfa) maps, an integrated map was obtained, containing 337 molecular and one morphological markers ordered in 17 linkage groups (LGs), linked between Mola and Tolfa. The integrated map covers 1,488.8 cM, with an average distance of 4.4 cM between markers. The map was aligned with already existing maps for artichoke, and 12 LGs were linked via 31 bridge markers. LG numbering has been proposed. A total of 124 EST-SSRs and two genes were mapped here for the first time, providing a framework for the construction of a functional map in artichoke. The establishment of a consensus map represents a necessary condition to plan a complete sequencing of the globe artichoke genome.

Genotyping-by-Sequencing in Vigna unguiculata Landraces and Its Utility for Assessing Taxonomic Relationships.

Genotyping by sequencing (GBS) was used to analyze relationships among cowpea and asparagus bean landraces from southern Italy and to assess the utility of this technology to study taxonomy in a wider panel, including V. unguiculata cultigroups, subspecies, and other Vigna species. The analysis of SNPs derived from GBS highlighted that, among the cowpea landraces, the African samples were separated from the other material, while, for the Italian landraces, a certain clustering depending on seed color/pattern was observed in the dendrogram. When examining the V. unguiculata species complex, a clear separation between the two groups of wild subspecies, i.e., the allogamous wild perennials and the perennial out/inbreds, could be observed, the former representing the more ancestral wild progenitors of V. unguiculata. The species V. vexillata appeared more closely related to V. unguiculata than to the other Vigna species analyzed.

Identification and characterization of protease inhibitors in Diplotaxis species.

PCR analysis of the genomes of two wild Brassicaceae plants, Diplotaxis muralis and Diplotaxis tenuifolia, demonstrated the presence of several genes coding for potential protease inhibitors, classifiable within the mustard inhibitor family (MSI). This is a small family of plant protease inhibitors named after the mustard trypsin inhibitor MTI-2, the first protease inhibitor characterized in Brassicaceae. From identified sequences two recombinant inhibitors were expressed in Pichia pastoris. In comparison with MTI-2, they show a reduced activity against bovine trypsin. However, when tested against trypsin-like proteases present in the guts of Helicoverpa zea larvae, the Diplotaxis inhibitors and MTI-2 show similar activities, indicating that the usually adopted procedure of reporting activity of plant protease inhibitors against bovine trypsin may lead to wrong estimation of their effect on insect proteases. This issue is of particular relevance when planning the use of PI genes for developing insect resistant plants.

The Use of Nitrogen and Its Regulation in Cereals: Structural Genes, Transcription Factors, and the Role of miRNAs.

Cereals and, especially, rice, maize, and wheat, are essential commodities, on which human nutrition is based. Expanding population and food demand have required higher production which has been achieved by increasing fertilization, and especially nitrogen supply to cereal crops. In fact, nitrogen is a crucial nutrient for the plant, but excessive use poses serious environmental and health issues. Therefore, increasing nitrogen use efficiency in cereals is of pivotal importance for sustainable agriculture. The main steps in the use of nitrogen are uptake and transport, reduction and assimilation, and translocation and remobilization. Many studies have been carried out on the genes involved in these phases, and on transcription factors regulating these genes. Lately, increasing attention has been paid to miRNAs responding to abiotic stress, including nutrient deficiency. Many miRNAs have been found to regulate transcription factors acting on the expression of specific genes for nitrogen uptake or remobilization. Recent studies on gene regulatory networks have also demonstrated that miRNAs can interact with several nodes in the network, functioning as key regulators in nitrogen metabolism.

Durum wheat genome highlights past domestication signatures and future improvement targets.

The domestication of wild emmer wheat led to the selection of modern durum wheat, grown mainly for pasta production. We describe the 10.45 gigabase (Gb) assembly of the genome of durum wheat cultivar Svevo. The assembly enabled genome-wide genetic diversity analyses revealing the changes imposed by thousands of years of empirical selection and breeding. Regions exhibiting strong signatures of genetic divergence associated with domestication and breeding were widespread in the genome with several major diversity losses in the pericentromeric regions. A locus on chromosome 5B carries a gene encoding a metal transporter (TdHMA3-B1) with a non-functional variant causing high accumulation of cadmium in grain. The high-cadmium allele, widespread among durum cultivars but undetected in wild emmer accessions, increased in frequency from domesticated emmer to modern durum wheat. The rapid cloning of TdHMA3-B1 rescues a wild beneficial allele and demonstrates the practical use of the Svevo genome for wheat improvement.