William C. Plaxton

The Functional Organization and Control of Plant Respiration

The respiratory pathways of glycolysis, the tricarboxylic acid (TCA) cycle, and mitochondrial electron transport chain (miETC) are central features of carbon metabolism and bioenergetics in aerobic organisms. Respiration is essential for growth, maintenance, and carbon balance of all plant cells. Although the majority of respiratory enzymes are common to all organisms, plant respiration has evolved as a complex metabolic network endowed with a wide variety of unique characteristics. Plants have the option of employing alternative enzymes that bypass several of the conventional steps in cytosolic glycolysis, the TCA cycle, and miETC. The extent and conditions under which these bypasses operate is the subject of intensive research. The highly flexible nature of respiratory metabolism in plants has likely evolved in response to the crucial biosynthetic role played by respiration beyond its role in ATP generation; both functions must proceed if plants are to survive under varying and often stressful environmental and nutritional conditions. Additional complexity arises due to the existence of tissue- and/or developmental-specific isozymes of many plant respiratory enzymes, as well as the extensive interactions between photosynthesis and respiration, and plastidic, cytosolic, and mitochondrial metabolism in general. Recent progress in biochemistry, physiology, cell biology, genomics, transcriptomics, proteomics, metabolomics, and in vivo flux analyses have resulted in exciting new insights into many aspects of plant respiratory metabolism. Experiments on transgenic or mutant plants possessing significantly elevated or reduced levels of respiratory enzymes are augmenting our understanding of the functions, organization, and control of plant respiration. Metabolic engineering of plant respiration is of significant practical interest as it provides both an important approach to enhancing crop yields, as well as a potential mechanism for mitigating global climate change due to elevated atmospheric CO 2 levels. Referee: Dr. Greg C. Vanlerberghe, University of Toronto at Scarborough, Department of Life Sciences and Department of Botany, 1265 Military Trail, Scarborough, ON Canada M1C 1A4

Metabolic Adaptations of Plant Respiration to Nutritional Phosphate Deprivation

Plants respond adaptively to orthophosphate (Pi) deprivation through the induction of alternative pathways of glycolysis and mitochondrial electron transport. These respiratory bypasses allow respiration to proceed in Pi-deficient plant cells because they negate the necessity for adenylates and Pi, both pools of which are severely depressed following nutritional Pi starvation.

Opportunities for improving phosphorus‐use efficiency in crop plants

Limitation of grain crop productivity by phosphorus (P) is widespread and will probably increase in the future. Enhanced P efficiency can be achieved by improved uptake of phosphate from soil (P-acquisition efficiency) and by improved productivity per unit P taken up (P-use efficiency). This review focuses on improved P-use efficiency, which can be achieved by plants that have overall lower P concentrations, and by optimal distribution and redistribution of P in the plant allowing maximum growth and biomass allocation to harvestable plant parts. Significant decreases in plant P pools may be possible, for example, through reductions of superfluous ribosomal RNA and replacement of phospholipids by sulfolipids and galactolipids. Improvements in P distribution within the plant may be possible by increased remobilization from tissues that no longer need it (e.g. senescing leaves) and reduced partitioning of P to developing grains. Such changes would prolong and enhance the productive use of P in photosynthesis and have nutritional and environmental benefits. Research considering physiological, metabolic, molecular biological, genetic and phylogenetic aspects of P-use efficiency is urgently needed to allow significant progress to be made in our understanding of this complex trait.

Metabolic Adaptations of Phosphate-Starved Plants

Orthophosphate (Pi) is an essential macronutrient that plays a central role in virtually all major metabolic processes in plants, particularly photosynthesis and respiration. Many metabolites are Pi monoesters, whereas the phosphoanhydride bonds of compounds such as ATP function to transfer energy

PHOSPHITE (PHOSPHOROUS ACID): ITS RELEVANCE IN THE ENVIRONMENT AND AGRICULTURE AND INFLUENCE ON PLANT PHOSPHATE STARVATION RESPONSE

Phosphites (H2PO3 −; Phi) are alkali metal salts of phosphorous acid [HPO(OH)2] that are being widely marketed either as an agricultural fungicide or as a superior source of plant phosphorus (P) nutrition. Published research conclusively indicates that Phi functions as an effective control agent for a number of crop diseases caused by various species of pathogenic pseudo fungi belonging to the genus Phytophthora. However, evidence that Phi can be directly used by plants as a sole source of nutritional P is lacking. When Phi is administered in such as way as to allow it to come into contact with bacteria, either associated with plant root systems or in the soil, then the oxidation of Phi to phosphate (HPO4 2−; Pi) may take place. By this indirect method Phi could thus become available to the plant as a P nutrient. The rates at which this occurs are slow, taking months or as much as a year, depending on the soil type. Phi is not without direct effects on plants itself, as Phi concentrations comparable to those required to control plant infection by pathogenic Phytophthora, or to restrict Phytophthora growth in sterile culture, are extremely phytotoxic to Pi-deprived, but not Pi-fertilized, plants. This is because Phi treatment negates the acclimation of plants to Pi deficiency by disrupting the induction of enzymes (e.g., acid phosphatase) and transporters (e.g., high-affinity plasmalemma Pi translocator) characteristic of their Pi starvation response. Thus, Phi intensifies the deleterious effects of P-deficiency by ‘tricking’ Pi-deprived plant cells into sensing that they are Pi-sufficient, when, in fact, their cellular Pi content is extremely low. The Phi anion appears to effectively obstruct the signal transduction pathway by which plants (and yeast) perceive and respond to Pi deprivation at the molecular level. The review concludes by citing concerns and recommendations regarding the significant input of Phi into food products and the environment that arises from its extensive use in agriculture and industry.

The remarkable diversity of plant PEPC (phosphoenolpyruvate carboxylase): recent insights into the physiological functions and post-translational controls of non-photosynthetic PEPCs.

PEPC [PEP (phosphoenolpyruvate) carboxylase] is a tightly controlled enzyme located at the core of plant C-metabolism that catalyses the irreversible β-carboxylation of PEP to form oxaloacetate and Pi. The critical role of PEPC in assimilating atmospheric CO(2) during C(4) and Crassulacean acid metabolism photosynthesis has been studied extensively. PEPC also fulfils a broad spectrum of non-photosynthetic functions, particularly the anaplerotic replenishment of tricarboxylic acid cycle intermediates consumed during biosynthesis and nitrogen assimilation. An impressive array of strategies has evolved to co-ordinate in vivo PEPC activity with cellular demands for C(4)-C(6) carboxylic acids. To achieve its diverse roles and complex regulation, PEPC belongs to a small multigene family encoding several closely related PTPCs (plant-type PEPCs), along with a distantly related BTPC (bacterial-type PEPC). PTPC genes encode ~110-kDa polypeptides containing conserved serine-phosphorylation and lysine-mono-ubiquitination sites, and typically exist as homotetrameric Class-1 PEPCs. In contrast, BTPC genes encode larger ~117-kDa polypeptides owing to a unique intrinsically disordered domain that mediates BTPCs tight interaction with co-expressed PTPC subunits. This association results in the formation of unusual ~900-kDa Class-2 PEPC hetero-octameric complexes that are desensitized to allosteric effectors. BTPC is a catalytic and regulatory subunit of Class-2 PEPC that is subject to multi-site regulatory phosphorylation in vivo. The interaction between divergent PEPC polypeptides within Class-2 PEPCs adds another layer of complexity to the evolution, physiological functions and metabolic control of this essential CO(2)-fixing plant enzyme. The present review summarizes exciting developments concerning the functions, post-translational controls and subcellular location of plant PTPC and BTPC isoenzymes.

The Fungicide Phosphonate Disrupts the Phosphate-Starvation Response in Brassica nigra Seedlings

The development of Brassica nigra seedlings over 20 d of growth was disrupted by the fungicide phosphonate (Phi) in a manner inversely correlated with nutritional inorganic phosphate (Pi) levels. The growth of Pi-sufficient (1.25 mM Pi) seedlings was suppressed when 10, but not 5, mM Phi was added to the nutrient medium. In contrast, the fresh weights and root:shoot ratios of Pi-limited (0.15 mM) seedlings were significantly reduced at 1.5 mM Phi, and they progressively declined to about 40% of control values as medium Phi concentration was increased to 10 mM. Intracellular Pi levels generally decreased in Phi-treated seedlings, and Phi accumulated in leaves and roots to levels up to 6- and 16-fold that of Pi in Pi-sufficient and Pi-limited plants, respectively. Extractable activities of the Pi-starvation-inducible enzymes phosphoenolpyruvate phosphatase and inorganic pyrophosphate-dependent phosphofructokinase were unaltered in Pi-sufficient seedlings grown on 5 or 10 mM Phi. However, when Pi-limited seedlings were grown on 1.5 to 10 mM Phi (a) the induction of phosphoenolpyruvate phosphatase and inorganic pyrophosphate-dependent phosphofructokinase activities by Pi limitation was reduced by 40 to 90%, whereas (b) soluble protein concentrations and the activities of the ATP-dependent phosphofructokinase and pyruvate kinase were unaffacted. It is concluded that Phi specifically interrupts processes involved in regulation of the Pi-starvation response in B. nigra.

Disruption of the phosphate-starvation response of oilseed rape suspension cells by the fungicide phosphonate

Abstract. The influence of the anti-fungal agent phosphonate (Phi) on the response of oilseed rape (Brassica napus L. cv. Jet Neuf ) cell suspensions to inorganic phosphate (Pi) starvation was examined. Subculture of the cells for 7 d in the absence of Pi increased acid phosphatase (APase; EC 3.1.3.2) and pyrophosphate (PPi)-dependent phosphofructokinase (PFP; EC 2.7.1.90) activities by 4.5- and 2.8-fold, respectively, and led to a 19-fold increase in Vmax and a 14-fold decrease in Km (Pi) for Pi uptake. Addition of 2 mM Phi to the nutrient media caused dramatic reductions in the growth and Pi content of the Pi-starved, but not Pi-sufficient cells, and largely abolished the Pi-starvation-dependent induction of PFP, APase, and the high-affinity plasmalemma Pi translocator. Immunoblotting indicated the cells contain three APase isoforms that are synthesized de novo following Pi stress, and that Phi treatment represses this process. Phosphonate treatment of Pi-starved cells significantly altered the relative extent of in-vivo 32P-labelling of polypeptides having Mrs of 66, 55, 45 and 40 kDa. However, Phi had no effect on the total adenylate pool of Pi-starved cells which was about 32% lower than that of Pi-sufficient cells by day 7. Soluble protein levels, and activities of pyruvate kinase (EC 2.7.1.40) and ATP-dependent phosphofructokinase (EC 2.7.1.11) were unaffected by Pi starvation and/or Phi treatment. The effects of Phi on the growth, and APase and PFP activities of Pi-starved B. napus seedlings were similar to those observed in the suspension cells. The results are consistent with the hypothesis that a primary site of Phi action in higher plants is at the level of the signal transduction chain by which plants perceive and respond to Pi stress at the molecular level.

Biochemical and molecular characterization of AtPAP26, a vacuolar purple acid phosphatase up-regulated in phosphate-deprived arabidopsis suspension cells and seedlings

A vacuolar acid phosphatase (APase) that accumulates during phosphate (Pi) starvation of Arabidopsis (Arabidopsis thaliana) suspension cells was purified to homogeneity. The final preparation is a purple APase (PAP), as it exhibited a pink color in solution (Amax = 520 nm). It exists as a 100-kD homodimer composed of 55-kD glycosylated subunits that cross-reacted with an anti-(tomato intracellular PAP)-IgG. BLAST analysis of its 23-amino acid N-terminal sequence revealed that this PAP is encoded by At5g34850 (AtPAP26; one of 29 PAP genes in Arabidopsis) and that a 30-amino acid signal peptide is cleaved from the AtPAP26 preprotein during its translocation into the vacuole. AtPAP26 displays much stronger sequence similarity to orthologs from other plants than to other Arabidopsis PAPs. AtPAP26 exhibited optimal activity at pH 5.6 and broad substrate selectivity. The 5-fold increase in APase activity that occurred in Pi-deprived cells was paralleled by a similar increase in the amount of a 55-kD anti-(tomato PAP or AtPAP26)-IgG immunoreactive polypeptide and a >30-fold reduction in intracellular free Pi concentration. Semiquantitative reverse transcription-PCR indicated that Pi-sufficient, Pi-starved, and Pi-resupplied cells contain similar amounts of AtPAP26 transcripts. Thus, transcriptional controls appear to exert little influence on AtPAP26 levels, relative to translational and/or proteolytic controls. APase activity and AtPAP26 protein levels were also up-regulated in shoots and roots of Pi-deprived Arabidopsis seedlings. We hypothesize that AtPAP26 recycles Pi from intracellular P metabolites in Pi-starved Arabidopsis. As AtPAP26 also exhibited alkaline peroxidase activity, a potential additional role in the metabolism of reactive oxygen species is discussed.

The Dual-Targeted Purple Acid Phosphatase Isozyme AtPAP26 Is Essential for Efficient Acclimation of Arabidopsis to Nutritional Phosphate Deprivation

Induction of intracellular and secreted acid phosphatases (APases) is a widespread response of orthophosphate (Pi)-starved (−Pi) plants. APases catalyze Pi hydrolysis from a broad range of phosphomonoesters at an acidic pH. The largest class of nonspecific plant APases is comprised of the purple APases (PAPs). Although the biochemical properties, subcellular location, and expression of several plant PAPs have been described, their physiological functions have not been fully resolved. Recent biochemical studies indicated that AtPAP26, one of 29 PAPs encoded by the Arabidopsis (Arabidopsis thaliana) genome, is the predominant intracellular APase, as well as a major secreted APase isozyme up-regulated by −Pi Arabidopsis. An atpap26 T-DNA insertion mutant lacking AtPAP26 transcripts and 55-kD immunoreactive AtPAP26 polypeptides exhibited: (1) 9- and 5-fold lower shoot and root APase activity, respectively, which did not change in response to Pi starvation, (2) a 40% decrease in secreted APase activity during Pi deprivation, (3) 35% and 50% reductions in free and total Pi concentration, respectively, as well as 5-fold higher anthocyanin levels in shoots of soil-grown −Pi plants, and (4) impaired shoot and root development when subjected to Pi deficiency. By contrast, no deleterious influence of AtPAP26 loss of function occurred under Pi-replete conditions, or during nitrogen or potassium-limited growth, or oxidative stress. Transient expression of AtPAP26-mCherry in Arabidopsis suspension cells verified that AtPAP26 is targeted to the cell vacuole. Our results confirm that AtPAP26 is a principal contributor to Pi stress-inducible APase activity, and that it plays an important role in the Pi metabolism of −Pi Arabidopsis.