Plastome engineering of ribulose-1,5-bisphosphate carboxylase/oxygenase in tobacco to form a sunflower large subunit and tobacco small subunit hybrid

PURPOSE: To assess the time-course of the relaxation times and the orientationally averaged water diffusion coefficient Doav in postnatal brain development. MATERIALS AND METHODS: Multisection maps of T1, T2, and the trace of the diffusion tensor (Trace[D] = 3 x Doav) were obtained in four kittens at eight time points. RESULTS: In the adult, Doav was about 700 micron 2/sec in both white and gray matter. In the newborn, Doav was 1,100-1,350 micron 2/sec in white matter and 1,000 micron 2/sec in gray matter. For all anatomic regions and time points, the correlation between Doav and 1/T2 was high (R2 = 0.87, P < .001). T1 showed a lower correlation with Doav and a higher sensitivity to myelinization than did T2. CONCLUSION: Although Doav shows dramatic changes in the maturing brain, the high correlation between Doav and T2 indicates that little additional information can be obtained by measuring this diffusion parameter during normal brain development. This contrasts with previous findings in brain ischemia, where Doav and T2 appear to be uncorrelated. After including the authors' data and published iontophoretic measurements in a simple model of diffusion in tissues, the authors suggest that the underlying mechanisms of Doav reduction in brain maturation and ischemia are different. Doav changes during development are mainly affected by events occurring in the cellular compartment, while changes in extracellular volume fraction and tortuosity, which are thought to determine the reduction in Doav during ischemia, are probably of secondary importance.     

Fragmentation of the large subunit of ribulose-1,5-bisphosphate carboxylase by reactive oxygen species occurs near Gly-329

The large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) in the illuminated lysates of wheat (Triticum aestivum L.) chloroplasts is broken down by reactive oxygen radicals into 37- and 16-kDa polypeptides. Analysis of the terminal amino acid residues of both fragments revealed that the C terminus of the 37-kDa fragment was Ser-328 and the N terminus of the 16-kDa fragment was Thr-330. Gly-329, which links the two fragments, was missing, suggesting that the fragmentation of the LSU in the lysates driven by oxygen-free radicals occurs at Gly-329. Purified rubisco, exposed to a hydroxyl radical-generating system, was also cleaved at the same site of the LSU. The cleavage site was positioned at the N-terminal end of the flexible loop (loop 6) within the beta/alpha-barrel domain, constituting the catalytic site of rubisco. The binding of a reaction intermediate analogue, 2-carboxyarabinitol 1,5-bisphosphate, to the active form of rubisco completely protected the enzyme from the fragmentation. The fragmentation was differentially affected by CO2, Mg2+, ribulose 1, 5-bisphosphate, or 2-carboxyarabinitol 1,5-bisphosphate. All these results indicate that the conformation of the catalytic site of the enzyme is involved as an important factor determining the breakdown of rubisco by reactive oxygen species. Reactive oxygen species generated at its catalytic site by a Fenton-type reaction may trigger the site-specific degradation of the LSU in the lysates of chloroplasts in the light.     

Cloning and sequencing of a form II ribulose-1,5-biphosphate carboxylase/oxygenase from the bacterial symbiont of the hydrothermal vent tubeworm Riftia pachyptila

The bacterial symbiont of the hydrothermal vent tubeworm fixes carbon via the Calvin-Benson cycle and has been shown previously to express a form II ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). The gene cbbM, which encodes this enzyme, has been cloned and sequenced. The gene has the highest identity with the cbbM gene from Rhodospirillum rubrum, and analysis of the inferred amino acid sequence reveals that all active-site residues are conserved. This is the first form II RubisCO cloned and sequenced from a chemoautotrophic symbiont and from a deep-sea organism.