Synthesis of 2¹-carboxy-D-arabinitol-1-phosphate in French bean (Phaseolus vulgaris L.): a search for precursors

Discs of French bean leaves were vacuum infiltrated with solutions containing C-14-labelled substances. The infiltrated discs were either transferred immediately to darkness or first illuminated for 2h and then transferred to darkness. After 6h in darkness the discs were extracted with buffer containing CO2, Mg2+ and additional ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco; EC 4.1.1.39). Protein in the extracts was separated from substances of low molecular weight by gel filtration and coagulated by heating to 100 degrees C. Coagulated protein was removed by centrifugation and cations in the supernatant solution were removed by ion exchange resin. The non-volatile anions in the resulting solutions, among which was 2(1)-carboxy-D-arabinitol-1-phosphate (CA1P), were separated by HPLC. The amount of CA1P was determined from the signal of a pulsed amperometric detector and its radioactivity by scintillation counting. Vacuum infiltration of [2(1)-C-14] 2(1)-carboxy-D-arabinitol (CA) resulted in 12.6% of the radioactivity in the leaf discs being in CA1P after 6 h in darkness and 21.6% when 2h light was given before the dark treatment. Where radioactive glucose, fructose, sucrose, hamamelose, glycerate, glycine or acetate were infiltrated, C-14 in CA1P was less than 1% of the total present after the dark period with or without a preceding period of light. Incorporation of C-14 from [C-14] CA into CA1P in darkness was strongly inhibited by 2,4-dinitrophenol and also to a lesser extent by tentoxin. With both inhibitors the main effect was a decreased uptake of the substrate. Illumination prior to darkness stimulated the incorporation of radioactivity from CA, glycine, glucose, sucrose, and hamamelose into CA1P in subsequent darkness. Unlike the other substrates, which were extensively metabolized, CA and hamamelose were converted to few products; CA was converted almost exclusively to CA1P and CA1P was a major product of hamamelose metabolism.