Engineering the expression level of cytosolic nucleoside diphosphate kinase in transgenic Solanum tuberosum roots alters growth,respiration and carbon metabolism

Nucleoside diphosphate kinase (NDPK) is a ubiquitous enzyme that catalyzes the transfer of the γ‐phosphate from a donor nucleoside triphosphate to an acceptor nucleoside diphosphate. In this study we used a targeted metabolomic approach and measurement of physiological parameters to report the effects of the genetic manipulation of cytosolic NDPK (NDPK1) expression on physiology and carbon metabolism in potato (Solanum tuberosum) roots. Sense and antisense NDPK1 constructs were introduced in potato using Agrobacterium rhizogenes to generate a population of root clones displaying a 40‐fold difference in NDPK activity. Root growth, O₂ uptake, flux of carbon between sucrose and CO₂, levels of reactive oxygen species and some tricarboxylic acid cycle intermediates were positively correlated with levels of NDPK1 expression. In addition, NDPK1 levels positively affected UDP‐glucose and cellulose contents. The activation state of ADP‐glucose pyrophosphorylase, a key enzyme in starch synthesis, was higher in antisense roots than in roots overexpressing NDPK1. Further analyses demonstrated that ADP‐glucose pyrophosphorylase was more oxidized, and therefore less active, in sense clones than antisense clones. Consequently, antisense NDPK1 roots accumulated more starch and the starch to cellulose ratio was negatively affected by the level of NDPK1. These data support the idea that modulation of NDPK1 affects the distribution of carbon between starch and cellulose biosynthetic pathways.     

A metabolic bypass of the triosephosphate isomerase reaction

Triosephosphate isomerase (TIM) catalyzes the interconversion of d-glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, an essential step in glycolytic and gluconeogenic metabolism. To uncover promiscuous isomerases embedded within the Escherichia coli genome, we searched for genes capable of restoring growth of a TIM-deficient bacterium under gluconeogenic conditions. Rather than discovering an isomerase, we selected yghZ, a gene encoding a member of the aldo-keto reductase superfamily. Here we show that YghZ catalyzes the stereospecific, NADPH-dependent reduction of l-glyceraldehyde 3-phosphate, the enantiomer of the TIM substrate. This transformation provides an alternate pathway to the formation of dihydroxyacetone phosphate.     

A possible role for pyrophosphate in the coordination of cytosolic and plastidial carbon metabolism within the potato tuber

The early stages of tuber development are characterized by cell division, high metabolic activity, and the predominance of invertase as the sucrose (Suc) cleaving activity. However, during the subsequent phase of starch accumulation the cleavage of Suc occurs primarily by the action of Suc synthase. The mechanism that is responsible for this switch in Suc cleaving activities is currently unknown. One striking difference between the invertase and Suc synthase mediated cleavage of Suc is the direct involvement of inorganic pyrophosphate (PPi) in the latter case. There is presently no convincing explanation of how the PPi required to support this process is generated in potato (Solanum tuberosum) tubers. The major site of PPi production in a maturing potato tubers is likely to be the reaction catalyzed by ADP-glucose pyrophosphorylase, the first committed step of starch biosynthesis in amyloplasts. We present data based on the analysis of the PPi levels in various transgenic plants altered in starch and Suc metabolism that support the hypothesis that PPi produced in the plastid is used to support cytosolic Suc breakdown and that PPi is an important coordinator of cytosolic and plastidial metabolism in potato tubers.     

Duplicated plastid and triplicated cytosolic isozymes of triosephosphate isomerase in maize (Zea mays L.)

We studied electrophoretic variation and inheritance of triosephosphate isomerase (TPI) isozymes in maize (Zea mays L.). In contrast to most diploid plants, in maize, TPI exists as multiple isozymes in both the plastid and cytosolic subcellular compartments. Phenotypes result from the overlay of two independent sets of isozymes and allozymes, representing the plastid (encoded by the nuclear genes Tpi1 and Tpi2) and cytosolic (encoded by Tpi3, Tpi4, and Tpi5) systems. All possible intragenic and intergenic dimeric enzymes are formed between polypeptides within each subcellular compartment. No heterodimers are formed between plastid and cytosolic polypeptides. Extensive surveys of accessions of land races and inbred lines revealed 22 allelic variants for the five loci. Most alleles have been formally validated by segregation analysis. We describe two null alleles at Tpi4, distinguished by their relative abilities to form intergenic heterodimers with polypeptides specified by Tpi3 and Tpi5. Linkage analyses and crosses with B-A translocation stocks were effective in determining the chromosome locations of all five loci. Duplicated genes for both the plastid and cytosolic isozymes were localized to genomic regions that possess numerous other redundant sequences. We placed Tpi1 on the long arm of chromosome 7, approximately 23 centimorgans (cM) distal to g11; we localized its duplicate--Tpi2--17 cM distal to v4 on the long arm of chromosome 2. The triplicate loci encoding cytosolic TPIs reside on chromosomes 3 and 8. Tpi4 is approximately equidistant (11 cM) from d1 and Lg3, near the centromere of chromosome 3. Tpi3 and Tpi5 are located on distal ends of the most poorly marked maize chromosome; Tpi3 is 29 cM distal to Idh 1 on 8L, and Tpi5 is on 8S or near the centromere on 8L. In contrast to most duplicated maize sequences, which often occur in parallel linkages on different chromosomes, Tpi3 and Tpi5 provide an example of intrachromosomal gene duplication. Several of the Tpi loci are located in sparsely mapped regions of the genome, and Tpi1 is the first isozyme marker for chromosome 7.     

Computational modeling of the catalytic reaction in triosephosphate isomerase

We present a comprehensive analysis of the catalytic cycle of the enzyme triosephosphate isomerase (TIM), including both the reactive chemistry and the catalytic loop and side-chain motions. Combining accurate mixed quantum mechanics/molecular mechanics (QM/MM) and protein structure prediction methods, we have modeled both the structural and chemical aspects of the reversible isomerization of dihydroxyacetone phosphate (DHAP) to d-glyceraldehyde 3-phosphate (GAP), for which there is a wealth of experimental data. The conjunction of this novel computational approach with the use of the recent near-atomic resolution TIM-DHAP Michaelis complex PDB structure, 1NEY.pdb, has enabled us to obtain robust qualitative and, where available, quantitative agreement with a wide range of experimental data. Among the principal conclusions that we are able to draw are the importance of the monoanionic (as opposed to dianioic) form of the substrate phosphate group in the catalytic cycle, detailed positioning and energetics of the key catalytic residues in the active-site, the flexible nature of Glu165, which favors its direct involvement in the formation of the enediol intermediate, energetics of the open and closed form of the catalytic loop region in the presence and absence of substrate, and quantitative reproduction of various experimentally measured reaction rates, typically to within approximately 1 kcal/mol. Our results are consistent with the available experimental data, and provide an initial picture as to why loop opening when GAP is the product has a higher barrier than when DHAP is the product.     

Proton diffusion in the active site of triosephosphate isomerase

The current model for hydrogen flow in the aldose-ketose isomerases is probably incorrect. Enzymes of this class are characterized by both hydrogen transfer and proton exchange in the interconversion of substrate and product. The transfer is believed to be due to the action of a unique basic residue in the active site. Exchange is presumed to occur by dissociation of the abstracted proton and reassociation from the medium prior to its transfer to the intermediate enediol on the way to product. Dissociation of a necessary proton from the intermediate state imposes limits on the overall catalytic rate depending on the pKa of the protonated base and the pH of the medium. A case in point is triose-P isomerase (TIM), where kcat is approximately 10(4) s-1. T-Labeled substrate is found to lose approximately 95% of its T to the medium when totally converted to product. Although the active site base is believed to be a glutamate of pKa = 3.9, the pH dependence of maximum velocity is known to be flat up to pH 10. The loss of hydrogen required to form product as indicated by isotope exchange must be restored completely at this high pH, requiring a base of very high pKa, or there must be some other explanation for the loss of isotope. The present study demonstrates the existence of a single proton on human and rabbit TIM and three protons on yeast TIM that rapidly exchange with the abstracted proton at the E.enediol state internal exchange. Exchange with the medium external exchange occurs from the enzyme after substrate or product has dissociated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antisense inhibition of cytosolic NADP-dependent isocitrate dehydrogenase in transgenic potato plants

Cytosolic NADP-dependent isocitrate dehydrogenase (cyt-NADP-ICDH; EC 1.1.1.42) has been suggested to play a major role in the production of 2-oxoglutarate, an important precursor for amino acid synthesis. Using an antisense RNA approach under the control of the cauliflower mosaic virus 35S promoter, transgenic potato plants were created in which NADP-ICDH activity was reduced to 8% of the wild-type level in leaves. Residual activity was almost completely due to mitochondrial and chloroplastic NADP-ICDH isoforms. Activity staining after non-denaturing polyacrylamide gel electrophoresis revealed the complete absence of a major activity band in leaves of antisense plants. No differences in growth or development, including flower formation and tuber yield, were observed between transgenic and wild-type plants. Photosynthesis and respiration were also unchanged. Levels of amino acids were the same in wild-type and cyt-NADP-ICDH antisense plants, even when accumulation of amino acids was induced by incubation of detached leaves in tap water in the dark (`induced senescence'). Consistent with a reduction in NADP-ICDH activity, however, were slight increases in the levels of isocitrate (up to 2.5-fold) and citrate (up to 2-fold). 2-Oxoglutarate was not reduced. Our data indicate that potato plants can cope with a severe reduction in cyt-NADP-ICDH activity without major shifts in growth and metabolism. Received: 28 July 1997 / Accepted: 3 November 1997     

Structural basis for the modulation of plant cytosolic triosephosphate isomerase activity by mimicry of redox‐based modifications

Reactive oxidative species (ROS) and S‐glutathionylation modulate the activity of plant cytosolic triosephosphate isomerases (cTPI). Arabidopsis thaliana cTPI (AtcTPI) is subject of redox regulation at two reactive cysteines that function as thiol switches. Here we investigate the role of these residues, AtcTPI‐Cys13 and At‐Cys218, by substituting them with aspartic acid that mimics the irreversible oxidation of cysteine to sulfinic acid and with amino acids that mimic thiol conjugation. Crystallographic studies show that mimicking AtcTPI‐Cys13 oxidation promotes the formation of inactive monomers by reposition residue Phe75 of the neighboring subunit, into a conformation that destabilizes the dimer interface. Mutations in residue AtcTPI‐Cys218 to Asp, Lys, or Tyr generate TPI variants with a decreased enzymatic activity by creating structural modifications in two loops (loop 7 and loop 6) whose integrity is necessary to assemble the active site. In contrast with mutations in residue AtcTPI‐Cys13, mutations in AtcTPI‐Cys218 do not alter the dimeric nature of AtcTPI. Therefore, modifications of residues AtcTPI‐Cys13 and AtcTPI‐Cys218 modulate AtcTPI activity by inducing the formation of inactive monomers and by altering the active site of the dimeric enzyme, respectively. The identity of residue AtcTPI‐Cys218 is conserved in the majority of plant cytosolic TPIs, this conservation and its solvent‐exposed localization make it the most probable target for TPI regulation upon oxidative damage by reactive oxygen species. Our data reveal the structural mechanisms by which S‐glutathionylation protects AtcTPI from irreversible chemical modifications and re‐routes carbon metabolism to the pentose phosphate pathway to decrease oxidative stress.     

A kinetic approach to the thermal inactivation of an immobilized triosephosphate isomerase

The process of thermal inactivation of triosephosphate isomerase covalently attached to a silica-based support activated with p-benzoquinone was found to be a complex one. At 50 degrees C, a characteristic activation preceding the thermal inactivation was observed. Following the intramolecular changes caused by heat, the values of K(M) and V(max) were determined during the activation. It was presumed that the complex thermal inactivation kinetics reflects the microheterogeneity of the immobilized enzyme molecules. The phosphate ion proved to be a better stabilizer than the substrate. (c) 1992 John Wiley & Sons, Inc.     

Inhibition of triosephosphate isomerase by phosphoenolpyruvate in the feedback-regulation of glycolysis

The inhibition of triosephosphate isomerase (TPI) in glycolysis by the pyruvate kinase (PK) substrate phosphoenolpyruvate (PEP) results in a newly discovered feedback loop that counters oxidative stress in cancer and actively respiring cells. The mechanism underlying this inhibition is illuminated by the co-crystal structure of TPI with bound PEP at 1.6 Å resolution, and by mutational studies guided by the crystallographic results. PEP is bound to the catalytic pocket of TPI and occludes substrate, which accounts for the observation that PEP competitively inhibits the interconversion of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. Replacing an isoleucine residue located in the catalytic pocket of TPI with valine or threonine altered binding of substrates and PEP, reducing TPI activity in vitro and in vivo. Confirming a TPI-mediated activation of the pentose phosphate pathway (PPP), transgenic yeast cells expressing these TPI mutations accumulate greater levels of PPP intermediates and have altered stress resistance, mimicking the activation of the PK–TPI feedback loop. These results support a model in which glycolytic regulation requires direct catalytic inhibition of TPI by the pyruvate kinase substrate PEP, mediating a protective metabolic self-reconfiguration of central metabolism under conditions of oxidative stress.     

Electrophilic catalysis in triosephosphate isomerase: the role of histidine-95

Electrophilic catalysis by histidine-95 in triosephosphate isomerase has been probed by using Fourier transform infrared spectroscopy and X-ray crystallography. The carbonyl stretching frequency of dihydroxyacetone phosphate bound to the wild-type enzyme is known to be 19 cm-1 lower (at 1713 cm-1) than that of dihydroxyacetone phosphate free in solution (at 1732 cm-1), and this decrease in stretching frequency has been ascribed to an enzymic electrophile that polarizes the substrate carbonyl group toward the transition state for the enolization. Infrared spectra of substrate bound to two site-directed mutants of yeast triosephosphate isomerase in which histidine-95 has been changed to glutamine or to asparagine show unperturbed carbonyl stretching frequencies between 1732 and 1742 cm-1. The lack of carbonyl polarization when histidine-95 is removed suggests that histidine-95 is indeed the catalytic electrophile, at least for dihydroxyacetone phosphate. Kinetic studies of the glutamine mutant (H95Q) have shown that the enzyme follows a subtly different mechanism of proton transfers involving only a single acid-base catalytic group. These findings suggest an additional role for histidine-95 as a general acid-base catalyst in the wild-type enzyme. The X-ray crystal structure of the H95Q mutant with an intermediate analogue, phosphoglycolohydroxamate, bound at the active site has been solved to 2.8-A resolution, and this structure clearly implicates glutamate-165, the catalytic base in the wild-type isomerase, as the sole acid-base catalyst for the mutant enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exogenous auxin affects ascorbate metabolism in roots of tomato seedlings

Ascorbate levels and redox states, as well as the activities of the enzymes of ascorbate metabolism, were analyzed in roots of tomato seedlings during the culture on a medium supplemented with auxin and compared to the control cultured on an auxin-free medium. Biochemical parameters were determined separately in the distal part of the root where the inhibitory effect of auxin on root elongation growth is observed and in the proximal half on the organ which reacts to auxin treatment with increased lateral root proliferation. ASC peroxidase activity was found to be stimulated by auxin treatment in the lateral-root forming part of the root. This effect was not observed in the distal part of the organ. On the other hand, ASC oxidase activity was raised by auxin exclusively in the distal part of the root. An inhibitory effect of auxin supplementation to the medium on ASC—reducing enzymes was observed. The dehydroascorbate reductase activity was found to be inhibited by auxin only in the proximal part, while the activity of monodehydroascorbate reductase in both, the proximal and distal parts of the root. Ascorbate content increased in roots during culture irrespective of the presence of auxin. However, auxin treatment resulted in higher DHA levels and more significant participation of DHA in the total ascorbate pool when compared to the control grown on the auxin-free medium. Similar to auxin, adding DHA to the culture medium stimulated lateral root formation and inhibited primary root elongation. In contrast to DHA, ASC treatment affected significantly neither lateral root formation nor primary root growth and partly reversed the stimulatory effect of IAA on root formation and the inhibitory effect on root elongation. These results suggest that auxin induced changes in ascorbate metabolism may be involved in developmental reactions in tomato roots.     

The role of water in the catalytic efficiency of triosephosphate isomerase

The structural basis for the effect of the S96P mutation in chicken triosephosphate isomerase (cTIM) has been analyzed using a combination of X-ray crystallography and Fourier transform infrared spectroscopy. The X-ray structure is that of the enzyme complexed with phosphoglycolohydroxamate (PGH), an intermediate analogue, solved at a resolution of 1.9 A. The S96P mutation was identified as a second-site reverent when catalytically crippled mutants, E165D and H95N, were subjected to random mutagenesis. The presence of the second mutation leads to enhanced activity over the single mutation. However, the effect of the S96P mutation alone is to decrease the catalytic efficiency of the enzyme. The crystal structures of the S96P double mutants show that this bulky proline side chain alters the water structure within the active-site cavity (E165D; ref 1) and prevents nonproductive binding conformations of the substrate (H95N; ref 2). Comparison of the S96P single mutant structure with those of the wild-type cTIM, those of the single mutants (E165D and H95N), and those of the double mutants (E165D/S96P and H95N/S96P) begins to address the role of the conserved serine residue at this position. The results indicate that the residue positions the catalytic base E165 optimally for polarization of the substrate carbonyl, thereby aiding in proton abstraction. In addition, this residue is involved in positioning critical water molecules, thereby affecting the way in which water structure influences activity.     

Transport and metabolism of raffinose family oligosaccharides in transgenic potato

Raffinose family oligosaccharides (RFOs) are involved in the storage and transport of carbon and serve as compatible solutes for protection against abiotic stresses like drought or cold. RFOs are usually transported in plant species that load sugars symplastically into the phloem. Loading probably occurs by a polymer trapping mechanism which establishes a concentration gradient of assimilates between the mesophyll and the vasculature. Transgenic approaches have demonstrated phloem transport of small molecules produced in the companion cells of apoplastic loading species, but these molecules have been non-native transport substances to plants. In this study, transgenic potato plants with constitutive or companion cell specific overexpression of galactinol synthase (GS) or GS plus raffinose synthase (RS) are characterized, which together provide new insights into the metabolism and transport of RFOs in plants. It is demonstrated that raffinose and galactinol are both transported in the phloem and that, whilst the effect of GS overexpression is promoter-independent, that of RS is dependent on the promoter used. The presence of significant amounts of galactinol in the phloem is shown and also that transgenic potato is unable to transport large amounts of raffinose despite high RS expression and substrate concentrations. These data indicate that there may be additional features of intermediary cells, the specialized companion cells of RFO transporting plants, required for significant RFO synthesis and transport that are currently not well-understood.     

Heterologous expression of AtPAP2 in transgenic potato influences carbon metabolism and tuber development

Changes in carbon flow and sink/source activities can affect floral, architectural, and reproductive traits of plants. In potato, overexpression (OE) of the purple acid phosphatase 2 of Arabidopsis (AtPAP2) resulted in earlier flowering, faster growth rate, increased tubers and tuber starch content, and higher photosynthesis rate. There was a significant change in sucrose, glucose and fructose levels in leaves, phloem and sink biomass of the OE lines, consistent with an increased expression of sucrose transporter 1 (StSUT1). Furthermore, the expression levels and enzyme activity of sucrose-phosphate synthase (SPS) were also significantly increased in the OE lines. These findings strongly suggest that higher carbon supply from the source and improved sink strength can improve potato tuber yield.     

Analyzing the effect of decreasing cytosolic triosephosphate isomerase on Solanum tuberosum hairy root cells using a kinetic–metabolic model

A kinetic–metabolic model of Solanum tuberosum hairy roots is presented in the interest of understanding the effect on the plant cell metabolism of a 90% decrease in cytosolic triosephosphate isomerase (cTPI, EC 5.3.1.1) expression by antisense RNA. The model considers major metabolic pathways including glycolysis, pentose phosphate pathway, and TCA cycle, as well as anabolic reactions leading to lipids, nucleic acids, amino acids, and structural hexoses synthesis. Measurements were taken from shake flask cultures for six extracellular nutrients (sucrose, fructose, glucose, ammonia, nitrate, and inorganic phosphate) and 15 intracellular compounds including sugar phosphates (G6P, F6P, R5P, E4P) and organic acids (PYR, aKG, SUCC, FUM, MAL) and the six nutrients. From model simulations and experimental data it can be noted that plant cell metabolism redistributes metabolic fluxes to compensate for the cTPI decrease, leading to modifications in metabolites levels. Antisense roots showed increased exchanges between the pentose phosphate pathway and the glycolysis, an increased oxygen uptake and growth rate. Biotechnol. Bioeng. 2013; 110: 924–935. © 2012 Wiley Periodicals, Inc.     

Bacterial inoculation of maize affects carbon allocation to roots and carbon turnover in the rhizosphere

To assess the influence of bacteria inoculation on carbon flow through maize plant and rhizosphere,¹⁴C allocation after¹⁴CO₂ application to shoots over a 5-day period was determined. Plants were grown on C- and N-free quartz sand in two-compartment pots, separating root and shoot space. While one treatment remained uninoculated, treatments two and three were inoculated withPantoea agglomerans (D5/23) andPseudomonas fluorescens (Ps I A12), respectively, five days after planting. Bacterial inoculation had profound impacts on carbon distribution within the system. Root/rhizosphere respiration was increased and more carbon was allocated to roots of plants being inoculated. After five days of¹⁴CO₂ application, more ethanol-soluble substances were found in roots of inoculated treatments and lower rhizodeposition indicated intensive C turnover in the rhizosphere. In both inoculated treatments the intensity of photosynthesis measured as net-CO₂-assimilation rates were increased when compared to the uninoculated plants. However, high C turnover in the rhizosphere reduced shoot growth of D5/23 inoculated plants, with no effect on shoot growth of Ps I A12 inoculated plants. A separation of labeled compounds in roots and rhizodeposition revealed that neutral substances (sugars) constituted the largest fraction. The relative fractions of sugars, amino acids and organic acids in roots and rhizodeposition suggest that amino acid exudation was particularly stimulated by bacterial inoculation and that turnover of this substance group is high in the rhizosphere.