Exploring the genetic landscape of nitrogen uptake in durum wheat: genome-wide characterization and expression profiling of NPF and NRT2 gene families

Nitrate uptake by plants primarily relies on two gene families: Nitrate transporter 1/peptide transporter (NPF) and Nitrate transporter 2 (NRT2). Here, we extensively characterized the NPF and NRT2 families in the durum wheat genome, revealing 211 NPF and 20 NRT2 genes. The two families share many Cis Regulatory Elements (CREs) and Transcription Factor binding sites, highlighting a partially overlapping regulatory system and suggesting a coordinated response for nitrate transport and utilization. Analyzing RNA-seq data from 9 tissues and 20 cultivars, we explored expression profiles and co-expression relationships of both gene families. We observed a strong correlation between nucleotide variation and gene expression within the NRT2 gene family, implicating a shared selection mechanism operating on both coding and regulatory regions. Furthermore, NPF genes showed highly tissue-specific expression profiles, while NRT2s were mainly divided in two co-expression modules, one expressed in roots (NAR2/NRT3 dependent) and the other induced in anthers and/ovaries during maturation. Our evidences confirmed that the majority of these genes were retained after small-scale duplication events, suggesting a neo- or sub-functionalization of many NPFs and NRT2s. Altogether, these findings indicate that the expansion of these gene families in durum wheat could provide valuable genetic variability useful to identify NUE-related and candidate genes for future breeding programs in the context of low-impact and sustainable agriculture

Transcriptome changes induced by Arbuscular mycorrhizal symbiosis in leaves of durum wheat (Triticum durum Desf.) promote higher salt tolerance

The salinity of soil is a relevant environmental problem around the world, with climate change raising its relevance, particularly in arid and semiarid areas. Arbuscular Mycorrhizal Fungi (AMF) positively affect plant growth and health by mitigating biotic and abiotic stresses, including salt stress. The mechanisms through which these benefits manifest are, however, still unclear. This work aimed to identify key genes involved in the response to salt stress induced by AMF using RNA-Seq analysis on durum wheat (Triticum turgidum L. subsp. durum Desf. Husn.). Five hundred sixty-three differentially expressed genes (DEGs), many of which involved in pathways related to plant stress responses, were identified. The expression of genes involved in trehalose metabolism, RNA processing, vesicle trafficking, cell wall organization, and signal transduction was significantly enhanced by the AMF symbiosis. A downregulation of genes involved in both enzymatic and non-enzymatic oxidative stress responses as well as amino acids, lipids, and carbohydrates metabolisms was also detected, suggesting a lower oxidative stress condition in the AMF inoculated plants. Interestingly, many transcription factor families, including WRKY, NAC, and MYB, already known for their key role in plant abiotic stress response, were found differentially expressed between treatments. This study provides valuable insights on AMF-induced gene expression modulation and the beneficial effects of plant-AMF interaction in durum wheat under salt stress

Genetic differentiation of the Capparis spinosa group in the Mediterranean area

The Capparis spinosa group is represented in the Mediterranean by a complex of taxa widespread in North Africa, the Middle East, and southern Europe. The taxonomy of this group used to be based on morphological characters with little work on the genetics of the group, and there is still much to be learned about its evolutionary history and diversification. We sampled 431 individuals of two subspecies and five varieties of C. spinosa and analysed them using highly informative EST-SSR markers to evaluate the population genetic diversity, structure and differentiation of the species in the Mediterranean. In addition, comparisons with the genetic profiles of C. spinosa subsp. cartilaginea, the putative ancestral taxon were made to investigate the phylogeographic history and possible gene flow across taxa. Integrated Bayesian approaches showed: i) a high divergence among C. spinosa subsp. spinosa var. canescens, C. spinosa subsp. spinosa var. aegyptia and the three varieties belonging to C. spinosa subsp. rupestris (var. rupestris, var. ovata and var. myrtifolia), with a clear separation between var. aegyptia and var. canescens which allows to consider var. aegyptia as a subspecies of C. spinosa; ii) a significant correlation between genetic divergence and geographic distance between the five varieties studied; iii) that the different varieties in the Mediterranean may have been derived from C. spinosa subsp. cartilaginea. Further genomic investigations are required to confirm our results. However, the findings presented allows us to suggest the genus Capparis can be considered a model for the study of the gene flow and differentiation in species occurring in a wide range of habitats

Bioactive Metabolite Survey of Actinobacteria Showing Plant Growth Promoting Traits to Develop Novel Biofertilizers

The use of chemical fertilizers and pesticides has caused harmful impacts on the environment with the increase in economic burden. Biofertilizers are biological products containing living microorganisms capable of improving plant growth through eco-friendly mechanisms. In this work, three actinobacterial strains Streptomyces violaceoruber, Streptomyces coelicolor, and Kocuria rhizophila were characterized for multiple plant growth promoting (PGP) traits such as indole acetic acid production, phosphate solubilization, N-2-fixation, and drought and salt tolerance. Then, these strains were investigated for their secreted and cellular metabolome, revealing a rich arsenal of bioactive molecules, including antibiotics and siderophores, with S. violaceoruber being the most prolific strain. Furthermore, the in vivo assays, performed on tomato (Solanum lycopersicum L.), resulted in an improved germination index and the growth of seedlings from seeds treated with PGP actinobacteria, with a particular focus on S. violaceoruber cultures. In particular, this last strain, producing volatile organic compounds having antimicrobial activity, was able to modulate volatilome and exert control on the global DNA methylation of tomato seedlings. Thus, these results, confirming the efficacy of the selected actinobacteria strains in promoting plant growth and development by producing volatile and non-volatile bioactive molecules, can promote eco-friendly alternatives in sustainable agriculture

Integrated omics approach reveals the molecular pathways activated in tomato by Kocuria rhizophila, a soil plant growth-promoting bacterium

Plant microbial biostimulants application has become a promising and eco-friendly agricultural strategy to improve crop yields, reducing chemical inputs for more sustainable cropping systems. The soil dwelling bacterium Kocuria rhizophila was previously characterized as Plant Growth Promoting Bacteria (PGPB) for its multiple PGP traits, such as indole-3-acetic acid production, phosphate solubilization capability and salt and drought stress tolerance. Here, we evaluated by a multi-omics approach, the PGP activity of K. rhizophila on tomato, revealing the molecular pathways by which it promotes plant growth. Transcriptomic analysis showed several up-regulated genes mainly related to amino acid metabolism, cell wall organization, lipid and secondary metabolism, together with a modulation in the DNA methylation profile, after PGPB inoculation. In agreement, proteins involved in photosynthesis, cell division, and plant growth were highly accumulated by K. rhizophila. Furthermore, "amino acid and peptides", "monosaccharides", and "TCA" classes of metabolites resulted the most affected by PGPB treatment, as well as dopamine, a catecholamine neurotransmitter mediating plant growth through S-adenosylmethionine decarboxylase (SAMDC), a gene enhancing the vegetative growth, up-regulated in tomato by K. rhizophila treatment. Interestingly, eight gene modules well correlated with differentially accumulated proteins (DAPs) and metabolites (DAMs), among which two modules showed the highest correlation with nine proteins, including a nucleoside diphosphate kinase, and cytosolic ascorbate peroxidase, as well as with several amino acids and metabolites involved in TCA cycle. Overall, our findings highlighted that sugars and amino acids, energy regulators, involved in tomato plant growth, were strongly modulated by the K. rhizophila-plant interaction

Identification and characterization of nitrate-related genes in durum wheat (Triticum turgidum L. subsp. durum Desf.) to improve Nitrogen Use Efficiency

The increasing foods demand caused by the increasing world population posed new challenges to the modern agriculture. In recent years, many strategies have been adopted to cope with these needs and satisfy these demands. Among these, the improvement of agronomic techniques and plant selection and breeding are crucial for a sustainable improvement of crop production. Nitrogen (N) is an essential nutrient for plant growth and development due to its important role in several biochemical pathways, constituting 2-5 % of plant dry matter. The excessive use of N fertilizer led to high environmental pollution with impacts on both ecosystems and human health. Therefore, N inputs reduction is one of the most important challenges to develop more sustainable agricultural systems. The selection of genotypes with a high Nitrogen Use Efficiency (NUE) is crucial to achieve this goal. Indeed, NUE is a complex trait governed by both environmental and genetic factors and their interaction. To reach a deep understanding of the molecular mechanisms involved in NUE and their role in plant selection for the complex trait is a common goal for crops. Wheat is one of the most important cereal crops worldwide due to its adaptability to a wide range of environments. Durum wheat (Triticum turgidum L. subsp. durum (Desf.) Husn. syn. Triticum durum) account for almost 5% of the total world wheat production with more than half belonging to the Mediterranean area. The recent complete durum wheat genome assembly report allows for a deeper and easier genetic investigation of more complex traits and their regulation, including NUE-related genes. A better understanding of the induction and the regulation of N-related genes might assist both breeding molecular assisted programs and the selection of novel high NUE genotypes. The high genome duplication and its being allopolyploid as well as the large size of the durum wheat genome pose further challenges for the identification of putative target genes. The aim of this work was the identification of key genes involved in N-related pathways responsible for NUE differences between genotypes and the characterization of transcriptional patterns underlying nitrate uptake, remobilization, and assimilation dynamics and their regulatory network. To achieve these goals, nine genotypes, chosen for their large variability in terms of plant growth habits, grain yield potential, and year of release were adopted for NUE evaluation (Chapter 2). Here, we focused on the identification of durum wheat genotypes highly contrasting for NUE. A first trial was performed in pots and in a controlled environment, using labeled 15N fertilizer mainly to quantify Nitrogen Uptake Efficiency (NUpE). The second trial, performed in field with high and low N supply, showed high variability for the complex trait allowing us to select four genotypes (Senatore Cappelli, Orizzonte, Antalis, and Appio) characterized for contrasting NUE. These genotypes were then used for a comparative transcriptomic analysis in a time-course design under different nitrogen availability. We further focused on the characterization of the two main gene families involved in the nitrate transport, NPF (formerly, NRT1) and NRT2, in the durum wheat genome (Chapter 3). NPF and NRT2 families are of large importance for nitrate uptake from the soil, its translocation and remobilization in different plant tissues as well as for nitrate signaling. Here, for the first time, we identified two-hundreds eleven (211) Triticum durum NPF (TdNPF) and twenty (20) TdNRT2s, providing a deep annotation of their protein sequences and conserved domains. We further described their evolutionary relationships and the putative Transcription Factors (TFs) involved in their regulation, mainly MYB and MYC and ABA related TFs for TdNRT2s and TdNPFs, respectively. High salinity in soil also affects the availability of nutrients, their uptake, translocation or portioning within the plant. Thus, we also investigated the salt-stress tolerance aided by Arbuscular Mycorrhizal Fungi (AMF) in durum wheat comparing AMF-inoculated and uninoculated plants (Chapter 4). AMF hyphae have been reported to supply up to 25% plant N, enhancing N uptake through a higher membrane stability. The study of AMF-induced genes might help to detect key genes involved in stress-response and nutrient uptake. In our experiment, the expression of genes involved in trehalose metabolism, RNA processing, vesicle trafficking, cell wall organization, and signal transduction was significantly enhanced by the AMF fungi symbiosis. Furthermore, many transcription factors, including WRKY, NAC, and MYB, known for their key role in plant abiotic stress response appeared differentially expressed between treatments. We further described the co-expression relationship between these genes and how these may affect the plant stress response. Finally, the four genotypes previously selected for their contrasting NUE, were adopted for a high-depth comparative RNA sequencing to describe the expression profiles in response to both high and low N supply to detect genotype-specific expression patterns potentially involved in their different NUE (Chapter 5). We were able to precisely supply nitrate in both high and low concentrations by using a hydroponic system, focusing on both short- (8h) and long-term (96h) plant responses to N supply. To cluster Differential Expressed Genes (DEGs) and identify functional pathways involved in nitrate metabolism, a Weighted Genes Co-expression Analysis (WGCNA) were carried out able also to detect modules of co-expression among genotypes and treatments. The comparison between contrasting genotypes allowed us to select candidate genes related to high NUE and its components. The analysis was performed on both root and shoot to break down the expression patterns involved in N uptake from the soil as well as N translocation and assimilation. Forty and thirty-four thousand DEGs were detected in root and shoot, respectively, clustered in 21 and 28 co-expression modules that were functionally characterized using GO terms enrichments. Genes involved in peptide biosynthesis, transmembrane transport, translation, oxoacid and carboxylic acid metabolisms were highly induced in root in response to N treatments whereas genes involved in photosynthesis, isoprenoid and terpenoid biosynthesis, lipid, carbohydrate and oxoacid metabolism were up-regulated in shoot. Furthermore, TFs and regulatory genes (such as Protein Kinases) involved in the N response were characterized. We also detected genotype specific expression variation inside the co-expression modules and among TdNPFs and TdNRT2s. In detail, the two older varieties, Cappelli and Appio showed a higher and faster induction of many genes related to oxidative stress response, transmembrane transport and amino acid transport as well as many TdNPFs, mainly in root. This study provided new insights into the molecular mechanisms of durum wheat in response to N supply and revealed many functional pathways involved in N uptake, translocation and assimilation in durum wheat. Furthermore, the utilization of contrasting genotypes for NUE aided the identification of several candidate genes for improving the complex trait in durum wheat

Morphological and Physiological Root Traits and Their Relationship with Nitrogen Uptake in Wheat Varieties Released from 1915 to 2013

Identifying genotypes with a greater ability to absorb nitrogen (N) may be important to reducing N loss in the environment and improving the sustainability of agricultural systems. This study extends the knowledge of variability among wheat genotypes in terms of morphological or physiological root traits, N uptake under conditions of low soil N availability, and in the amount and rapidity of the use of N supplied with fertilizer. Nine genotypes of durum wheat were chosen for their different morpho-phenological characteristics and year of their release. The isotopic tracer 15N was used to measure the fertilizer N uptake efficiency. The results show that durum wheat breeding did not have univocal effects on the characteristics of the root system (weight, length, specific root length, etc.) or N uptake capacity. The differences in N uptake among the studied genotypes when grown in conditions of low N availability appear to be related more to differences in uptake efficiency per unit of weight and length of the root system than to differences in the morphological root traits. The differences among the genotypes in the speed and the ability to take advantage of the greater N availability, determined by N fertilization, appear to a certain extent to be related to the development of the root system and the photosynthesizing area. This study highlights some variability within the species in terms of the development, distribution, and efficiency of the root system, which suggests that there may be sufficient grounds for improving these traits with positive effects in terms of adaptability to difficult environments and resilience to climate change

Burrowers from the Past: Mitochondrial Signatures of Ordovician Bivalve Infaunalization

Bivalves and gastropods are the two largest classes of extant molluscs. Despite sharing a huge number of features, they do not share a key ecological one: gastropods are essentially epibenthic, although most bivalves are infaunal. However, this is not the ancestral bivalve condition; Cambrian forms were surface crawlers and only during the Ordovician a fundamental infaunalization process took place, leading to bivalves as we currently know them. This major ecological shift is linked to the exposure to a different redox environoments (hypoxic or anoxic) and with the Lower Devonian oxygenation event. We investigated selec- tive signatures on bivalve and gastropod mitochondrial genomes with respect to a time calibrated mitochondrial phylogeny by means of dN/dS ratios. We were able to detect 1) a major signal of directional selection between the Ordovician and the Lower Devonian for bivalve mitochondrial Complex I, and 2) an overall higher directional selective pressure on bivalve Complex V with respect to gastropods. These and other minor dN/dS patterns and timings are discussed, showing that the Ordovician infaunalization event left heavy traces in bivalve mitochondrial genomes

HERMES: an Improved Method to Test Mitochondrial Genome Molecular Synapomorphies among Clades.

Mitochondrial chromosomes have diversified among eukaryotes and many different architectures and features are now acknowledged for this genome. Here we present the improved HERMES index, which can measure and quantify the amount of molecular change experienced by mitochondrial genomes. We test the improved approach with ten molecular phylogenetic studies based on complete mitochondrial genomes, representing six bilaterian Phyla. In most cases, HERMES analysis spotted out clades or single species with peculiar molecular synapomorphies, allowing to identify phylogenetic and ecological patterns. The software presented herein handles linear, circular, and multi-chromosome genomes, thus widening the HERMES scope to the complete eukaryotic domain