Enzymic Activities Associated with the Ability of Aerial and Submerged Forms of Littorella uniflora (L.) Aschers to perform CAM

ABSRACT: Groenhof, A. C, Smirnoff, N. and Bryant, J. A. 1988. Enzymicactivities associated with the ability of aerial and submergedforms of Littorella uniflora (L.) Aschers to perform CAM.—J.exp. Bot. 39: 353-361. The submerged form of Littorella uniflora shows a full CAM modeof photosynthesis as shown by diel acid fluctuations and elevatedactivities (in comparison to non-submerged leaves) of the enzymesphosphoenolpyruvate carboxylase (PEPC) and NADP-malic enzyme.Non-submerged plants exhibit no diel fluctuations of acidityand no changes in activity of NADP-malic enzyme or PEPC. PEPCactivity is low and NADP-malic enzyme is not detectable. Furthercharacterization of PEPC extracted from submerged plants duringthe light and dark periods of a diel cycle shows that the enzymeextracted in the dark is more active. In addition, the enzymeshows a decrease in K_m (PEP) and an increase in V_max in thepresence of glucose-6-phosphate, whilst in the presence of malateK_m (PEP) is increased and V_max decreased; this response to malateis only observed in the light and at pH 7.2. Molecular weightdeterminations using a Sephacryl S-300 column show that theenzyme extracted from plants during the dark period has an apparentmol. wt. of 375 KDa and the enzyme extracted from plants duringthe light period has an apparent mol. wt. of 307 KDa. Key words: Littorella uniflora (shoreweed), Crassulacean acid metabolism, PEP carboxylase, malic enzyme     

THE BIOCHEMISTRY OF NITRIFYING MICROORGANISMS

• 1 Biological nitrification is mediated primarily by two genera of bacteria, Nitrosomonas and its marine form Nitrosocystis, oxidizing ammonia to nitrite, and Nitrobacter, converting nitrite into nitrate. These are chemoautotrophic organisms since they usually derive their energy for growth by oxidizing these inorganic nitrogen compounds and their carbon from carbon dioxide, carbonates or bicarbonates.
• 2 The morphology and structure of these Gram-negative bacteria studied by electron microscopy show numerous intracellular membranes reminiscent of those in photosynthetic bacteria and blue-green algae. These structures may therefore be associated with the production of ATP.
• 3 The bacteria are difficult to grow in pure cultures in sufficient amounts for biochemical work since their generation time is around 10 hr. and the yields are only about one hundredth of those obtained with heterotrophic bacteria. Thus in continuous cultures great care must be taken to avoid ‘wash-out’ of the cells. Since Nitrosomonas and Nitrosocystis produce copious amounts of nitrous acid, which would eventually retard growth, pH stat units are used to titrate the cultures continuously with a solution of sodium carbonate, to hold the pH around 7–8.
• 4 The respiratory chain which is associated with cell membranes, contains flavin, quinones and many cytochromes linking to oxygen as a terminal acceptor. In Nitro-somonas-Nitrosocytis hydroxylamine is oxidized by the electron transfer chain and in Nitrobacter nitrous acid is utilized. The ammonia-oxidizing system, which in Nitrosomonas probably resides near the cell surface, does not appear to survive cell breakage. During the oxidation of hydroxylamine and nitrous acid by the respiratory chains, a phosphorylation occurs but the P/O ratios around 0–30 are low. There is little energy reserve material in the cells, possibly β-hydroxybutyrate and some metaphosphates and as soon as the oxidative processes are impaired the cells cease dividing.
• 5 Chemoautotrophic bacteria have a novel way of producing reduced nicotinamide adenine dinucleotide (NADH). This involves a reversal of electron flow from reduced cytochrome c to nicotinamide adenine dinucleotide (NAD) that is energy-dependent, thus requiring adenosine triphosphate.
• 6 Reductase enzymes, nitrate, nitrite and hydroxylamine reductases in Nitrobacter and nitrite and hydroxylamine reductases in Nitrosomonas, have been described. They appear to be readily extracted in soluble form and are probably assimilatory enzymes since ¹⁶N labelled nitrate, nitrite and hydroxylamine respectively in Nitrobacter and the last two in Nitrosomonas are readily incorporated into cell nitrogen. It has been suggested that a particulate nitrate reductase in Nitrobacter is coupled to the synthesis of adenosine triphosphate but adequate experimental evidence for this concept has not been produced.
• 7 Some recent observations with Nitrobacter suggest that it grows on acetate, deriving all its energy and carbon skeletons from this source but the mean generation time for the bacterium is unchanged. Under these conditions the carbon dioxide fixing enzymes of the pentose pathway are suppressed. This then is a case of facultative chemoautotrophy but there is no increase in the biosynthesis of the TCA enzymes. Whether this is a widespread phenomenon in other chemoautotrophic bacteria remains to be established. If this does prove to be the case it would aid their survival in a variety of habitats and extend their distribution in soils and seas.
• 8 The carbon dioxide fixing enzymes of the pentose pathway are found in the soluble parts of the cells. The major route is via the carboxydismutase system with only a small incorporation via the phosphoenolpyruvate carboxylase enzyme. Enzymes of the tricarboxylic acid cycle have low activities compared with those in heterotrophs and this overall slow metabolism, rather than the lack of a specific enzyme such as NADH oxidase, may well account for the slow growth of these bacteria. Although there is very active glutamic dehydrogenase in Nitrosomonas that utilizes ammonia, the enzyme has a very small activity in Nitrobacter. This poses a problem of the route of incorporation of nitrite nitrogen into cell nitrogen in the latter bacterium.
• 9 A few heterotrophic fungi have been described which oxidize ammonia to nitrate but their activity is small compared with that of the nitrifying bacteria.
• 10 It is concluded that the nitrifying bacteria which have many novel biochemical features not met with in other organisms merit further study.

     

Secular distribution of Burgess-Shale-type preservation

Burgess-Shale-type preservation is defined as a taphonomic pathway involving the exceptional organic preservation of non-mineralizing organisms in fully marine siliciclastic sediments. In the Phanerozoic it occurs widely in Lower and Middle Cambrian sequences but subsequently disappears as a significant taphonomic mode. The hypothesis that this distribution derives solely from a secular increase in the depth of bioturbation is falsified: low bioturbation indices do not prevent the rapid enzymatic degradation of organic structure, nor do they account for the conspicuous absence of comparable preservation during the Vendian. An earlier, Late Riphean (ca. 750–850 Ma), interval of enhanced organic-walled fossil preservation suggests a long-term recurrence in Burgess-Shale-type taphonomy that is independent of metazoan activity. A model based on the potentially powerful anti-enzymatic and/or stabilizing effects of clay minerals on organic molecules is proposed to account for Burgess-Shale-type preservation. Long-term changes in average clay mineralogies and the ocean chemistry that determines their interaction with organic molecules are likely to have induced the pronounced secular distribution of these fossil biotas, while regional variations in tectonism, weathering, etc., explain their non-uniform geographic distribution; the close correlation between exceptional, organic-walled fossil preservation and volcano-genic sedimentation in Tertiary lake deposits provides a compelling analogue. Recognition of a temporal control on Burgess-Shale-type preservation constrains the evolutionary scenarios that can be drawn from such biotas; significantly, neither the initial rate of appearance, nor the ultimate fate of Burgess-Shale-type taxa can be directly assessed. □ Taphonomy, exceptional preservation, organic preservation, fossil Lagerstätten, Burgess Shale, clay mineralogy, clay-organic interactions, secular change, Cambrian, Proterozoic.