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 solutionscontaining ¹⁴C-labelled substances. The infiltrated discs wereeither transferred immediately to darkness or first illuminatedfor 2 h and then transferred to darkness. After 6 h in darknessthe discs were extracted with buffer containing CO₂, Mg²⁺ andadditional ribulose-1, 5-bisphosphate carboxylase/oxygenase(Rubisco; EC 4.1.1.39 [EC] ). Protein in the extracts was separatedfrom substances of low molecular weight by gel filtration andcoagulated by heating to 100C. Coagulated protein was removedby centrifugation and cations in the supernatant solution wereremoved by ion exchange resin. The non-volatile anions in theresulting solutions, among which was 2¹-carboxy-D-arabinitol-1-phosphate(CA1P), were separated by HPLC. The amount of CA1P was determinedfrom the signal of a pulsed amperometric detector and its radioactivityby scintillation counting. Vacuum infiltration of [2¹–¹⁴C]2¹-carboxy-D-arabinitol (CA) resulted in 12.6% of the radioactivityin the leaf discs being in CA1P after 6 h in darkness and 21.6%when 2 h light was given before the dark treatment. Where radioactiveglucose, fructose, sucrose, hamamelose, glycerate, glycine oracetate were infiltrated, ¹⁴C in CA1P was less than 1% of thetotal present after the dark period with or without a precedingperiod of light. Incorporation of ¹⁴C from [¹⁴C] CA into CA1Pin darkness was strongly inhibited by 2,4-dinitrophenol andalso to a lesser extent by tentoxin. With both inhibitors themain effect was a decreased uptake of the substrate. Illuminationprior to darkness stimulated the incorporation of radioactivityfrom CA, glycine, glucose, sucrose, and hamamelose into CA1Pin subsequent darkness. Unlike the other substrates, which wereextensively metabolized, CA and hamamelose were converted tofew products; CA was converted almost exclusively to CA1P andCA1P was a major product of hamamelose metabolism. Key words: CA1P, Phaseolus vulgaris, precursors, synthesis     

Neuronal competition determines the spatial pattern of neuropeptide expression by identified neurons of the leech.

Staining adult and embryonic leech ventral nerve cords with antibodies raised against the molluscan neuropeptides small cardioactive peptide B (SCP) and FMRFamide results in segment-specific and bilaterally asymmetric patterns of cell staining. One immunoreactive neuron, the RAS interneuron, is present in only four rostral segmental ganglia, while another, the CAS interneuron, is restricted to the four most caudal abdominal ganglia and tail. In addition to their segment-specific distributions, only one RAS or CAS cell is found in each segmental ganglion, and they alternate sides between adjacent ganglia (either L-R-L-R or R-L-R-L) with a fidelity of about 95%. This paper utilizes cell deletion techniques to investigate the determination of the asymmetric and alternating pattern of RAS and CAS neurons. We show that developmentally equivalent RAS and CAS homologs are present on both sides of the appropriate ganglia, and that within each ganglion one of the initially paired homologs loses the ability to assume the immunoreactive RAS or CAS fate 2-3 days after axonogenesis has begun. These experiments suggest that there is a competitive interaction between bilateral homologs which ensures that only one mature RAS/CAS neuron is formed per ganglion, and that contralateral RAS/CAS neurons are not required in the same or adjacent ganglia for the determination of the RAS or CAS developmental pathways. Nerve cord transections between ganglia in the CAS domain can alter the spatial pattern of CAS neuron determination, confirming that both bilateral homologs retain the ability to express neuropeptide until late embryonic stages, and suggesting that the alternating pattern of RAS/CAS cells requires communication between adjacent ganglia through the longitudinal connectives.