Net biosynthesis of l-AA in Arabidopsis cell suspension cultures in the presence of 15 mm (final concentration) of various substrates. Aliquots (400 μL) of cell culture were periodically removed and assayed for l-AA and DHA by HPLC, as described in “Materials and Methods.” Rates of l-AA biosynthesis were calculated from linear regression analysis of the increase in intracellular total l-AA levels. The rates of l-AA biosynthesis relative to the control incubation (1.0), were 2.7, 8.5, 29.5, 48.4, and 64.0, for d-glucuronolactone, l-GuL, MeGalUA, l-GL, and l-Gal, respectively. Absolute rates of l-AA biosynthesis are expressed in nmol total l-AA/400 μL aliquot cell suspension. On average 400 μL of cell suspension contains 48.7 mg fresh weight cells, equivalent to approximately 1.07 × 10⁶ cells. ⋄, L-Gal; □, L-GL; ▵, MeGalUA; ×, L-Gul; *, D-GlcUL; ○, control.

Biosynthesis of l-AA by crude, gel-filtrated cell extracts of Arabidopsis cell suspension culture. Cells extracted with 1 mL/g fresh weight extraction buffer as described in “Materials and Methods,” and reactions started by the addition of 5 mm (final concentration) l-GL (○) or l-Gal (L-Gal/NAD; □) or of 25 mm l-GuL (▵). Incubations with 5 mm l-Gal also contained 0.125 mm NAD (final concentration). Aliquots of 20 μL of reaction mixture were periodically removed and the reaction stopped by the addition of an equal volume of 6% metaphosphoric acid/1 mm EDTA containing 2.5 mm DTT. l-AA formed was quantitated by capillary electrophoresis as described in “Materials and Methods.” ×, Control/NAD.

Localization of l-GL-dependent (gray bars; GLDH) and l-GuL-dependent (black bars; GuL) l-AA biosynthetic activities. After cell debris were removed, crude Arabidopsis cell suspension extracts were separated into mitochondrial and soluble fractions by centrifugation. The rates of l-AA biosynthesis were then quantitated by HPCE following the addition of substrate, as described in “Materials and Methods.” Rates of l-AA biosynthesis are expressed in nmol l-AA formed min⁻¹ g⁻¹ fresh weight of cells (gfw).

Generalized scheme situating possible alternate routes of l-AA biosynthesis within plant hexose metabolism. Compounds marked with an asterisk were used as substrates in incubation experiments with an Arabidopsis cell suspension culture (see text). l-GulA, l-gulonic acid; d-GlcUL, d-glucuronolactone. Enzymes catalyzing the individual numbered reactions are given below. 1, Hexokinase (EC 2.7.1.1) also catalyzes reaction 11; 2, Glc-6-P isomerase (EC 5.3.1.9); 3, Man-6-P isomerase (EC 5.3.1.8); 4, phosphomannomutase (EC 5.4.2.8); 5, Man-1-P guanylyltransferase (EC 2.7.7.22); 6, GDP-Man 3,5-epimerase (EC. 5.1.3.18); 7, hydrolase; 8, sugar phosphatase (EC 3.1.3.23); 9, l-Gal 1-dehydrogenase; 10, l-galactono-1,4-lactone dehydrogenase (EC 1.3.2.3); 11, d-Man kinase/hexokinase (EC 2.7.1.1); 12, phosphoglucomutase (EC 5.4.2.2); 13, UTP-Glc-1-P uridylyl transferase (EC 2.7.7.9); 14, UDP-d-Glc dehydrogenase (EC 1.1.1.22); 15, UDP-glucuronate 4-epimerase (EC 5.1.3.6); 16, glucuronate-1-P uridylyltransferase (EC 2.7.7.44); 17, d-glucuronokinase (EC 2.7.1.43); 18, d-glucuronate (hexuronate) reductase (EC 1.1.1.19) may also catalyze reaction 27; 19/28, aldonolactonase (EC 3.1.1.17); 20, l-gulono-1,4-lactone oxidase/dehydrogenase; 21, spontaneous lactonization, or uronolactonase activity (EC 3.1.1.19); 22, (spontaneous) methyl esterase activity; 23, glucuronolactone reductase activity (EC 1.1.1.20); 24, l-galactono-1,4-lactone 3-epimerase; 25, galacturonate-1-P uridylyltransferase; 26, galacturonokinase (EC 2.7.1.44); 27, hexuronate (d-galacturonate) reductase; 28/19, aldonolactonase; 29, (spontaneous) methylesterase activity; 30, myoinositol 1-P synthase (EC 5.5.1.4); 31, myoinositol 1-P monophosphatase (EC 3.1.3.25); 32, myoinositol oxygenase (EC 1.13.99.1); 33, d-galactokinase (EC 2.7.1.6); 34, UTP-hexose 1-P uridylyltransferase (EC 2.7.7.10); 35, UDP-Glc 4-epimerase (EC 5.1.3.2); 36, Suc synthase (EC 2.4.1.13); 37, fructokinase (EC 2.7.1.4).