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.

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.

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.

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.

Assessment of dry eye in a GVHD murine model: Approximation through tear osmolarity measurement.

Dry eye disease is one of the most frequent pathological events that take place in the course of the graft versus host disease (GVHD), and is the main cause of deterioration in quality of life for patients. Thus, demonstration of dry eye signs in murine models of oGVHD is crucial for the validation of these models for the study of the disease. Given the increasing evidence that tear osmolarity is an important player of dry eye disease, our purpose in this study was to validate the use of a reliable method to assess tear osmolarity in mice: the electrical impedance method. Then, we wanted to test its utility with an oGVHD model. Tear volume assessment was also performed, using the phenol red thread test. We found differences in tear osmolarity in mice that received a transplant with cells from bone marrow and spleen (the GVHD group) when compared with mice that only received bone marrow cells (the BM group) at day 7 (362 ± 8 mOsm/l and 345 ± 9 mOsm/l respectively; P < 0.01) and day 21 (348 ± 19 mOsm/l vs. 326 ± 15 mOsm/l; P < 0.05). We found also differences in tear volume at day 14 (2.30 ± 0.61 mm in oGVHD group and 2.89 ± 0.62 mm in BM group; P = 0.06) and at day 21 (2.10 ± 0.30 mm in oGVHD group and 2.89 ± 0.32 mm in BM group; P < 0.01). Besides this, we observed reduction in epithelial thickness between the GVHD and BM groups (37.0 ± 6.2 µm and 43.6 ± 3.3 µm respectively; P < 0.05). These data show the usefulness of the electrical impedance method to measure tear osmolarity in mice. We can also conclude that this oGVHD model mimics the tear film alterations found in human dry eye disease, what contributes to give relevance to this model for the study of GVHD.

Metabolic and Transcriptional Analysis of Durum Wheat Responses to Elevated CO2 at Low and High Nitrate Supply.

Elevated [CO2] (eCO2) can lead to photosynthetic acclimation and this is often intensified by low nitrogen (N). Despite intensive studies of plant responses to eCO2, the regulation mechanism of primary metabolism at the whole-plant level in interaction with [Formula: see text] supply remains unclear. We examined the metabolic and transcriptional responses triggered by eCO2 in association with physiological-biochemical traits in flag leaves and roots of durum wheat grown hydroponically in ambient and elevated [CO2] with low (LN) and high (HN) [Formula: see text] supply. Multivariate analysis revealed a strong interaction between eCO2 and [Formula: see text] supply. Photosynthetic acclimation induced by eCO2 in LN plants was accompanied by an increase in biomass and carbohydrates, and decreases of leaf organic N per unit area, organic acids, inorganic ions, Calvin-Benson cycle intermediates, Rubisco, nitrate reductase activity, amino acids and transcripts for N metabolism, particularly in leaves, whereas [Formula: see text] uptake was unaffected. In HN plants, eCO2 did not decrease photosynthetic capacity or leaf organic N per unit area, but induced transcripts for N metabolism, especially in roots. In conclusion, the photosynthetic acclimation in LN plants was associated with an inhibition of leaf [Formula: see text] assimilation, whereas up-regulation of N metabolism in roots could have mitigated the acclimatory effect of eCO2 in HN plants.

Functional and transcriptional characterization of a barley mutant with impaired photosynthesis.

Chemical mutagenesis induces variations that may assist in the identification of targets for adaptation to growth under atmospheric CO2 enrichment. The aim of this work was to characterize the limitations causing reduced photosynthetic capacity in G132 mutagenized barley (Hordeum vulgare L. cv. Graphic) grown in a glasshouse. Compared to the wild type (WT) G132 showed increased transcript levels for the PSII light harvesting complex, but lower levels of chlorophyll, transcripts for protochlorophyllide oxidoreductase A and psbQ, and PSII quantum efficiency in young leaves. Rubisco limitation had an overriding influence on G132 photosynthesis, and was due to strong and selective decreases in Rubisco protein and activity. These reductions were accompanied by enhanced Rubisco transcripts, but increased levels of a Rubisco degradation product. G132 showed lower levels of carbohydrates, amino acids and corresponding transcripts, and proteins, but not of nitrate. Many of the measured parameters recovered in the mutant as development progressed, or decreased less than in the WT, indicating that senescence was delayed. G132 had a longer growth period than the WT and similar final plant dry matter. The reduced resource investment in Rubisco of G132 may prove useful for studies on barley adaptation to elevated CO2 and climate change.

Photosynthesis-dependent/independent control of stomatal responses to CO2 in mutant barley with surplus electron transport capacity and reduced SLAH3 anion channel transcript.

The mechanisms of stomatal sensitivity to CO2 are yet to be fully understood. The role of photosynthetic and non-photosynthetic factors in stomatal responses to CO2 was investigated in wild-type barley (Hordeum vulgare var. Graphic) and in a mutant (G132) with decreased photochemical and Rubisco capacities. The CO2 and DCMU responses of stomatal conductance (gs), gas exchange, chlorophyll fluorescence and levels of ATP, with a putative transcript for stomatal opening were analysed. G132 had greater gs than the wild-type, despite lower photosynthesis rates and higher intercellular CO2 concentrations (Ci). The mutant had Rubisco-limited photosynthesis at very high CO2 levels, and higher ATP contents than the wild-type. Stomatal sensitivity to CO2 under red light was lower in G132 than in the wild-type, both in photosynthesizing and DCMU-inhibited leaves. Under constant Ci and red light, stomatal sensitivity to DCMU inhibition was higher in G132. The levels of a SLAH3-like slow anion channel transcript, involved in stomatal closure, decreased sharply in G132. The results suggest that stomatal responses to CO2 depend partly on the balance of photosynthetic electron transport to carbon assimilation capacities, but are partially regulated by the CO2 signalling network. High gs can improve the adaptation to climate change in well-watered conditions.

Quantitative RT-PCR Platform to Measure Transcript Levels of C and N Metabolism-Related Genes in Durum Wheat: Transcript Profiles in Elevated [CO2] and High Temperature at Different Levels of N Supply.

Only limited public transcriptomics resources are available for durum wheat and its responses to environmental changes. We developed a quantitative reverse transcription-PCR (qRT-PCR) platform for analysing the expression of primary C and N metabolism genes in durum wheat in leaves (125 genes) and roots (38 genes), based on available bread wheat genes and the identification of orthologs of known genes in other species. We also assessed the expression stability of seven reference genes for qRT-PCR under varying environments. We therefore present a functional qRT-PCR platform for gene expression analysis in durum wheat, and suggest using the ADP-ribosylation factor as a reference gene for qRT-PCR normalization. We investigated the effects of elevated [CO(2)] and temperature at two levels of N supply on C and N metabolism by combining gene expression analysis, using our qRT-PCR platform, with biochemical and physiological parameters in durum wheat grown in field chambers. Elevated CO(2) down-regulated the photosynthetic capacity and led to the loss of N compounds, including Rubisco; this effect was exacerbated at low N. Mechanistically, the reduction in photosynthesis and N levels could be associated with a decreased transcription of the genes involved in photosynthesis and N assimilation. High temperatures increased stomatal conductance, and thus did not inhibit photosynthesis, even though Rubisco protein and activity, soluble protein, leaf N, and gene expression for C fixation and N assimilation were down-regulated. Under a future scenario of climate change, the extent to which C fixation capacity and N assimilation are down-regulated will depend upon the N supply.