Temperature responses of Rubisco from Paniceae grasses provide opportunities for improving C3 photosynthesis.

Enhancing the catalytic properties of the CO2-fixing enzyme Rubisco is a target for improving agricultural crop productivity. Here, we reveal extensive diversity in the kinetic response between 10 and 37 °C by Rubisco from C3 and C4 species within the grass tribe Paniceae. The CO2 fixation rate (kcatc) for Rubisco from the C4 grasses with nicotinamide adenine dinucleotide (NAD) phosphate malic enzyme (NADP-ME) and phosphoenolpyruvate carboxykinase (PCK) photosynthetic pathways was twofold greater than the kcatc of Rubisco from NAD-ME species across all temperatures. The declining response of CO2/O2 specificity with increasing temperature was less pronounced for PCK and NADP-ME Rubisco, which would be advantageous in warmer climates relative to the NAD-ME grasses. Modelled variation in the temperature kinetics of Paniceae C3 Rubisco and PCK Rubisco differentially stimulated C3 photosynthesis relative to tobacco above and below 25 °C under current and elevated CO2. Amino acid substitutions in the large subunit that could account for the catalytic variation among Paniceae Rubisco are identified; however, incompatibilities with Paniceae Rubisco biogenesis in tobacco hindered their mutagenic testing by chloroplast transformation. Circumventing these bioengineering limitations is critical to tailoring the properties of crop Rubisco to suit future climates

Positively selected amino acid replacements within the RuBisCO enzyme of oak trees are associated with ecological adaptations

Phylogenetic analysis by maximum likelihood (PAML) has become the standard approach to study positive selection at the molecular level, but other methods may provide complementary ways to identify amino acid replacements associated with particular conditions. Here, we compare results of the decision tree (DT) model method with ones of PAML using the key photosynthetic enzyme RuBisCO as a model system to study molecular adaptation to particular ecological conditions in oaks (Quercus). We sequenced the chloroplast rbcL gene encoding RuBisCO large subunit in 158 Quercus species, covering about a third of the global genus diversity. It has been hypothesized that RuBisCO has evolved differentially depending on the environmental conditions and leaf traits governing internal gas diffusion patterns. Here, we show, using PAML, that amino acid replacements at the residue positions 95, 145, 251, 262 and 328 of the RuBisCO large subunit have been the subject of positive selection along particular Quercus lineages associated with the leaf traits and climate characteristics. In parallel, the DT model identified amino acid replacements at sites 95, 219, 262 and 328 being associated with the leaf traits and climate characteristics, exhibiting partial overlap with the results obtained using PAML

Coevolution of amino acid residues in the key photosynthetic enzyme Rubisco

<p>Abstract</p> <p>Background</p> <p>One of the key forces shaping proteins is coevolution of amino acid residues. Knowing which residues coevolve in a particular protein may facilitate our understanding of protein evolution, structure and function, and help to identify substitutions that may lead to desired changes in enzyme kinetics. Rubisco, the most abundant enzyme in biosphere, plays an essential role in the process of carbon fixation through photosynthesis, thus facilitating life on Earth. This makes Rubisco an important model system for studying the dynamics of protein fitness optimization on the evolutionary landscape. In this study we investigated the selective and coevolutionary forces acting on large subunit of land plants Rubisco using Markov models of codon substitution and clustering approaches applied to amino acid substitution histories.</p> <p>Results</p> <p>We found that both selection and coevolution shape Rubisco, and that positively selected and coevolving residues have their specifically favored amino acid composition and pairing preference. The mapping of these residues on the known Rubisco tertiary structures showed that the coevolving residues tend to be in closer proximity with each other compared to the background, while positively selected residues tend to be further away from each other. This study also reveals that the residues under positive selection or coevolutionary force are located within functionally important regions and that some residues are targets of both positive selection and coevolution at the same time.</p> <p>Conclusion</p> <p>Our results demonstrate that coevolution of residues is common in Rubisco of land plants and that there is an overlap between coevolving and positively selected residues. Knowledge of which Rubisco residues are coevolving and positively selected could be used for further work on structural modeling and identification of substitutions that may be changed in order to improve efficiency of this important enzyme in crops.</p

Molecular Adaptation of rbcL in the Heterophyllous Aquatic Plant Potamogeton

Heterophyllous aquatic plants show marked phenotypic plasticity. They adapt to environmental changes by producing different leaf types: submerged, floating and terrestrial leaves. By contrast, homophyllous plants produce only submerged leaves and grow entirely underwater. Heterophylly and submerged homophylly evolved under selective pressure modifying the species-specific optima for photosynthesis, but little is known about the evolutionary outcome of habit. Recent evolutionary analyses suggested that rbcL, a chloroplast gene that encodes a catalytic subunit of RuBisCO, evolves under positive selection in most land plant lineages. To examine the adaptive evolutionary process linked to heterophylly or homophylly, we analyzed positive selection in the rbcL sequences of ecologically diverse aquatic plants, Japanese Potamogeton.Phylogenetic and maximum likelihood analyses of codon substitution models indicated that Potamogeton rbcL has evolved under positive Darwinian selection. The positive selection has operated specifically in heterophyllous lineages but not in homophyllous ones in the branch-site models. This suggests that the selective pressure on this chloroplast gene was higher for heterophyllous lineages than for homophyllous lineages. The replacement of 12 amino acids occurred at structurally important sites in the quaternary structure of RbcL, two of which (residue 225 and 281) were identified as potentially under positive selection.Our analysis did not show an exact relationship between the amino acid replacements and heterophylly or homophylly but revealed that lineage-specific positive selection acted on the Potamogeton rbcL. The contrasting ecological conditions between heterophyllous and homophyllous plants have imposed different selective pressures on the photosynthetic system. The increased amino acid replacement in RbcL may reflect the continuous fine-tuning of RuBisCO under varying ecological conditions

Rubisco evolution in C₄ eudicots: an analysis of Amaranthaceae sensu lato.

BACKGROUND: Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyses the key reaction in the photosynthetic assimilation of CO₂. In C₄ plants CO₂ is supplied to Rubisco by an auxiliary CO₂-concentrating pathway that helps to maximize the carboxylase activity of the enzyme while suppressing its oxygenase activity. As a consequence, C₄ Rubisco exhibits a higher maximum velocity but lower substrate specificity compared with the C₃ enzyme. Specific amino-acids in Rubisco are associated with C₄ photosynthesis in monocots, but it is not known whether selection has acted on Rubisco in a similar way in eudicots. METHODOLOGY/PRINCIPAL FINDINGS: We investigated Rubisco evolution in Amaranthaceae sensu lato (including Chenopodiaceae), the third-largest family of C₄ plants, using phylogeny-based maximum likelihood and Bayesian methods to detect Darwinian selection on the chloroplast rbcL gene in a sample of 179 species. Two Rubisco residues, 281 and 309, were found to be under positive selection in C₄ Amaranthaceae with multiple parallel replacements of alanine by serine at position 281 and methionine by isoleucine at position 309. Remarkably, both amino-acids have been detected in other C₄ plant groups, such as C₄ monocots, illustrating a striking parallelism in molecular evolution. CONCLUSIONS/SIGNIFICANCE: Our findings illustrate how simple genetic changes can contribute to the evolution of photosynthesis and strengthen the hypothesis that parallel amino-acid replacements are associated with adaptive changes in Rubisco

Terfenadine induces apoptosis and autophagy in melanoma cells through ROS-dependent and -independent mechanisms

Previously we found that terfenadine, an H1 histamine receptor antagonist, acts as a potent apoptosis inducer in melanoma cells through modulation of Ca2+ homeostasis. In this report, focusing our attention on the apoptotic mechanisms activated by terfenadine, we show that this drug can potentially activate distinct intrinsic signaling pathways depending on culture conditions. Serum-deprived conditions enhance the cytotoxic effect of terfenadine and caspase-4 and -2 are activated upstream of caspase-9. Moreover, although we found an increase in ROS levels, the apoptosis was ROS independent. Conversely, terfenadine treatment in complete medium induced ROS-dependent apoptosis. Caspase-4, -2, and -9 were simultaneously activated and p73 and Noxa induction were involved. ROS inhibition prevented p73 and Noxa expression but not p53 and p21 expression, suggesting a role for Noxa in p53-independent apoptosis in melanoma cells. Finally, we found that terfenadine induced autophagy, that can promote apoptosis. These findings demonstrate the great potential of terfenadine to kill melanoma cells through different cellular signaling pathways and could contribute to define new therapeutic strategies in melanoma

Response of the photosynthetic apparatus to a flowering-inductive period by water stress in Citrus

The photosynthetic responses to a flowering-inductive water-stress period and recovery were studied and compared in two Citrus species. Under greenhouse conditions, Fino lemon and Owari satsuma trees were subjected to moderate (-2 MPa at predawn) and severe (-3 MPa) water stress levels and were re-watered after 60 days. Vegetative growth was inhibited during the stress assays, and strong defoliation levels were reported, especially in Fino lemon. In both species, bud sprouting was induced after re-watering. Flowers and vegetative shoots developed in Owari satsuma after a drought period, and the development was independent of the stress level. In Fino lemon, vegetative shoots and flowers were primarily formed after moderate and severe stress, respectively. The photosynthetic rate and stomatal conductance were reduced by water stress, and a marked increase in water-use efficiency at the moderate water deficit level was observed. Nevertheless, the photosynthetic apparatus was not damaged, since the maximum quantum yield, photosynthetic pigment concentrations and Rubisco level and activity did not change. Furthermore, the measured malonyldialdehyde (MDA) and peroxidase activity indicated that oxidative stress was not specifically triggered by water stress in our study. Therefore, the gas exchange, fluorescence and biochemical parameters suggested that diffusional limitations to photosynthesis predominated in both of the studied Citrus species, and explained the rapid recovery of the photosynthetic parameters after rehydration. The net CO 2 fixation rate and stomatal conductance were recovered within 24 h in Fino lemon, whereas 3 days were required in Owari satsuma. This suggests the presence of some metabolic limitations in the latter species. 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Exploring the three PIPs and three TIPs of grapevine for transport of water and atypical substrates through heterologous expression in aqy-null yeast

Aquaporins are membrane channels that facilitate the transport of water and other small molecules across the cellular membranes. We examined the role of six aquaporins of Vitis vinifera (cv. Touriga nacional) in the transport of water and atypical substrates (other than water) in an aqy-null strain of Saccharomyces cerevisiae. Their functional characterization for water transport was performed by stopped-flow fluorescence spectroscopy. The evaluation of permeability coefficients (Pf) and activation energies (Ea) revealed that three aquaporins (VvTnPIP2;1, VvTnTIP1;1 and VvTnTIP2;2) are functional for water transport, while the other three (VvTnPIP1;4, VvTnPIP2;3 and VvTnTIP4;1) are non-functional. TIPs (VvTnTIP1;1 and VvTnTIP2;2) exhibited higher water permeability than VvTnPIP2;1. All functional aquaporins were found to be sensitive to HgCl2, since their water conductivity was reduced (24–38%) by the addition of 0.5 mM HgCl2. Expression of Vitis aquaporins caused different sensitive phenotypes to yeast strains when grown under hyperosmotic stress generated by KCl or sorbitol. Our results also indicate that Vitis aquaporins are putative transporters of other small molecules of physiological importance. Their sequence analyses revealed the presence of signature sequences for transport of ammonia, boron, CO2, H2O2 and urea. The phenotypic growth variations of yeast cells showed that heterologous expression of Vitis aquaporins increased susceptibility to externally applied boron and H2O2, suggesting the contribution of Vitis aquaporins in the transport of these speciesinfo:eu-repo/semantics/publishedVersio

Microarray analysis and scale-free gene networks identify candidate regulators in drought-stressed roots of loblolly pine (P. taeda L.)

<p>Abstract</p> <p>Background</p> <p>Global transcriptional analysis of loblolly pine (<it>Pinus taeda </it>L.) is challenging due to limited molecular tools. PtGen2, a 26,496 feature cDNA microarray, was fabricated and used to assess drought-induced gene expression in loblolly pine propagule roots. Statistical analysis of differential expression and weighted gene correlation network analysis were used to identify drought-responsive genes and further characterize the molecular basis of drought tolerance in loblolly pine.</p> <p>Results</p> <p>Microarrays were used to interrogate root cDNA populations obtained from 12 genotype × treatment combinations (four genotypes, three watering regimes). Comparison of drought-stressed roots with roots from the control treatment identified 2445 genes displaying at least a 1.5-fold expression difference (false discovery rate = 0.01). Genes commonly associated with drought response in pine and other plant species, as well as a number of abiotic and biotic stress-related genes, were up-regulated in drought-stressed roots. Only 76 genes were identified as differentially expressed in drought-recovered roots, indicating that the transcript population can return to the pre-drought state within 48 hours. Gene correlation analysis predicts a scale-free network topology and identifies eleven co-expression modules that ranged in size from 34 to 938 members. Network topological parameters identified a number of central nodes (hubs) including those with significant homology (E-values ≤ 2 × 10^-30) to 9-cis-epoxycarotenoid dioxygenase, zeatin O-glucosyltransferase, and ABA-responsive protein. Identified hubs also include genes that have been associated previously with osmotic stress, phytohormones, enzymes that detoxify reactive oxygen species, and several genes of unknown function.</p> <p>Conclusion</p> <p>PtGen2 was used to evaluate transcriptome responses in loblolly pine and was leveraged to identify 2445 differentially expressed genes responding to severe drought stress in roots. Many of the genes identified are known to be up-regulated in response to osmotic stress in pine and other plant species and encode proteins involved in both signal transduction and stress tolerance. Gene expression levels returned to control values within a 48-hour recovery period in all but 76 transcripts. Correlation network analysis indicates a scale-free network topology for the pine root transcriptome and identifies central nodes that may serve as drivers of drought-responsive transcriptome dynamics in the roots of loblolly pine.</p

Mountain strongholds for woody angiosperms during the Late Pleistocene in SE Iberia

Mediterranean mountains played an essential role during glacial periods as vegetation refugia. The SE Iberia Late Pleistocene woody angiosperm fossil and floristic evidences are reviewed in the context of phylogeographical studies aiming to identify (i) spatial patterns related to woody angiosperms glacial survival, (ii) structural and functional characteristics of montane refugia, and (iii) gaps in knowledge on the woody angiosperm patterns of survival in Mediterranean mountains. The distribution of palaeobotanical data for SE Iberia refugia has been found to be taphonomically biased due to the scarcity of available and/or studied high-altitude Late Pleistocene sites. However, Siles Lake data together with floristic inference provide evidences for woody angiosperms’ survival in a high-altitude Mediterranean area. The main features boosting survival at montane contexts are physiographic complexity and water availability. Phylogeography studies have mainly been conducted at a continental scale. Although they cohere with palaeobotanical data to a broad scale, a general lack of sampling of SE Iberian range-edge populations, as well as misconceptions about the origin of the populations sampled, impede to infer the proper location of woody angiosperms’ mountain refugia and their importance in the post-glacial European colonisation. We conclude that floristic, geobotanical, palaeobotanical, ethnographical and genetic evidence should be merged to gain a deeper understanding on the role played by Mediterranean mountains as glacial refugia in order to explain the current distribution of many plants and the large biodiversity levels encountered in Mediterranean mountain areas. This is hallmark for effective and efficient conservation and management