The mechanism of rhythmic ethylene production in sorghum. The role of phytochrome B and simulated shading.

Mutant sorghum (Sorghum bicolor [L.] Moench) deficient in functional phytochrome B exhibits reduced photoperiodic sensitivity and constitutively expresses a shade-avoidance phenotype. Under relatively bright, high red:far-red light, ethylene production by seedlings of wild-type and phytochrome B-mutant cultivars progresses through cycles in a circadian rhythm; however, the phytochrome B mutant produces ethylene peaks with approximately 10 times the amplitude of the wild type. Time-course northern blots show that the mutant's abundance of the 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase mRNA SbACO2 is cyclic and is commensurate with ethylene production, and that ACC oxidase activity follows the same pattern. Both SbACO2 abundance and ACC oxidase activity in the wild-type plant are very low under this regimen. ACC levels in the two cultivars did not demonstrate fluctuations coincident with the ethylene produced. Simulated shading caused the wild-type plant to mimic the phenotype of the mutant and to produce high amplitude rhythms of ethylene evolution. The circadian feature of the ethylene cycle is conditionally present in the mutant and absent in the wild-type plant under simulated shading. SbACO2 abundance in both cultivars demonstrates a high-amplitude diurnal cycle under these conditions; however, ACC oxidase activity, although elevated, does not exhibit a clear rhythm correlated with ethylene production. ACC levels in both cultivars show fluctuations corresponding to the ethylene rhythm previously observed. It appears that at least two separate mechanisms may be involved in generating high-amplitude ethylene rhythms in sorghum, one in response to the loss of phytochrome B function and another in response to shading.

Ethylene Biosynthesis during Aerenchyma Formation in Roots of Maize Subjected to Mechanical Impedance and Hypoxia.

Germinated maize (Zea mays L.) seedlings were enclosed in modified triaxial cells in an artificial substrate and exposed to oxygen deficiency stress (4% oxygen, hypoxia) or to mechanical resistance to elongation growth (mechanical impedance) achieved by external pressure on the artificial substrate, or to both hypoxia and impedance simultaneously. Compared with controls, seedlings that received either hypoxia or mechanical impedance exhibited increased rates of ethylene evolution, greater activities of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, ACC oxidase, and cellulase, and more cell death and aerenchyma formation in the root cortex. Effects of hypoxia plus mechanical impedance were strongly synergistic on ethylene evolution and ACC synthase activity; cellulase activity, ACC oxidase activity, or aerenchyma formation did not exhibit this synergism. In addition, the lag between the onset of stress and increases in both ACC synthase activity and ethylene production was shortened by 2 to 3 h when mechanical impedance or impedance plus hypoxia was applied compared with hypoxia alone. The synergistic effects of hypoxia and mechanical impedance and the earlier responses to mechanical impedance than to hypoxia suggest that different mechanisms are involved in the promotive effects of these stresses on maize root ethylene biosynthesis.

Transport and Metabolism of 1-Aminocyclopropane-1-carboxylic Acid in Sunflower (Helianthus annuus L.) Seedlings.

Transport and metabolism of [2,3-(14)C] 1-aminocyclopropane-1-carboxylic acid (ACC) from roots to shoots in 4-day-old sunflower (Helianthus annuus L.) seedlings were studied. [(14)C]ACC was detected in, and (14)C(2)H(4) was evolved from, shoots 0.5 hours after [(14)C]ACC was supplied to roots. Ethylene emanation from the shoots returned to normal levels after 6 hours. The roots showed a similar pattern, although at 24 hours ethylene emanation was still slightly higher than in those plants that did not receive ACC. [(14)C]N-malonyl-ACC (MACC) was detected in both tissues at all times sampled. [(14)C]MACC levels surpassed [(14)C]ACC levels in the shoot at 2 hours, whereas [(14)C]MACC levels in the root remained below [(14)C]ACC levels until 6 hours, after which they were higher. Thin-layer chromatography analysis identified [(14)C] ACC in 1-hour shoot extracts, and [(14)C]MACC was identified in root tissues at 1 and 12 hours after treatment. [(14)C]ACC and [(14)C] MACC in the xylem sap of treated seedlings were identified by thin-layer chromatography. Xylem transport of [(14)C]ACC in treated seedlings, and transport of ACC in untreated seedlings, was confirmed by gas chromatography-mass spectrometry. Some evidence for the presence of [(14)C]MACC in xylem sap in [(14)C]ACC-treated seedlings is presented. A substantial amount of radioactivity in both ACC and MACC fractions was detected leaking from the roots over 24 hours. A second radiolabeled volatile compound was trapped in a CO(2)-trapping solution but not in mercuric perchlorate. Levels of this compound were highest after the peak of ACC levels and before peak MACC levels in both tissues, suggesting that an alternate pathway of ACC metabolism was operating in this system.

Acetyl-coenzyme A carboxylase from the developing endosperm of Ricinus communis. I. Isolation and characterization.

Acetyl-coenzyme A carboxylase has been purified from the plastids of developing castor oil seeds. High concentrations of the enzyme are required for stability as well as the presence of dithiothreitol, glycerol, bicarbonate, Triton X-100, and polyvinyl-pyrrolidone. It has a molecular weight of approximately 528,000 and appears to be membrane associated. Acetyl-CoA carboxylase is active over a wide pH range with an optimum at 8.0. Arrhenius plots are biphasic. The enzyme displays normal Michaelis-Menten kinetics with limiting Michaelis constants of KATP, 0.1 mM; KHCO-3, 3.0 mM; and Kacetyl-CoA, 0.05 mM. Monovalent cations, such as K+ and Cs+, exert a small activating effect on the enzyme while a divalent cation, Mn2+ or Mg2+, is essential for activity. The enzyme does not appear to be highly regulated by cellular metabolites.

Acetyl-coenzyme A carboxylase from the developing endosperm of Ricinus communis. II. A two-site kinetic mechanism.

The reaction kinetics of acetyl-coenzyme A carboxylase purified from developing castor oil seeds have been examined. On the basis of the substrate interaction and product inhibition results, a hybrid ping-pong mechanism is proposed. This type of mechanism demands that the active site of the enzyme be separated into two functionally distinct catalytic sites. The carboxybiotin intermediate formed at one site by the hydrolysis of ATP swings to the second site where acetyl-CoA is carboxylated to form malonyl-CoA. This hybrid rapid-equilibrium random bi bi uni uni ping-pong mechanism which includes the formation of three abortive complexes, E . HCO-3 . ADP, E . HCO-3 . Pi and E . Pi . Pi, is analogous to the hybrid ping-pong mechanism previously described for methylmalonyl-CoA transcarboxylase.