Differential Efficacy of Small Molecules Dynasore and Mdivi-1 for the Treatment of Dry Eye Epitheliopathy or as a Countermeasure for Nitrogen Mustard Exposure of the Ocular Surface.

The ocular surface comprises the wet mucosal epithelia of the cornea and conjunctiva, the associated glands, and the overlying tear film. Epitheliopathy is the common pathologic outcome when the ocular surface is subjected to oxidative stress. Whether different stresses act via the same or different mechanisms is not known. Dynasore and dyngo-4a, small molecules developed to inhibit the GTPase activity of classic dynamins DNM1, DNM2, and DNM3, but not mdivi-1, a specific inhibitor of DNM1L, protect corneal epithelial cells exposed to the oxidant tert-butyl hydroperoxide (tBHP). Here we report that, while dyngo-4a is the more potent inhibitor of endocytosis, dynasore is the better cytoprotectant. Dynasore also protects corneal epithelial cells against exposure to high salt in an in vitro model of dysfunctional tears in dry eye. We now validate this finding in vivo, demonstrating that dynasore protects against epitheliopathy in a mouse model of dry eye. Knockdown of classic dynamin DNM2 was also cytoprotective against tBHP exposure, suggesting that dynasore's effect is at least partially on target. Like tBHP and high salt, exposure of corneal epithelial cells to nitrogen mustard upregulated the unfolded protein response and inflammatory markers, but dynasore did not protect against nitrogen mustard exposure. In contrast, mdivi-1 was cytoprotective. Interestingly, mdivi-1 did not inhibit the nitrogen mustard-induced expression of inflammatory cytokines. We conclude that exposure to tBHP or nitrogen mustard, two different oxidative stress agents, cause corneal epitheliopathy via different pathologic pathways. SIGNIFICANCE STATEMENT: Results presented in this paper, for the first time, implicate the dynamin DNM2 in ocular surface epitheliopathy. The findings suggest that dynasore could serve as a new topical treatment for dry eye epitheliopathy and that mdivi-1 could serve as a medical countermeasure for epitheliopathy due to nitrogen mustard exposure, with potentially increased efficacy when combined with anti-inflammatory agents and/or UPR modulators.

Characterization of Cell Surface Glycan Profiles in Human and Mouse Corneas Using Lectin Microarrays.

The advent of high-throughput sequencing technologies has facilitated the profiling of glycosylation genes at a single-cell level in complex biological systems, but the significance of these gene signatures to the composition of the glycocalyx remains ambiguous. Here, we used lectin microarrays to characterize the composition of cell surface glycans in human and mouse corneas and determine its relationship to single-cell transcriptomic data. Our results identify a series of cell surface glycan signatures that are unique to the different cell types of the human cornea and that correlate, to a certain extent, with the transcriptional expression of glycosylation genes. These include pathways involved in the biosynthesis of O-glycans in epithelial cells and core fucose on stromal and endothelial cell surfaces. Moreover, we show that human and mouse corneas display some structural differences in terms of cell surface glycan composition. These results could provide insights into the specialized function of individual cell types in the cornea and foster the identification of novel cornea-specific biomarkers.

Roles unveiled for membrane-associated mucins at the ocular surface using a Muc4 knockout mouse model.

Membrane-associated mucins (MAMs) are proposed to play critical roles at the ocular surface; however, in vivo evidence has been lacking. Here we investigate these roles by phenotyping of a Muc4 KO mouse. Histochemical analysis for expression of the beta-galactosidase transgene replacing Muc4 revealed a spiraling ribbon pattern across the corneal epithelium, consistent with centripetal cell migration from the limbus. Depletion of Muc4 compromised transcellular barrier function, as evidenced by an increase in rose bengal staining. In addition, the corneal surface was less smooth, consistent with disruption of tear film stability. While surface cells presented with well-developed microprojections, an increase in the number of cells with fewer microprojections was observed. Moreover, an increase in skin-type keratin K10 and a decrease in transcription factor Pax6 was observed, suggesting an incipient transdifferentiation. Despite this, no evidence of inflammatory dry eye disease was apparent. In addition, Muc4 had no effect on signaling by toll-like receptor Tlr4, unlike reports for MUC1 and MUC16. Results of this study provide the first in vivo evidence for the role of MAMs in transcellular barrier function, tear film stability, apical epithelial cell architecture, and epithelial mucosal differentiation at the ocular surface.

Dynasore Protects Corneal Epithelial Cells Subjected to Hyperosmolar Stress in an In Vitro Model of Dry Eye Epitheliopathy.

Epitheliopathy at the ocular surface is a defining sign of dry eye disease, a common disorder that affects 10% to 30% of the world's population. Hyperosmolarity of the tear film is one of the main drivers of pathology, with subsequent endoplasmic reticulum (ER) stress, the resulting unfolded protein response (UPR), and caspase-3 activation implicated in the pathway to programmed cell death. Dynasore, is a small molecule inhibitor of dynamin GTPases that has shown therapeutic effects in a variety of disease models involving oxidative stress. Recently we showed that dynasore protects corneal epithelial cells exposed to the oxidant tBHP, by selective reduction in expression of CHOP, a marker of the UPR PERK branch. Here we investigated the capacity of dynasore to protect corneal epithelial cells subjected to hyperosmotic stress (HOS). Similar to dynasore's capacity to protect against tBHP exposure, dynasore inhibits the cell death pathway triggered by HOS, protecting against ER stress and maintaining a homeostatic level of UPR activity. However, unlike with tBHP exposure, UPR activation due to HOS is independent of PERK and mostly driven by the UPR IRE1 branch. Our results demonstrate the role of the UPR in HOS-driven damage, and the potential of dynasore as a treatment to prevent dry eye epitheliopathy.

Galectins Differentially Regulate the Surface Glycosylation of Human Monocytes.

Monocytes are circulating blood cells that rapidly mobilize to inflamed sites where they serve diverse effector functions shaped in part by microenvironmental cues. The establishment of specific glycosylation patterns on the immune cell glycocalyx is fundamental to direct the inflammatory response, but relatively little is known about the mechanisms whereby the microenvironment controls this process. Here, we report that galectins differentially participate in remodeling the surface glycosylation of human primary CD14+CD16- monocytes under proinflammatory conditions. Using a lectin array on biotinylated protein, we found that the prototypic galectin-1 negatively influenced the expression of galactose epitopes on the surface of monocytic cells. On the other hand, the tandem-repeat galectin-8 and, to a certain extent, the chimeric galectin-3 promoted the expression of these residues. Jacalin flow cytometry and pull-down experiments further demonstrated that galectin-8 causes a profound upregulation of mucin-type O-glycosylation in cell surface proteins from primary monocytes and THP-1 cells. Overall, these results highlight the emerging role of the galectin signature on inflamed tissues and provide new insights into the contribution of extracellular galectins to the composition of the glycocalyx in human monocytes.

Fungal and ciliate protozoa are the main rumen microbes associated with methane emissions in dairy cattle.

BACKGROUND: Mitigating the effects of global warming has become the main challenge for humanity in recent decades. Livestock farming contributes to greenhouse gas emissions, with an important output of methane from enteric fermentation processes, mostly in ruminants. Because ruminal microbiota is directly involved in digestive fermentation processes and methane biosynthesis, understanding the ecological relationships between rumen microorganisms and their active metabolic pathways is essential for reducing emissions. This study analysed whole rumen metagenome using long reads and considering its compositional nature in order to disentangle the role of rumen microbes in methane emissions. RESULTS: The ß-diversity analyses suggested a subtle association between methane production and overall microbiota composition (0.01 < R2 < 0.02). Differential abundance analysis identified 36 genera and 279 KEGGs as significantly associated with methane production (Padj < 0.05). Those genera associated with high methane production were Eukaryota from Alveolata and Fungi clades, while Bacteria were associated with low methane emissions. The genus-level association network showed 2 clusters grouping Eukaryota and Bacteria, respectively. Regarding microbial gene functions, 41 KEGGs were found to be differentially abundant between low- and high-emission animals and were mainly involved in metabolic pathways. No KEGGs included in the methane metabolism pathway (ko00680) were detected as associated with high methane emissions. The KEGG network showed 3 clusters grouping KEGGs associated with high emissions, low emissions, and not differentially abundant in either. A deeper analysis of the differentially abundant KEGGs revealed that genes related with anaerobic respiration through nitrate degradation were more abundant in low-emission animals. CONCLUSIONS: Methane emissions are largely associated with the relative abundance of ciliates and fungi. The role of nitrate electron acceptors can be particularly important because this respiration mechanism directly competes with methanogenesis. Whole metagenome sequencing is necessary to jointly consider the relative abundance of Bacteria, Archaea, and Eukaryota in the statistical analyses. Nutritional and genetic strategies to reduce CH4 emissions should focus on reducing the relative abundance of Alveolata and Fungi in the rumen. This experiment has generated the largest ONT ruminal metagenomic dataset currently available.

Screening for Higher Grain Yield and Biomass among Sixty Bread Wheat Genotypes Grown under Elevated CO2 and High-Temperature Conditions.

Global warming will inevitably affect crop development and productivity, increasing uncertainty regarding food production. The exploitation of genotypic variability can be a promising approach for selecting improved crop varieties that can counteract the adverse effects of future climate change. We investigated the natural variation in yield performance under combined elevated CO2 and high-temperature conditions in a set of 60 bread wheat genotypes (59 of the 8TH HTWSN CIMMYT collection and Gazul). Plant height, biomass production, yield components and phenological traits were assessed. Large variations in the selected traits were observed across genotypes. The CIMMYT genotypes showed higher biomass and grain yield when compared to Gazul, indicating that the former performed better than the latter under the studied environmental conditions. Principal component and hierarchical clustering analyses revealed that the 60 wheat genotypes employed different strategies to achieve final grain yield, highlighting that the genotypes that can preferentially increase grain and ear numbers per plant will display better yield responses under combined elevated levels of CO2 and temperature. This study demonstrates the success of the breeding programs under warmer temperatures and the plants' capacity to respond to the concurrence of certain environmental factors, opening new opportunities for the selection of widely adapted climate-resilient wheat genotypes.

Genotypic Variability on Grain Yield and Grain Nutritional Quality Characteristics of Wheat Grown under Elevated CO2 and High Temperature.

The progressive rise in atmospheric CO2 concentrations and temperature associated with climate change is predicted to have a major impact on the productivity and quality of food crops. Therefore, food security is highly dependent on climate change. Following a survey with 60 bread wheat genotypes, here we investigated the genetic variation in grain yield and nutritional quality among 10 of these genotypes grown under elevated CO2 and temperature. With this purpose, the biomass production, grain yield-related traits, the grain concentration of starch, total protein, phenolic compounds, and mineral nutrients, together with the total antioxidant capacity, were determined. Variation among genotypes was found for almost all the studied traits. Higher grain and ear numbers were associated with increased grain yield but decreased grain total protein concentration and minerals such as Cu, Fe, Mg, Na, P, and Zn. Mineral nutrients were mainly associated with wheat biomass, whereas protein concentration was affected by plant biomass and yield-related traits. Associations among different nutrients and promising nutrient concentrations in some wheat genotypes were also found. This study demonstrates that the exploration of genetic diversity is a powerful approach, not only for selecting genotypes with improved quality, but also for dissecting the effect of the environment on grain yield and nutritional composition.

Membrane-associated mucins of the human ocular surface in health and disease.

Mucins are a family of high molecular weight, heavily-glycosylated proteins produced by wet epithelial tissues, including the ocular surface epithelia. Densely-packed O-linked glycan chains added post-translationally confer the biophysical properties of hydration, lubrication, anti-adhesion and repulsion. Membrane-associated mucins (MAMs) are the distinguishing components of the mucosal glycocalyx. At the ocular surface, MAMs maintain wetness, lubricate the blink, stabilize the tear film, and create a physical barrier to the outside world. In addition, it is increasingly appreciated that MAMs function as cell surface receptors that transduce information from the outside to the inside of the cell. Recently, our team published a comprehensive review/perspectives article for molecular scientists on ocular surface MAMs, including previously unpublished data and analyses on two new genes MUC21 and MUC22, as well as new MAM functions and biological roles, comparing human and mouse (PMID: 31493487). The current article is a refocus for the audience of The Ocular Surface. First, we update the gene and protein information in a more concise form, and include a new section on glycosylation. Next, we discuss biological roles, with some new sections and further updating from our previous review. Finally, we provide a new chapter on MAM involvement in ocular surface disease. We end this with discussion of an emerging mechanism responsible for damage to the epithelia and their mucosal glycocalyces: the unfolded protein response (UPR). The UPR offers a novel target for therapeutic intervention.

Dynasore protects ocular surface mucosal epithelia subjected to oxidative stress by maintaining UPR and calcium homeostasis.

The mucosal epithelia of the ocular surface protect against external threats to the eye. Using a model of human stratified corneal epithelial cells with mucosal differentiation, we previously demonstrated that a small molecule inhibitor of dynamin GTPases, dynasore, prevents damage to cells and their transcellular barriers when subjected to oxidative stress. Investigating mechanisms, we now report the novel finding that dynasore acts by maintaining Ca+2 homeostasis, thereby inhibiting the PERK branch of the unfolded protein response (UPR) that promotes cell death. Dynasore was found to protect mitochondria by preventing mitochondrial permeability transition pore opening (mPTP), but, unlike reports using other systems, this was not mediated by dynamin family member DRP1. Necrostatin-1, an inhibitor of RIPK1 and lytic forms of programmed cell death, also inhibited mPTP opening and further protected the plasma membrane barrier. Significantly, necrostatin-1 did not protect the mucosal barrier. Oxidative stress increased mRNA for sXBP1, a marker of the IRE1 branch of the UPR, and CHOP, a marker of the PERK branch. It also stimulated phosphorylation of eIF2α, the upstream regulator of CHOP, as well as an increase in intracellular Ca2+. Dynasore selectively inhibited the increase in PERK branch markers, and also prevented the increase intracellular Ca2+ in response to oxidative stress. The increase in PERK branch markers were also inhibited when cells were treated with the cell permeable Ca2+ chelator, BAPTA-AM. To our knowledge, this is the first time that dynasore has been shown to have an effect on the UPR and suggests therapeutic applications.

Membrane-associated mucins of the ocular surface: New genes, new protein functions and new biological roles in human and mouse.

The mucosal glycocalyx of the ocular surface constitutes the point of interaction between the tear film and the apical epithelial cells. Membrane-associated mucins (MAMs) are the defining molecules of the glycocalyx in all mucosal epithelia. Long recognized for their biophysical properties of hydration, lubrication, anti-adhesion and repulsion, MAMs maintain the wet ocular surface, lubricate the blink, stabilize the tear film and create a physical barrier to the outside world. However, it is increasingly appreciated that MAMs also function as cell surface receptors that transduce information from the outside to the inside of the cell. A number of excellent review articles have provided perspective on the field as it has progressed since 1987, when molecular cloning of the first MAM was reported. The current article provides an update for the ocular surface, placing it into the broad context of findings made in other organ systems, and including new genes, new protein functions and new biological roles. We discuss the epithelial tissue-equivalent with mucosal differentiation, the key model system making these advances possible. In addition, we make the first systematic comparison of MAMs in human and mouse, establishing the basis for using knockout mice for investigations with the complexity of an in vivo system. Lastly, we discuss findings from human genetics/genomics, which are providing clues to new MAM roles previously unimagined. Taken together, this information allows us to generate hypotheses for the next stage of investigation to expand our knowledge of MAM function in intracellular signaling and roles unique to the ocular surface.

An "omic" approach to Pyrocystis lunula: New insights related with this bioluminescent dinoflagellate.

Pyrocystis lunula (Schutt) is a photoautotrophic dinoflagellate without armored form, frequently found in marine environments. Today, there are several biotechnological applications derived from the bioluminescent system of this species. From a post-genomic perspective, in order to have a starting point for studying the proteome of P. lunula, an "omics" approach (transcriptomics-proteomics) was assessed using fresh microalgae samples. A total of 80,874,825 raw reads were generated (11,292,087,505 bp; 55.82% GC) by mRNA sequencing. Very high-quality sequences were assembled into 414,295 contigs (219,203,407 bp; 55.38% GC) using Trinity software, generating a comprehensive reference transcriptome for this species. Then, a P. lunula proteome was inferred and further employed for its analysis on this species. A total of 17,461 peptides were identified, yielding 3182 protein identification hits, including 175 novel proteins. The identified proteins were further categorized according to functional description and gene ontology classification. SIGNIFICANCE: The major contribution of the present work is making available a reference transcriptome and proteome of P. lunula, that is now accessible for the research community, and a functional description of the 3182 proteins inferred from the transcriptome, including 175 novel proteins, which have already been deposited in the ProteomeXchange and NCBI SRA databases, respectively. In addition to this, a series of important factors related to the bioluminescent system and the regulation of gene expression, were identified and described.

Proteomic study of the membrane components of signalling cascades of Botrytis cinerea controlled by phosphorylation.

Protein phosphorylation and membrane proteins play an important role in the infection of plants by phytopathogenic fungi, given their involvement in signal transduction cascades. Botrytis cinerea is a well-studied necrotrophic fungus taken as a model organism in fungal plant pathology, given its broad host range and adverse economic impact. To elucidate relevant events during infection, several proteomics analyses have been performed in B. cinerea, but they cover only 10% of the total proteins predicted in the genome database of this fungus. To increase coverage, we analysed by LC-MS/MS the first-reported overlapped proteome in phytopathogenic fungi, the "phosphomembranome" of B. cinerea, combining the two most important signal transduction subproteomes. Of the 1112 membrane-associated phosphoproteins identified, 64 and 243 were classified as exclusively identified or overexpressed under glucose and deproteinized tomato cell wall conditions, respectively. Seven proteins were found under both conditions, but these presented a specific phosphorylation pattern, so they were considered as exclusively identified or overexpressed proteins. From bioinformatics analysis, those differences in the membrane-associated phosphoproteins composition were associated with various processes, including pyruvate metabolism, unfolded protein response, oxidative stress response, autophagy and cell death. Our results suggest these proteins play a significant role in the B. cinerea pathogenic cycle.

Subconjunctival injection of mesenchymal stromal cells protects the cornea in an experimental model of GVHD.

PURPOSE: To evaluate the therapeutic effect of subconjunctival injection of human mesenchymal stromal cells (hMSCs) in the cornea of mice with graft versus host disease (GVHD). METHODS: GVHD was induced in mice after hematopoietic stem cell transplantation (HSCT) between MHC-mismatched mouse strains. Subconjunctival injection of hMSCs was applied at day 10 post-HSCT. Infiltration of CD3+ cells in the cornea and epithelial alterations were analyzed by immunofluorescence. Tear was assessed using the PRT test and TearLab Osmolarity System. qPCR was used to evaluate changes in cytokines, Pax6 and Sprr1b expression. To evaluate the effect of irradiation, we analyzed the expression of these genes in TBI mice. RESULTS: Immune cell invasion occurs in mice with GVHD, as shown by the presence of CD3+ cells in the cornea. Interestingly, eyes treated with hMSC did not present CD3+ cells. Tear osmolarity was increased in GVHD eyes, but not in treated eyes. TNFa expression was highly increased in all corneas except in Control and treated eyes. Pax6 in corneal epithelium showed a similar pattern in GVHD and Control mice, and its gene expression was enhanced in GVHD corneas. In contrast, Pax6 was reduced in GVHD + MSC corneas. We also found an increase in SPRR1B staining in GVHD eyes that was lower in GVHD + MSC mice, demonstrating that corneal keratinization is less frequent after treatment with hMSC. CONCLUSIONS: The treatment with hMSCs by subconjunctival injection is effective in reducing corneal inflammation and squamous metaplasia in ocular GVHD (oGVHD). Local treatment with hMSCs is a promising strategy for oGVHD.

De Novo Transcriptome Analysis of Durum Wheat Flag Leaves Provides New Insights Into the Regulatory Response to Elevated CO2 and High Temperature.

Global warming is becoming a significant problem for food security, particularly in the Mediterranean basin. The use of molecular techniques to study gene-level responses to environmental changes in non-model organisms is increasing and may help to improve the mechanistic understanding of durum wheat response to elevated CO2 and high temperature. With this purpose, we performed transcriptome RNA sequencing (RNA-Seq) analyses combined with physiological and biochemical studies in the flag leaf of plants grown in field chambers at ear emergence. Enhanced photosynthesis by elevated CO2 was accompanied by an increase in biomass and starch and fructan content, and a decrease in N compounds, as chlorophyll, soluble proteins, and Rubisco content, in association with a decline of nitrate reductase and initial and total Rubisco activities. While high temperature led to a decline of chlorophyll, Rubisco activity, and protein content, the glucose content increased and starch decreased. Furthermore, elevated CO2 induced several genes involved in mitochondrial electron transport, a few genes for photosynthesis and fructan synthesis, and most of the genes involved in secondary metabolism and gibberellin and jasmonate metabolism, whereas those related to light harvesting, N assimilation, and other hormone pathways were repressed. High temperature repressed genes for C, energy, N, lipid, secondary, and hormone metabolisms. Under the combined increases in atmospheric CO2 and temperature, the transcript profile resembled that previously reported for high temperature, although elevated CO2 partly alleviated the downregulation of primary and secondary metabolism genes. The results suggest that there was a reprogramming of primary and secondary metabolism under the future climatic scenario, leading to coordinated regulation of C-N metabolism towards C-rich metabolites at elevated CO2 and a shift away from C-rich secondary metabolites at high temperature. Several candidate genes differentially expressed were identified, including protein kinases, receptor kinases, and transcription factors.

Valorisation of the microalgae Nannochloropsis gaditana biomass by proteomic approach in the context of circular economy.

Nannochloropsis gaditana is a non-flagellated microalgae that has been widely used for different purposes, mostly related with the industrial production of biofuels or aquiculture. However, in order to increase the economic viability of the obtained microalgae biomass from a production plant coupled to a coal power plant, a proteomic approach was initiated by using fresh and atomized microalgae samples, as the main used commercial forms. Above 51,000 high quality spectra were obtained per sample in the MS/MS analysis of whole proteome of N. gaditana, yielding above 7,500 peptides, leading the identification of 1,950 proteins, from the N. gaditana protein database, where 655 proteins were presented in all the replicates. The identified proteins were categorized according to gene ontology classification by molecular function and biological process. In this study, it has been described the first proteomic analysis of the microalgae N. gaditana under industrial conditions containing an important number of identified proteins. A significative presence of proteins with a potential role in different agri-food and biomedical applications was detected and studied being the core of future N. gaditana research to expand the current biotechnological applications of this microalga. SIGNIFICANCE OF THE STUDY: Three quarters of the planet earth correspond to seas and oceans, however its potential biotechnological use is still unknown. We described the first proteomic description of the microalgae N. gaditana under industrial conditions. Following the spirit of the EU initiatives of blue growth and the statements of circular economy, CO2 waste from a coal plant power has been transformed in a resource for microalgae biomass production, common product presentations were evaluated by proteomic, and its potential use of identified proteins in Agri-food and Biomedicine has been revealed.

On the path to extinction: Inbreeding and admixture in a declining grey wolf population.

Allee effects reduce the viability of small populations in many different ways, which act synergistically to lead populations towards extinction vortexes. The Sierra Morena wolf population, isolated in the south of the Iberian Peninsula and composed of just one or few packs for decades, represents a good example of how diverse threats act additively in very small populations. We sequenced the genome of one of the last wolves identified (and road-killed) in Sierra Morena and that of another wolf in the Iberian Wolf Captive Breeding Program and compared them with other wolf and dog genomes from around the world (including two previously published genome sequences from northern Iberian wolves). The results showed relatively low overall genetic diversity in Iberian wolves, but diverse population histories including past introgression of dog genes. The Sierra Morena wolf had an extraordinarily high level of inbreeding and long runs of homozygosity, resulting from the long isolation. In addition, about one-third of the genome was of dog origin. Despite the introgression of dog genes, heterozygosity remained low because of continued inbreeding after several hybridization events. The results thus illustrate the case of a small and isolated wolf population where the low population density may have favoured hybridization and introgression of dog alleles, but continued inbreeding may have resulted in large chromosomal fragments of wolf origin completely disappearing from the population, and being replaced by chromosomal fragments of dog origin. The latest population surveys suggest that this population may have gone extinct.

New insights into the impacts of elevated CO2, nitrogen, and temperature levels on the regulation of C and N metabolism in durum wheat using network analysis.

The use of correlation networks and hierarchical cluster analysis provides a framework to organize and study the coordination of parameters such as genes, metabolites, proteins and physiological parameters. We have analyzed 142 traits from primary C and N metabolism, including biochemical and gene expression analyses, in a range of 32 different growth conditions (various [CO2] levels, temperatures, N supplies, growth stages and experimental methods). To test the integration of primary metabolism, particularly under climate change, we investigated which C and N metabolic traits and transcript levels are correlated in durum wheat flag leaves using a correlation network and a hierarchical cluster analysis. There was a high amount of positive correlation between traits involved in a wide range of biological processes, suggesting a close and intricate coordination between C-N metabolisms at the biochemical and transcriptional levels. Transcript levels for genes related to N uptake and assimilation were especially coexpressed with genes belonging to the respiratory pathway, highlighting the coordination between the synthesis of organic N compounds and provision of energy and C skeletons. Also involved in this coordination were Rubisco and nitrate reductase activities, which play a key role in the regulation of plant metabolism. Carbohydrate accumulation was linked with a down-regulation of photosynthetic and N metabolism genes and nitrate reductase activity. Based on the degree of connectivity between nodes, network exploration facilitated the identification of some traits that may be biologically relevant during plant abiotic stress tolerance, as most of them are involved in limiting steps of plant metabolism.

Improved responses to elevated CO2 in durum wheat at a low nitrate supply associated with the upregulation of photosynthetic genes and the activation of nitrate assimilation.

Elevated CO2 often leads to photosynthetic acclimation, and N availability may alter this response. We investigated whether the coordination of shoot-root N assimilation by elevated CO2 may help to optimize the whole-plant N allocation and maximize photosynthesis in hydroponically-grown durum wheat at two NO3- supplies in interaction with plant development. Transcriptional and biochemical analyses were performed on flag leaves and roots. At anthesis, the improved photosynthetic acclimation response to elevated CO2 at low N was associated with increased Rubisco, chlorophyll and amino acid contents, and upregulation of genes related to their biosynthesis, light reactions and Calvin-Benson cycle, while a decrease was recorded at high N. Despite the decrease in carbohydrates with elevated CO2 at low N and the increase at high N, a stronger upward trend in leaf NR activity was found at low rather than high N. The induction of N recycling-related genes was accompanied by an amino acids decline at high N. At the grain-filling stage, the photosynthetic acclimation to elevated CO2 at high N was associated with the downregulation of both N assimilation, mainly in roots, and photosynthetic genes. At low N, enhanced root N assimilation partly compensated for slower shoot N assimilation and maximized photosynthetic capacity.