A new MVA ancestor-derived oncolytic vaccinia virus induces immunogenic tumor cell death and robust antitumor immune responses.

Vaccinia viruses (VACVs) are versatile therapeutic agents and different features of various VACV strains allow for a broad range of therapeutic applications. Modified VACV Ankara (MVA) is a particularly altered VACV strain that is highly immunogenic, incapable of replicating in mammalian hosts, and broadly used as a safe vector for vaccination. Alternatively, Western Reserve (WR) or Copenhagen (Cop) are VACV strains that efficiently replicate in cancer cells and, therefore, are used to develop oncolytic viruses. However, the immune evasion capacity of WR or Cop hinders their ability to elicit antitumor immune responses, which is crucial for efficacy in the clinic. Here, we describe a new VACV strain named Immune-Oncolytic VACV Ankara (IOVA), which combines efficient replication in cancer cells with induction of immunogenic tumor cell death (ICD). IOVA was engineered from an MVA ancestor and shows superior cytotoxicity in tumor cells. In addition, the IOVA genome incorporates mutations that lead to massive fusogenesis of tumor cells, which contributes to improved antitumor effects. In syngeneic mouse tumor models, the induction of ICD results in robust antitumor immunity directed against tumor neo-epitopes and eradication of large established tumors. These data present IOVA as an improved immunotherapeutic oncolytic vector.

Biological basis of extensive pleiotropy between blood traits and cancer risk.

BACKGROUND: The immune system has a central role in preventing carcinogenesis. Alteration of systemic immune cell levels may increase cancer risk. However, the extent to which common genetic variation influences blood traits and cancer risk remains largely undetermined. Here, we identify pleiotropic variants and predict their underlying molecular and cellular alterations. METHODS: Multivariate Cox regression was used to evaluate associations between blood traits and cancer diagnosis in cases in the UK Biobank. Shared genetic variants were identified from the summary statistics of the genome-wide association studies of 27 blood traits and 27 cancer types and subtypes, applying the conditional/conjunctional false-discovery rate approach. Analysis of genomic positions, expression quantitative trait loci, enhancers, regulatory marks, functionally defined gene sets, and bulk- and single-cell expression profiles predicted the biological impact of pleiotropic variants. Plasma small RNAs were sequenced to assess association with cancer diagnosis. RESULTS: The study identified 4093 common genetic variants, involving 1248 gene loci, that contributed to blood-cancer pleiotropism. Genomic hotspots of pleiotropism include chromosomal regions 5p15-TERT and 6p21-HLA. Genes whose products are involved in regulating telomere length are found to be enriched in pleiotropic variants. Pleiotropic gene candidates are frequently linked to transcriptional programs that regulate hematopoiesis and define progenitor cell states of immune system development. Perturbation of the myeloid lineage is indicated by pleiotropic associations with defined master regulators and cell alterations. Eosinophil count is inversely associated with cancer risk. A high frequency of pleiotropic associations is also centered on the regulation of small noncoding Y-RNAs. Predicted pleiotropic Y-RNAs show specific regulatory marks and are overabundant in the normal tissue and blood of cancer patients. Analysis of plasma small RNAs in women who developed breast cancer indicates there is an overabundance of Y-RNA preceding neoplasm diagnosis. CONCLUSIONS: This study reveals extensive pleiotropism between blood traits and cancer risk. Pleiotropism is linked to factors and processes involved in hematopoietic development and immune system function, including components of the major histocompatibility complexes, and regulators of telomere length and myeloid lineage. Deregulation of Y-RNAs is also associated with pleiotropism. Overexpression of these elements might indicate increased cancer risk.

Assessment of Growth, Yield, and Nutrient Uptake of Mediterranean Tomato Landraces in Response to Salinity Stress.

Salinity is a major stress factor that compromises vegetable production in semi-arid climates such as the Mediterranean. The accumulation of salts in the soil can be attributed to limited water availability, which can be exacerbated by changes in rainfall patterns and rising temperatures. These factors can alter soil moisture levels and evaporation rates, ultimately leading to an increase in soil salinity, and, concomitantly, the extent to which crop yield is affected by salinity stress is considered cultivar-dependent. In contrast to tomato hybrids, tomato landraces often exhibit greater genetic diversity and resilience to environmental stresses, constituting valuable resources for breeding programs seeking to introduce new tolerance mechanisms. Therefore, in the present study, we investigated the effects of mild salinity stress on the growth, yield, and nutritional status of sixteen Mediterranean tomato landraces of all size types that had been pre-selected as salinity tolerant in previous screening trials. The experiment was carried out in the greenhouse facilities of the Laboratory of Vegetable Production at the Agricultural University of Athens. To induce salinity stress, plants were grown hydroponically and irrigated with a nutrient solution containing NaCl at a concentration that could maintain the NaCl level in the root zone at 30 mM, while the non-salt-treated plants were irrigated with a nutrient solution containing 0.5 mM NaCl. Various plant growth parameters, including dry matter content and fruit yield (measured by the number and weight of fruits per plant), were evaluated to assess the impact of salinity stress. In addition, the nutritional status of the plants was assessed by determining the concentrations of macro- and micronutrients in the leaves, roots, and fruit of the plants. The key results of this study reveal that cherry-type tomato landraces exhibit the highest tolerance to salinity stress, as the landraces 'Cherry-INRAE (1)', 'Cherry-INRAE (3)', and 'Cherry-INRAE (4)' did not experience a decrease in yield when exposed to salinity stress. However, larger landraces such as 'de Ramellet' also exhibit mechanisms conferring tolerance to salinity, as their yield was not compromised by the stress applied. The identified tolerant and resistant varieties could potentially be used in breeding programs to develop new varieties and hybrids that are better adapted to salinity-affected environments. The identification and utilization of tomato varieties that are adapted to salinity stress is an important strategy for promoting agriculture sustainability, particularly in semi-arid regions where salinity stress is a major challenge.

How Important Are Functional and Developmental Constraints on Phenotypic Evolution? An Empirical Test with the Stomatal Anatomy of Flowering Plants.

AbstractQuantifying the relative contribution of functional and developmental constraints on phenotypic variation is a long-standing goal of macroevolution, but it is often difficult to distinguish different types of constraints. Alternatively, selection can limit phenotypic (co)variation if some trait combinations are generally maladaptive. The anatomy of leaves with stomata on both surfaces (amphistomatous) present a unique opportunity to test the importance of functional and developmental constraints on phenotypic evolution. The key insight is that stomata on each leaf surface encounter the same functional and developmental constraints but potentially different selective pressures because of leaf asymmetry in light capture, gas exchange, and other features. Independent evolution of stomatal traits on each surface imply that functional and developmental constraints alone likely do not explain trait covariance. Packing limits on how many stomata can fit into a finite epidermis and cell size-mediated developmental integration are hypothesized to constrain variation in stomatal anatomy. The simple geometry of the planar leaf surface and knowledge of stomatal development make it possible to derive equations for phenotypic (co)variance caused by these constraints and compare them with data. We analyzed evolutionary covariance between stomatal density and length in amphistomatous leaves from 236 phylogenetically independent contrasts using a robust Bayesian model. Stomatal anatomy on each surface diverges partially independently, meaning that packing limits and developmental integration are not sufficient to explain phenotypic (co)variation. Hence, (co)variation in ecologically important traits like stomata arises in part because there is a limited range of evolutionary optima. We show how it is possible to evaluate the contribution of different constraints by deriving expected patterns of (co)variance and testing them using similar but separate tissues, organs, or sexes.

The influence of grafting on crops' photosynthetic performance.

In a near scenario of climate change where stress-derived limitations on crops' yield by affecting plant gas-exchange are expected, grafting may become a cheap and easy technique to improve crops photosynthetic performance and water-use efficiency. Inconsistent data of the effect of rootstocks over gas-exchange can be found in literature, being necessary an integrative analysis of the effect of grafting over photosynthetic parameters. With this aim, we present a compilation of the effect of graft on the net CO2 assimilation rate (AN) and other photosynthetic parameters across different species with agronomic interest. No differences were observed in any photosynthetic parameter between non-grafted and self-grafted plants under non-stress conditions. However, differences were found depending on the used rootstock, particularly for the intrinsic water-use efficiency (WUEi). We observed that variations in AN induced by rootstocks were related to changes in both diffusive and biochemical parameters. Under drought or salt stress, different photosynthetic performances were observed depending on the rootstock, although the high variability among studies promted to remarkable results. Overall, we observed that grafting can be a useful technique to improve plant photosynthetic performance, and therefore, crop yield and WUE, and that the rootstock selection for a target environment is determinant for the variations in photosynthesis.

Leaf size as a key determinant of contrasting growth patterns in closely related Limonium (Plumbaginaceae) species.

This study aims to analyze the importance of leaf size on plant growth capacity among an array of closely related Limonium species, and its impact on the underlying determinants of growth reduction under extreme water deficit conditions. To do so, thirteen Balearic Limonium species with contrasting leaf size were grown under long-term well-watered (WW) and severe water-deficit (WD) conditions in a common garden experiment. Fundamental growth traits were measured, including relative growth rate (RGR), net assimilation rate (NAR), leaf area ratio (LAR), leaf mass area (LMA) and leaf mass ratio (LMR). WD promoted small changes in leaf size, and species with larger leaves had higher RGR than species with smaller leaves, irrespective of the water treatment. Most RGR variation across species and treatments was explained by NAR, with comparatively much lower importance of LAR. The factorization of LAR underlying components denoted the importance of LMA in explaining RGR, whereas the impact of LMR on RGR was negligible in Limonium. Further, species with larger leaves had higher water consumption but also higher water use efficiency, especially under WD. Therefore, contrary to general trends in species from dry environments, increased leaf size is linked to increased growth capacity and also increased water use efficiency across closely related Limonium species.

Mediterranean Long Shelf-Life Landraces: An Untapped Genetic Resource for Tomato Improvement.

The Mediterranean long shelf-life (LSL) tomatoes are a group of landraces with a fruit remaining sound up to 6-12 months after harvest. Most have been selected under semi-arid Mediterranean summer conditions with poor irrigation or rain-fed and thus, are drought tolerant. Besides the convergence in the latter traits, local selection criteria have been very variable, leading to a wide variation in fruit morphology and quality traits. The different soil characteristics and agricultural management techniques across the Mediterranean denote also a wide range of plant adaptive traits to different conditions. Despite the notorious traits for fruit quality and environment adaptation, the LSL landraces have been poorly exploited in tomato breeding programs, which rely basically on wild tomato species. In this review, we describe most of the information currently available for Mediterranean LSL landraces in order to highlight the importance of this genetic resource. We focus on the origin and diversity, the main selective traits, and the determinants of the extended fruit shelf-life and the drought tolerance. Altogether, the Mediterranean LSL landraces are a very valuable heritage to be revalued, since constitutes an alternative source to improve fruit quality and shelf-life in tomato, and to breed for more resilient cultivars under the predicted climate change conditions.

The ratio of trichomes to stomata is associated with water use efficiency in Solanum lycopersicum (tomato).

Trichomes are specialised structures that originate from the aerial epidermis of plants, and play key roles in the interaction between the plant and the environment. In this study we investigated the trichome phenotypes of four lines selected from the Solanum lycopersicum × Solanum pennellii introgression line (IL) population for differences in trichome density, and their impact on plant performance under water-deficit conditions. We performed comparative analyses at morphological and photosynthetic levels of plants grown under well-watered (WW) and also under water-deficit (WD) conditions in the field. Under WD conditions, we observed higher trichome density in ILs 11-3 and 4-1, and lower stomatal size in IL 4-1 compared with plants grown under WW conditions. The intrinsic water use efficiency (WUEi ) was higher under WD conditions in IL 11-3, and the plant-level water use efficiency (WUEb ) was also higher in IL 11-3 and in M82 for WD plants. The ratio of trichomes to stomata (T/S) was positively correlated with WUEi and WUEb , indicating an important role for both trichomes and stomata in drought tolerance in tomato, and offering a promising way to select for improved water use efficiency of major crops.

Coordination between leaf CO2 diffusion and Rubisco properties allows maximizing photosynthetic efficiency in Limonium species.

High photosynthetic efficiency intrinsically demands tight coordination between traits related to CO2 diffusion capacity and leaf biochemistry. Although this coordination constitutes the basis of existing mathematical models of leaf photosynthesis, it has been barely explored among closely related species, which could reveal rapid adaptation clues in the recent past. With this aim, we characterized the photosynthetic capacity of 12 species of Limonium, possessing contrasting Rubisco catalytic properties, grown under optimal (WW) and extreme drought conditions (WD). The availability of CO2 at the site of carboxylation (Cc ) determined the photosynthetic capacity of Limonium under WD, while both diffusional and biochemical components governed the photosynthetic performance under WW. The variation in the in vivo caboxylation efficiency correlated with both the concentration of active Rubisco sites and the in vitro-based properties of Rubisco, such as the maximum carboxylase turnover rate (kcatc ) and the Michaelis-Menten constant for CO2 (Kc ). Notably, the results confirmed the hypothesis of coordination between the CO2 offer and demand functions of photosynthesis: those Limonium species with high total leaf conductance to CO2 have evolved towards increased velocity (i.e. higher kcatc ), at the penalty of lower affinity for CO2 (i.e. lower specificity factor, Sc/o ).

Weak coordination between leaf structure and function among closely related tomato species.

Theory predicts that natural selection should favor coordination between leaf physiology, biochemistry and anatomical structure along a functional trait spectrum from fast, resource-acquisitive syndromes to slow, resource-conservative syndromes. However, the coordination hypothesis has rarely been tested at a phylogenetic scale most relevant for understanding rapid adaptation in the recent past or for the prediction of evolutionary trajectories in response to climate change. We used a common garden to examine genetically based coordination between leaf traits across 19 wild and cultivated tomato taxa. We found weak integration between leaf structure (e.g. leaf mass per area) and physiological function (photosynthetic rate, biochemical capacity and CO2 diffusion), even though all were arrayed in the predicted direction along a 'fast-slow' spectrum. This suggests considerable scope for unique trait combinations to evolve in response to new environments or in crop breeding. In particular, we found that partially independent variation in stomatal and mesophyll conductance may allow a plant to improve water-use efficiency without necessarily sacrificing maximum photosynthetic rates. Our study does not imply that functional trait spectra, such as the leaf economics spectrum, are unimportant, but that many important axes of variation within a taxonomic group may be unique and not generalizable to other taxa.

Effects of long-term individual and combined water and temperature stress on the growth of rice, wheat and maize: relationship with morphological and physiological acclimation.

This study evaluates the long-term individual and combined effects of high temperature (HT) and water deficit (WD) stress on plant growth, leaf gas-exchange and water use efficiency in cultivars of the three most important crops worldwide, rice, wheat and maize. Total plant biomass (Bt ) accumulation decreased under all treatments, being the combined HT-WD treatment the most detrimental in all three species. Although decreases in Bt correlated with adjustments in biomass allocation patterns (i.e. the leaf area ratio), most of the variation observed in Bt was explained by changes in leaf gas exchange parameters. Thus, integrated values of leaf carbon balance obtained from daily course measurements of photosynthesis and respiration were better predictors of plant growth than the instantaneous measurements of leaf gas exchange. Leaf water use efficiency, assessed both by gas exchange and carbon isotope measurements, was negatively correlated with Bt under WD, but not under the combined WD and HT treatment. A comparative analysis of the negative effects of single and combined stresses on the main parameters showed an additive component for WD and HT in rice and maize, in contrast to wheat. Overall, the results of the specific cultivars included in the study suggest that the species native climate plays a role shaping the species acclimation potential to the applied stresses. In this regard, wheat, originated in a cold climate, was the most affected species, which foretells a higher affectation of this crop due to climate change.

Environmentally driven evolution of Rubisco and improved photosynthesis and growth within the C3 genus Limonium (Plumbaginaceae).

Carbon assimilation by most ecosystems requires ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Its kinetic parameters are likely to have evolved in parallel with intracellular CO2 availability, with the result that faster forms of Rubisco occur in species with CO2 -concentrating mechanisms. The Rubisco catalytic properties were determined and evaluated in relation to growth and carbon assimilation capacity in Mediterranean Limonium species, inhabiting severe stress environments. Significant kinetic differences between closely related species depended on two amino acid substitutions at functionally important residues 309 and 328 within the Rubisco large subunit. The Rubisco of species facing the largest CO2 restrictions during drought had relatively high affinity for CO2 (low Michaelis-Menten constant for CO2 Kc) but low maximum rates of carboxylation (kcatc), while the opposite was found for species that maintained higher CO2 concentrations under similar conditions. Rubisco kinetic characteristics were correlated with photosynthetic rate in both well-watered and drought-stressed plants. Moreover, the drought-mediated decrease in plant biomass accumulation was consistently lower in species with higher Rubisco carboxylase catalytic efficiency (kcatc/Kc). The present study is the first demonstration of Rubisco adaptation during species diversification within closely related C3 plants, revealing a direct relationship between Rubisco molecular evolution and the biomass accumulation of closely related species subjected to unfavourable conditions.

Expanding knowledge of the Rubisco kinetics variability in plant species: environmental and evolutionary trends.

The present study characterizes the kinetic properties of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from 28 terrestrial plant species, representing different phylogenetic lineages, environmental adaptations and photosynthetic mechanisms. Our findings confirm that past atmospheric CO(2)/O(2) ratio changes and present environmental pressures have influenced Rubisco kinetics. One evolutionary adaptation to a decreasing atmospheric CO(2)/O(2) ratio has been an increase in the affinity of Rubisco for CO(2) (Kc falling), and a consequent decrease in the velocity of carboxylation (kcat (c)), which in turn has been ameliorated by an increase in the proportion of leaf protein accounted by Rubisco. The trade-off between K(c) and k(cat)(c) was not universal among the species studied and deviations from this relationship occur in extant forms of Rubisco. In species adapted to particular environments, including carnivorous plants, crassulacean acid metabolism species and C(3) plants from aquatic and arid habitats, Rubisco has evolved towards increased efficiency, as demonstrated by a higher k(cat)(c)/K(c) ratio. This variability in kinetics was related to the amino acid sequence of the Rubisco large subunit. Phylogenetic analysis identified 13 residues under positive selection during evolution towards specific Rubisco kinetic parameters. This crucial information provides candidate amino acid replacements, which could be implemented to optimize crop photosynthesis under a range of environmental conditions.

Photosynthesis limitations in three fern species.

Maximum photosynthesis rates in ferns are generally lower than those of seed plants, but little is known about the limiting factors, which are crucial to understand the evolution of photosynthesis in land plants. To address this issue, a gas exchange/chlorophyll fluorescence analysis was performed in three fern species spanning high phylogenetic range within Polypodiopsida (Osmunda regalis, Blechnum gibbum and Nephrolepis exaltata) to determine their maximum net photosynthesis (AN ), stomatal (gs ) and mesophyll (gm ) conductances to CO2 , and the maximum velocity of carboxylation (Vc,max ). The in vitro Rubisco specificity factor (SC /O ) was also determined. All three species had values for SC /O similar to those typical of seed plants, but values of AN , gs , gm and Vc,max were within the lowest range of those observed in seed plants. In addition, gs was unresponsive to light and CO2 , as already described in other fern species. On the contrary, gm varied with changes CO2 . A quantitative photosynthesis limitation analysis suggested that early land plants (ferns) presented not only stomatal limitations-which were less adjustable to the environment-but also restricted gm and Vc,max , resulting in limited maximum photosynthesis rates.

Leaf responses to drought stress in Mediterranean accessions of Solanum lycopersicum: anatomical adaptations in relation to gas exchange parameters.

In a previous study, important acclimation to water stress was observed in the Ramellet tomato cultivar (TR) from the Balearic Islands, related to an increase in the water-use efficiency through modifications in both stomatal (g(s)) and mesophyll conductances (g(m)). In the present work, the comparison of physiological and morphological traits between TR accessions grown with and without water stress confirmed that variability in the photosynthetic capacity was mostly explained by differences in the diffusion of CO2 through stomata and leaf mesophyll. Maximization of gm under both treatments was mainly achieved through adjustments in the mesophyll thickness and porosity and the surface area of chloroplasts exposed to intercellular airspace (S(c)). In addition, the lower g(m) /S(c) ratio for a given porosity in drought-acclimated plants suggests that the decrease in gm was due to an increased cell wall thickness. Stomatal conductance was also affected by drought-associated changes in the morphological properties of stomata, in an accession and treatment-dependent manner. The results confirm the presence of advantageous physiological traits in the response to drought stress in Mediterranean accessions of tomato, and relate them to particular changes in the leaf anatomical properties, suggesting specific adaptive processes operating at the leaf anatomical level.

Physiological and morphological adaptations in relation to water use efficiency in Mediterranean accessions of Solanum lycopersicum.

The physiological traits underlying the apparent drought resistance of 'Tomàtiga de Ramellet' (TR) cultivars, a population of Mediterranean tomato cultivars with delayed fruit deterioration (DFD) phenotype and typically grown under non-irrigation conditions, are evaluated. Eight different tomato accessions were selected and included six TR accessions, one Mediterranean non-TR accession (NTR(M)) and a processing cultivar (NTR(O)). Among the TR accessions two leaf morphology types, normal divided leaves and potato-leaf, were selected. Plants were field grown under well-watered (WW) and water-stressed (WS) treatments, with 30 and 10% of soil water capacity, respectively. Accessions were clustered according to the leaf type and TR phenotype under WW and WS, respectively. Correlation among parameters under the different water treatments suggested that potential improvements in the intrinsic water-use efficiency (A(N)/g(s)) are possible without negative impacts on yield. Under WS TR accessions displayed higher A(N)/g(s), which was not due to differences in Rubisco-related parameters, but correlated with the ratio between the leaf mesophyll and stomatal conductances (g(m)/g(s)). The results confirm the existence of differential traits in the response to drought stress in Mediterranean accessions of tomato, and demonstrate that increases in the g(m)/g(s) ratio would allow improvements in A(N)/g(s) in horticultural crops.