Decreasing the Mitochondrial Synthesis of Malate in Potato Tubers Does Not Affect Plastidial Starch Synthesis, Suggesting That the Physiological Regulation of ADPglucose Pyrophosphorylase Is Context Dependent

Modulation of the malate content of tomato (Solanum lycopersicum) fruit by altering the expression of mitochondrially localized enzymes of the tricarboxylic acid cycle resulted in enhanced transitory starch accumulation and subsequent effects on postharvest fruit physiology. In this study, we assessed whether such a manipulation would similarly affect starch biosynthesis in an organ that displays a linear, as opposed to a transient, kinetic of starch accumulation. For this purpose, we used RNA interference to down-regulate the expression of fumarase in potato (Solanum tuberosum) under the control of the tuber-specific B33 promoter. Despite displaying similar reductions in both fumarase activity and malate content as observed in tomato fruit expressing the same construct, the resultant transformants were neither characterized by an increased flux to, or accumulation of, starch, nor by alteration in yield parameters. Since the effect in tomato was mechanistically linked to derepression of the reaction catalyzed by ADP-glucose pyrophosphorylase, we evaluated whether the lack of effect on starch biosynthesis was due to differences in enzymatic properties of the enzyme from potato and tomato or rather due to differential subcellular compartmentation of reductant in the different organs. The results are discussed in the context both of current models of metabolic compartmentation and engineering

Identification and characterization of metabolite quantitative trait loci in tomato leaves and comparison with those reported for fruits and seeds

Introduction: To date, most studies of natural variation and metabolite quantitative trait loci (mQTL) in tomato have focused on fruit metabolism, leaving aside the identification of genomic regions involved in the regulation of leaf metabolism. Objective: This study was conducted to identify leaf mQTL in tomato and to assess the association of leaf metabolites and physiological traits with the metabolite levels from other tissues. Methods: The analysis of components of leaf metabolism was performed by phenotypying 76 tomato ILs with chromosome segments of the wild species Solanum pennellii in the genetic background of a cultivated tomato (S. lycopersicum) variety M82. The plants were cultivated in two different environments in independent years and samples were harvested from mature leaves of non-flowering plants at the middle of the light period. The non-targeted metabolite profiling was obtained by gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). With the data set obtained in this study and already published metabolomics data from seed and fruit, we performed QTL mapping, heritability and correlation analyses. Results: Changes in metabolite contents were evident in the ILs that are potentially important with respect to stress responses and plant physiology. By analyzing the obtained data, we identified 42 positive and 76 negative mQTL involved in carbon and nitrogen metabolism. Conclusions: Overall, these findings allowed the identification of S. lycopersicum genome regions involved in the regulation of leaf primary carbon and nitrogen metabolism, as well as the association of leaf metabolites with metabolites from seeds and fruits.Fil: Nunes Nesi, Adriano. Max Planck Institute Of Molecular Plant Physiology; Alemania. Universidade Federal de Viçosa.; BrasilFil: Alseekh, Saleh. Center Of Plant Systems Biology And Biotechnology; Bulgaria. Max Planck Institute Of Molecular Plant Physiology; AlemaniaFil: de Oliveira Silva, Franklin Magnum. Universidade Federal de Viçosa.; BrasilFil: Omranian, Nooshin. Max Planck Institute Of Molecular Plant Physiology; Alemania. Center Of Plant Systems Biology And Biotechnology; BulgariaFil: Lichtenstein, Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Mirnezhad, Mohammad. Leiden University; Países BajosFil: Romero González, Roman R.. Leiden University; Países BajosFil: Sabio y Garcia, Julia Veronica. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Conte, Mariana. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Leiss, Kirsten A.. Leiden University; Países BajosFil: Klinkhamer, Peter G. L.. Leiden University; Países BajosFil: Nikoloski, Zoran. University of Potsdam; Alemania. Max Planck Institute of Molecular Plant Physiology; AlemaniaFil: Carrari, Fernando Oscar. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Fernie, Alisdair R.. Max Planck Institute of Molecular Plant Physiology; Alemania. Center of Plant System Biology and Biotechnology; Bulgari

Extending the ecological distribution of Desmonostoc genus: proposal of Desmonostoc salinum sp. nov., a novel Cyanobacteria from a saline–alkaline lake

Cyanobacteria is an ancient phylum of oxygenic photosynthetic microorganisms found in almost all environments of Earth. In recent years, the taxonomic placement of some cyanobacterial strains, including those belonging to the genus Nostoc sensu lato, have been reevaluated by means of a polyphasic approach. Thus, 16S rRNA gene phylogeny and 16S–23S internal transcribed spacer (ITS) secondary structures coupled with morphological, ecological and physiological data are considered powerful tools for a better taxonomic and systematics resolution, leading to the description of novel genera and species. Additionally, underexplored and harsh environments, such as saline–alkaline lakes, have received special attention given they can be a source of novel cyanobacterial taxa. Here, a filamentous heterocytous strain, Nostocaceae CCM-UFV059, isolated from Laguna Amarga, Chile, was characterized applying the polyphasic approach; its fatty acid profile and physiological responses to salt (NaCl) were also determined. Morphologically, this strain was related to morphotypes of the Nostoc sensu lato group, being phylogenetically placed into the typical cluster of the genus Desmonostoc. CCM-UFV059 showed identity of the 16S rRNA gene as well as 16S–23S secondary structures that did not match those from known described species of the genus Desmonostoc, as well as distinct ecological and physiological traits. Taken together, these data allowed the description of the first strain of a member of the genus Desmonostoc from a saline–alkaline lake, named Desmonostoc salinum sp. nov., under the provisions of the International Code of Nomenclature for algae, fungi and plants. This finding extends the ecological coverage of the genus Desmonostoc, contributing to a better understanding of cyanobacterial diversity and systematics

Mild reductions in cytosolic NADP-dependent isocitrate dehydrogenase activity result in lower amino acid contents and pigmentation without impacting growth

Transgenic tomato (Solanum lycopersicum) plants were generated targeting the cytosolic NADP-dependent isocitrate dehydrogenase gene (SlICDH1) via the RNA interference approach. The resultant transformants displayed a relatively mild reduction in the expression and activity of the target enzyme in the leaves. However, biochemical analyses revealed that the transgenic lines displayed a considerable shift in metabolism, being characterized by decreases in the levels of the TCA cycle intermediates, total amino acids, photosynthetic pigments, starch and NAD(P)H. The plants showed little change in photosynthesis with the exception of a minor decrease in maximum photosynthetic efficiency (Fv/Fm), and a small decrease in growth compared to the wild type. These results reveal that even small changes in cytosolic NADP-dependent isocitrate dehydrogenase activity lead to noticeable alterations in the activities of enzymes involved in primary nitrate assimilation and in the synthesis of 2-oxoglutarate derived amino acids. These data are discussed within the context of current models for the role of the various isoforms of isocitrate dehydrogenase within plant amino acid metabolism

Influence of mitochondrial genome rearrangement on cucumber leaf carbon and nitrogen metabolism

The MSC16 cucumber (Cucumis sativus L.) mitochondrial mutant was used to study the effect of mitochondrial dysfunction and disturbed subcellular redox state on leaf day/night carbon and nitrogen metabolism. We have shown that the mitochondrial dysfunction in MSC16 plants had no effect on photosynthetic CO2 assimilation, but the concentration of soluble carbohydrates and starch was higher in leaves of MSC16 plants. Impaired mitochondrial respiratory chain activity was associated with the perturbation of mitochondrial TCA cycle manifested, e.g., by lowered decarboxylation rate. Mitochondrial dysfunction in MSC16 plants had different influence on leaf cell metabolism under dark or light conditions. In the dark, when the main mitochondrial function is the energy production, the altered activity of TCA cycle in mutated plants was connected with the accumulation of pyruvate and TCA cycle intermediates (citrate and 2-OG). In the light, when TCA activity is needed for synthesis of carbon skeletons required as the acceptors for NH4+ assimilation, the concentration of pyruvate and TCA intermediates was tightly coupled with nitrate metabolism. Enhanced incorporation of ammonium group into amino acids structures in mutated plants has resulted in decreased concentration of organic acids and accumulation of Glu

Seasonal changes in levels of chlorophyll, nitrate and total soluble carbohydrates of brachiaria decumbens in a long-term silvopastoral system.

Our objective was to quantify the levels of chlorophyll (Chl), nitrate and total soluble carbohydrates in leaf blades of Brachiaria decumbens grown in the understory of a silvopastoral system (SPS) under intense shade and compare them to those grown in an open pasture (OP), with B. decumbens under full sunlight. Two treatments, corresponding to the specific production system (SPS and OP) were evaluated over four seasons: spring 2016, summer 2017, autumn 2017, and winter 2017. In the SPS, we observed an increase in the levels of chlorophyll and nitrate, and a reduction in total soluble carbohydrates relative to those in OP. This pattern of response was also influenced by the seasons, with higher levels of chlorophyll and nitrate during seasons with greater availability of resources. In contrast, total soluble carbohydrates were lower in spring and summer and higher in autumn and winter. Our results demonstrated that intense light restriction (70% shading) in an SPS reduces the levels of total soluble carbohydrates and increases the levels of chlorophyll and nitrate, which are directly related to the productivity, longevity, and nutritional value of the forage.WCCLF 2021. Evento online

The mitochondrial NAD+ transporter (NDT1) plays important roles in cellular NAD+ homeostasis in \u3ci\u3eArabidopsis thaliana\u3c/i\u3e

Nicotinamide adenine dinucleotide (NAD+) is an essential coenzyme required for all living organisms. In eukaryotic cells, the final step of NAD+ biosynthesis is exclusively cytosolic. Hence, NAD+ must be imported into organelles to support their metabolic functions. Three NAD+ transporters belonging to the mitochondrial carrier family (MCF) have been biochemically characterized in plants. AtNDT1 (At2g47490), focus of the current study, AtNDT2 (At1g25380), targeted to the inner mitochondrial membrane, and AtPXN (At2g39970), located in the peroxisomal membrane. Although AtNDT1 was presumed to reside in the chloroplast membrane, subcellular localization experiments with green fluorescent protein (GFP) fusions revealed that AtNDT1 locates exclusively in the mitochondrial membrane in stably transformed Arabidopsis plants. To understand the biological function of AtNDT1 in Arabidopsis, three transgenic lines containing an antisense construct of AtNDT1 under the control of the 35S promoter alongside a T-DNA insertional line were evaluated. Plants with reduced AtNDT1 expression displayed lower pollen viability, silique length, and higher rate of seed abortion. Furthermore, these plants also exhibited an increased leaf number and leaf area concomitant with higher photosynthetic rates and higher levels of sucrose and starch. Therefore, lower expression of AtNDT1 was associated with enhanced vegetative growth but severe impairment of the reproductive stage. These results are discussed in the context of the mitochondrial localization of AtNDT1 and its important role in the cellular NAD+ homeostasis for both metabolic and developmental processes in plants

Systematic Analysis of Stability Patterns in Plant Primary Metabolism

Metabolic networks are characterized by complex interactions and regulatory mechanisms between many individual components. These interactions determine whether a steady state is stable to perturbations. Structural kinetic modeling (SKM) is a framework to analyze the stability of metabolic steady states that allows the study of the system Jacobian without requiring detailed knowledge about individual rate equations. Stability criteria can be derived by generating a large number of structural kinetic models (SK-models) with randomly sampled parameter sets and evaluating the resulting Jacobian matrices. Until now, SKM experiments applied univariate tests to detect the network components with the largest influence on stability. In this work, we present an extended SKM approach relying on supervised machine learning to detect patterns of enzyme-metabolite interactions that act together in an orchestrated manner to ensure stability. We demonstrate its application on a detailed SK-model of the Calvin-Benson cycle and connected pathways. The identified stability patterns are highly complex reflecting that changes in dynamic properties depend on concerted interactions between several network components. In total, we find more patterns that reliably ensure stability than patterns ensuring instability. This shows that the design of this system is strongly targeted towards maintaining stability. We also investigate the effect of allosteric regulators revealing that the tendency to stability is significantly increased by including experimentally determined regulatory mechanisms that have not yet been integrated into existing kinetic models

A candidate gene survey of quantitative trait loci affecting chemical composition in tomato fruit

In tomato, numerous wild-related species have been demonstrated to be untapped sources of valuable genetic variability, including pathogen-resistance genes, nutritional, and industrial quality traits. From a collection of S. pennellii introgressed lines, 889 fruit metabolic loci (QML) and 326 yield-associated loci (YAL), distributed across the tomato genome, had been identified previously. By using a combination of molecular marker sequence analysis, PCR amplification and sequencing, analysis of allelic variation, and evaluation of co-response between gene expression and metabolite composition traits, the present report, provides a comprehensive list of candidate genes co-localizing with a subset of 106 QML and 20 YAL associated either with important agronomic or nutritional characteristics. This combined strategy allowed the identification and analysis of 127 candidate genes located in 16 regions of the tomato genome. Eighty-five genes were cloned and partially sequenced, totalling 45 816 and 45 787 bases from S. lycopersicum and S. pennellii, respectively. Allelic variation at the amino acid level was confirmed for 37 of these candidates. Furthermore, out of the 127 gene-metabolite co-locations, some 56 were recovered following correlation of parallel transcript and metabolite profiling. Results obtained here represent the initial steps in the integration of genetic, genomic, and expressional patterns of genes co-localizing with chemical compositional traits of the tomato fruit

Multi-level engineering facilitates the production of phenylpropanoid compounds in tomato

Phenylpropanoids comprise an important class of plant secondary metabolites. A number of transcription factors have been used to upregulate-specific branches of phenylpropanoid metabolism, but by far the most effective has been the fruit-specific expression of AtMYB12 in tomato, which resulted in as much as 10% of fruit dry weight accumulating as flavonols and hydroxycinnamates. We show that AtMYB12 not only increases the demand of flavonoid biosynthesis but also increases the supply of carbon from primary metabolism, energy and reducing power, which may fuel the shikimate and phenylalanine biosynthetic pathways to supply more aromatic amino acids for secondary metabolism. AtMYB12 directly binds promoters of genes encoding enzymes of primary metabolism. The enhanced supply of precursors, energy and reducing power achieved by AtMYB12 expression can be harnessed to engineer high levels of novel phenylpropanoids in tomato fruit, offering an effective production system for bioactives and other high value ingredients