Milium lendigerum

The metabolism of potato (Solanum tuberosum) roots constitutively over-and underexpressing hexokinase (HK, EC 2.7.1.1) was examined. An 11-fold variation in HK activity resulted in altered root growth, with antisense roots growing better than sense roots. Quantification of sugars, organic acids and amino acids in transgenic roots demonstrated that the manipulation of HK activity had very little effect on the intracellular pools of these metabolites. However, adenylate and free Pi levels were negatively affected by an increase in HK activity. The flux control coefficient of HK over the phosphorylation of glucose was measured for the first time in plants. Its value varied with HK level. It reached 1.71 at or below normal HK activity value and was much lower (0.32) at very high HK levels. Measurements of glycolytic flux and O-2 uptake rates demonstrated that the differences in glucose phosphorylation did not affect significantly glycolytic and respiratory metabolism. We hypothesized that these results could be explained by the existence of a futile cycle between the pools of hexose-Ps and carbohydrates. This view is supported by several lines of evidence. Firstly, activities of enzymes capable of catalyzing these reactions were detected in roots, including a hexose-P phosphatase. Secondly, metabolic tracer experiments using C-14-glucose as precursor showed the formation of C-14-fructose and C-14-sucrose. We conclude that futile cycling of hexose-P could be partially responsible for the differences in energetic status in roots with high and low HK activity and possibly cause the observed alterations in growth in transgenic roots. The involvement of HK and futile cycles in the control of glucose-6P metabolism is discussed

Total extractible HK activity and protein levels in transgenic potato root clones.

<p>Specific activities were measured with Glc (GK activity, a) and Fru (FK activity, b) in antisense (AS), control (Ctrl) and sense (S) clones. Values are means ± SE from three to ten separate extractions. Immunoblot analysis (c) of HK protein levels in potato root clones. Immunodetection was carried out with affinity-purified IgGs raised against recombinant ScHK2. Each lane was loaded with 6.5 µg of protein. The names of the clones at the bottom of the bar graph also identify the lanes in panel (c).</p

Pi pools and intracellular pHs.

<p>Pi quantification was done using a colorimetric assay. Intracellular Pi pools and pHs were measured by <i>in vivo</i>³¹P NMR. Root cultures (0.5–0.8 g FW) were inserted into a 10 mm NMR tube perfused with oxygenated MS medium. Spectra were recorded over periods of 1 h on a Varian Unity Inova 400 MHz NMR spectrometer.</p

Distribution of radioactivity in different fractions after feeding two antisense (AS213, AS301), one control (Ctrl3) and two sense (S101, S111) root clones with [U-¹⁴C]Glc.

<p>Following incubation with the radioactive tracer, radioactive CO₂ trapped in KOH was counted. Roots were extracted in 80% (v/v) ethanol and the extract fractionated into neutral, acidic and basic fractions. Aliquots of each fraction were counted. The radioactivity in the insoluble fraction represents the ethanol insoluble residue. Data are mean ± SE of 5 to 7 labeling experiments.</p

Distribution of radioactivity in different fractions after feeding an antisense (AS213), a control (Ctrl3) and a sense (S101) root clones with [U-¹⁴C]Suc.

<p>Following incubation with the radioactive tracer, radioactive CO₂ trapped in KOH was counted. Roots were extracted in 80% (v/v) ethanol and the extract fractionated into neutral, acidic and basic fractions. Aliquots of each fraction were counted. The radioactivity in the insoluble fraction represents the ethanol insoluble residue. Data are mean ± SE of 3 to 4 labeling experiments.</p

Amino acid contents in potato roots with altered HK specific activities.

<p>Levels of Glu (a), Gln (b), Arg (c), γ-aminobutyric acid (GABA) (d), His (e), Pro (f), Trp (g), Tyr (h), Phe (i), Ala (j), Leu (k), Val (l), Ile (m), Lys (n), Thr (o), Asp (p), Met (q), Ser (r) and Gly (s) were determined in root clones displaying a range of HK activities. Symbols used are: black circles, antisense; grey circles, control and grey triangles, sense clones. Y values are means ± SE from seven to twelve separate experiments, X values are from Fig. 1.</p

Measurements of metabolites, adenylates, and O₂ uptake in antisense (AS213, AS301), control (Ctrl3) and sense (S101, S111) clones.

<p>Data are mean ± SE for 9 to 20 independent measurements. Values marked with a particular superscript letter denote that they are significantly different from values marked with a different superscript letter on the same line (Student’s <i>t</i>-test, <i>P</i><0.05). Values that are not marked with a superscript letter are not significantly different between antisense, control and sense clones.</p

Effects of altered HK activity levels on potato root growth.

<p>a Representative pictures of antisense (AS301), control (Ctrl3) and sense (S107) clones grown for 14 d from a single 0.7 cm-long root tip. Total root length (b), tip number (c) and mean diameter (d) were assessed for antisense (black circles), control (grey circles) and sense (grey triangles) clones after 14 d of growth on solid MS medium. Y values are means ± SE of quadruplicates from two independent experiments, X values are from Fig. 1.</p

Specific activities of primary metabolism enzymes assayed in two antisense (AS213, AS301), one control (Ctrl3) and two sense (S101, S111) clones.

<p>Data are mean (mU mg⁻¹ protein) ± SE of quadruplicates from four to five independent experiments.</p

Organic acid contents in potato roots with altered HK specific activities.

<p>Levels of malate (a), isocitrate (b), fumarate (c) and shikimate (d) were quantified in root clones displaying a range of HK activities. Symbols used are: black circles, antisense; grey circles, control and grey triangles, sense clones.Y values are means ± SE from seven to twelve separate experiments, X values are from Fig. 1.</p