Pyrophosphate as an alternative energy currency in plants.

In the conditions of [Mg2+] elevation that occur, in particular, under low oxygen stress and are the consequence of the decrease in [ATP] and increase in [ADP] and [AMP], pyrophosphate (PPi) can function as an alternative energy currency in plant cells. In addition to its production by various metabolic pathways, PPi can be synthesized in the combined reactions of pyruvate, phosphate dikinase (PPDK) and pyruvate kinase (PK) by so-called PK/PPDK substrate cycle, and in the reverse reaction of membrane-bound H+-pyrophosphatase, which uses the energy of electrochemical gradients generated on tonoplast and plasma membrane. The PPi can then be consumed in its active forms of MgPPi and Mg2PPi by PPi-utilizing enzymes, which require an elevated [Mg2+]. This ensures a continuous operation of glycolysis in the conditions of suppressed ATP synthesis, keeping metabolism energy efficient and less dependent on ATP.

The uncoupling of respiration in plant mitochondria: keeping reactive oxygen and nitrogen species under control.

Plant mitochondrial respiration involves the operation of various alternative pathways. These pathways participate, both directly and indirectly, in the maintenance of mitochondrial functions though they do not contribute to energy production, being uncoupled from the generation of an electrochemical gradient across the mitochondrial membrane and thus from ATP production. Recent findings suggest that uncoupled respiration is involved in reactive oxygen species (ROS) and nitric oxide (NO) scavenging, regulation, and homeostasis. Here we discuss specific roles and possible functions of uncoupled mitochondrial respiration in ROS and NO metabolism. The mechanisms of expression and regulation of the NDA-, NDB- and NDC-type non-coupled NADH and NADPH dehydrogenases, the alternative oxidase (AOX), and the uncoupling protein (UCP) are examined in relation to their involvement in the establishment of the stable far-from-equilibrium state of plant metabolism. The role of uncoupled respiration in controlling the levels of ROS and NO as well as inducing signaling events is considered. Secondary functions of uncoupled respiration include its role in protection from stress factors and roles in biosynthesis and catabolism. It is concluded that uncoupled mitochondrial respiration plays an important role in providing rapid adaptation of plants to changing environmental factors via regulation of ROS and NO.

Optimization of nucleotide sugar supply for polysaccharide formation via thermodynamic buffering.

Plant polysaccharides (cellulose, hemicellulose, pectin, starch) are either direct (i.e. leaf starch) or indirect products of photosynthesis, and they belong to the most abundant organic compounds in nature. Although each of these polymers is made by a specific enzymatic machinery, frequently in different cell locations, details of their synthesis share certain common features. Thus, the production of these polysaccharides is preceded by the formation of nucleotide sugars catalyzed by fully reversible reactions of various enzymes, mostly pyrophosphorylases. These 'buffering' enzymes are, generally, quite active and operate close to equilibrium. The nucleotide sugars are then used as substrates for irreversible reactions of various polysaccharide-synthesizing glycosyltransferases ('engine' enzymes), e.g. plastidial starch synthases, or plasma membrane-bound cellulose synthase and callose synthase, or ER/Golgi-located variety of glycosyltransferases forming hemicellulose and pectin backbones. Alternatively, the irreversible step might also be provided by a carrier transporting a given immediate precursor across a membrane. Here, we argue that local equilibria, established within metabolic pathways and cycles resulting in polysaccharide production, bring stability to the system via the arrangement of a flexible supply of nucleotide sugars. This metabolic system is itself under control of adenylate kinase and nucleoside-diphosphate kinase, which determine the availability of nucleotides (adenylates, uridylates, guanylates and cytidylates) and Mg2+, the latter serving as a feedback signal from the nucleotide metabolome. Under these conditions, the supply of nucleotide sugars to engine enzymes is stable and constant, and the metabolic process becomes optimized in its load and consumption, making the system steady and self-regulated.

Magnesium Signaling in Plants.

Free magnesium (Mg2+) is a signal of the adenylate (ATP+ADP+AMP) status in the cells. It results from the equilibrium of adenylate kinase (AK), which uses Mg-chelated and Mg-free adenylates as substrates in both directions of its reaction. The AK-mediated primary control of intracellular [Mg2+] is finely interwoven with the operation of membrane-bound adenylate- and Mg2+-translocators, which in a given compartment control the supply of free adenylates and Mg2+ for the AK-mediated equilibration. As a result, [Mg2+] itself varies both between and within the compartments, depending on their energetic status and environmental clues. Other key nucleotide-utilizing/producing enzymes (e.g., nucleoside diphosphate kinase) may also be involved in fine-tuning of the intracellular [Mg2+]. Changes in [Mg2+] regulate activities of myriads of Mg-utilizing/requiring enzymes, affecting metabolism under both normal and stress conditions, and impacting photosynthetic performance, respiration, phloem loading and other processes. In compartments controlled by AK equilibrium (cytosol, chloroplasts, mitochondria, nucleus), the intracellular [Mg2+] can be calculated from total adenylate contents, based on the dependence of the apparent equilibrium constant of AK on [Mg2+]. Magnesium signaling, reflecting cellular adenylate status, is likely widespread in all eukaryotic and prokaryotic organisms, due simply to the omnipresent nature of AK and to its involvement in adenylate equilibration.

Biological thermodynamics: Ervin Bauer and the unification of life sciences and physics.

Biological systems operate toward the maximization of their self-maintenance and adaptability. This is achieved through the establishment of robust self-maintaining configurations acting as attractors resistant to external and internal perturbations. Ervin Bauer (1890-1938) was the first who formulated this essential thermodynamic constraint in the operation of biological systems, which he defined as the stable non-equilibrium state. The latter appears as the basic attractor relative to which biological organization is established. The stable non-equilibrium state represents a generalized cell energy status corresponding to efficient spatiotemporal organization of the fluxes of matter and energy and constantly reproducing the conditions of self-maintenance of metabolism and controlling the rates of major metabolic fluxes that follow thermodynamically and kinetically defined computational principles. This state is realized in the autopoietic structures having closed loops of causation based on the operation of biological codes. The principle of thermodynamic buffering determines the conditions for optimization of the fluxes of load and consumption in metabolism establishing the conditions of metabolic stable non-equilibrium. In developing and evolving biological systems, the principle of stable non-equilibrium is transformed into the principle of increasing external work, which is grounded in the hyper-restorative non-equilibrium dynamics. Bauer's concept of the stable non-equilibrium state puts thermodynamics into the frames of the internal biological causality governing self-maintenance and development of living systems. It can be defined as a relational theory of biological thermodynamics since the standard to which it refers represents the actual biological function rather than the abstract state of thermodynamic equilibrium.

Toward understanding the emergence of life: A dual function of the system of nucleotides in the metabolically closed autopoietic organization.

General structure of metabolism includes the reproduction of catalysts that govern metabolism. In this structure, the system becomes autopoietic in the sense of Maturana and Varela, and it is closed to efficient causation as defined by Robert Rosen. The autopoietic maintenance and operation of the catalysts takes place via the set of free nucleotides while the synthesis of catalysts occurs via the information encoded by the set of nucleotides arranged in polymers of RNA and DNA. Both energy charge and genetic information use the components of the same pool of nucleoside triphosphates, which is equilibrated by thermodynamic buffering enzymes such as nucleoside diphosphate kinase and adenylate kinase. This occurs in a way that the system becomes internally stable and metabolically closed, which initially could be realized at the level of ribozymes catalyzing basic metabolic reactions as well as own reproduction. The function of ATP, GTP, UTP, and CTP is dual, as these species participate both in the general metabolism as free nucleotides and in the transfer of genetic information via covalent polymerization to nucleic acids. The changes in their pools directly impact both bioenergetic pathways and nucleic acid turnover. Here we outline the concept of metabolic closure of biosystems grounded in the dual function of nucleotide coenzymes that serve both as energetic and informational molecules and through this duality generate the autopoietic performance and the ability for codepoietic evolutionary transformations of living systems starting from the emergence of prebiotic systems.

Citrate valve integrates mitochondria into photosynthetic metabolism.

While in heterotrophic cells and in darkness mitochondria serve as main producers of energy, during photosynthesis this function is transferred to chloroplasts and the main role of mitochondria in bioenergetics turns to be the balance of the level of phosphorylation of adenylates and of reduction of pyridine nucleotides to avoid over-energization of the cell and optimize major metabolic fluxes. This is achieved via the establishment and regulation of local equilibria of the tricarboxylic acid (TCA) cycle enzymes malate dehydrogenase and fumarase in one branch and aconitase and isocitrate dehydrogenase in another branch. In the conditions of elevation of redox level, the TCA cycle is transformed into a non-cyclic open structure (hemicycle) leading to the export of the tricarboxylic acid (citrate) to the cytosol and to the accumulation of the dicarboxylic acids (malate and fumarate). While the buildup of NADPH in chloroplasts provides operation of the malate valve leading to establishment of NADH/NAD+ ratios in different cell compartments, the production of NADH by mitochondria drives citrate export by establishing conditions for the operation of the citrate valve. The latter regulates the intercompartmental NADPH/NADP+ ratio and contributes to the biosynthesis of amino acids and other metabolic products during photosynthesis.

Thermodynamic buffering, stable non-equilibrium and establishment of the computable structure of plant metabolism.

The equilibria of coenzyme nucleotides and substrates established in plant cells generate simple rules that govern the plant metabolome and provide optimal conditions for the non-equilibrium fluxes of major metabolic processes such as ATP synthesis, CO2 fixation, and mitochondrial respiration. Fast and abundant enzymes, such as adenylate kinase, carbonic anhydrase or malate dehydrogenase, provide constant substrate flux for these processes. These "buffering" enzymes follow the Michaelis-Menten (MM) kinetics and operate near equilibrium. The non-equilibrium "engine" enzymes, such as ATP synthase, Rubisco or the respiratory complexes, follow the modified version of MM kinetics due to their high concentration and low concentration of their substrates. The equilibrium reactions serve as control gates for the non-equilibrium flux through the engine enzymes establishing the balance of the fluxes of load and consumption of metabolic components. Under the coordinated operation of buffering and engine enzymes, the concentrations of free and Mg-bound adenylates and of free Mg2+ are set, serving as feedback signals from the adenylate metabolome. Those are linked to various cell energetics parameters, including membrane potentials. Also, internal levels of reduced and oxidized pyridine nucleotides are established in the coordinated operation of malate dehydrogenase and respiratory components, with proton concentration as a feedback from pyridine nucleotide pools. Non-coupled pathways of respiration serve to equilibrate the levels of pyridine nucleotides, adenylates, and as a pH stat. This stable non-equilibrium organizes the fluxes of energy spatially and temporally, controlling the rates of major metabolic fluxes that follow thermodynamically and kinetically defined computational principles.