Demonstration of Both a Photosynthetic and a Nonphotosynthetic CO(2) Requirement for NH(4) Assimilation in the Green Alga Selenastrum minutum

Nitrogen-limited and nitrogen-sufficient cell cultures of Selenastrum minutum (Naeg.) Collins (Chlorophyta) were used to investigate the dependence of NH₄⁺ assimilation on exogenous CO₂. N-sufficient cells were only able to assimilate NH₄⁺ maximally in the presence of CO₂ and light. Inhibition of photosynthesis with 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron also inhibited NH₄⁺ assimilation. These results indicate that NH₄⁺ assimilation by N-sufficient cells exhibited a strict requirement for photosynthetic CO₂ fixation. N-limited cells assimilated NH₄⁺ both in the dark and in the light in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron, indicating that photosynthetic CO₂ fixation was not required for NH₄⁺ assimilation. Using CO₂ removal techniques reported previously in the literature, we were unable to demonstrate CO₂-dependent NH₄⁺ assimilation in N-limited cells. However, employing more stringent CO₂ removal techniques we were able to show a CO₂ dependence of NH₄⁺ assimilation in both the light and dark, which was independent of photosynthesis. The results indicate two independent CO₂ requirements for NH₄⁺ assimilation. The first is as a substrate for photosynthetic CO₂ fixation, whereas the second is a nonphoto-synthetic requirement, presumably as a substrate for the anaplerotic reaction catalyzed by phosphoenolpyruvate carboxylase.     

A major difference between the divergence patterns within the lines-1 families in mice and voles

L1 retroposons are represented in mice by subfamilies of interspersed sequences of varied abundance. Previous analyses have indicated that subfamilies are generated by duplicative transposition of a small number of members of the L1 family, the progeny of which then become a major component of the murine L1 population, and are not due to any active processes generating homology within preexisting groups of elements in a particular species. In mice, more than a third of the L1 elements belong to a clade that became active approximately 5 Mya and whose elements are > or = 95% identical. We have collected sequence information from 13 L1 elements isolated from two species of voles (Rodentia: Microtinae: Microtus and Arvicola) and have found that divergence within the vole L1 population is quite different from that in mice, in that there is no abundant subfamily of homologous elements. Individual L1 elements from voles are very divergent from one another and belong to a clade that began a period of elevated duplicative transposition approximately 13 Mya. Sequence analyses of portions of these divergent L1 elements (approximately 250 bp each) gave no evidence for concerted evolution having acted on the vole L1 elements since the split of the two vole lineages approximately 3.5 Mya; that is, the observed interspecific divergence (6.7%-24.7%) is not larger than the intraspecific divergence (7.9%-27.2%), and phylogenetic analyses showed no clustering into Arvicola and Microtus clades.      

Alternative Oxidase Activity in Tobacco Leaf Mitochondria (Dependence on Tricarboxylic Acid Cycle-Mediated Redox Regulation and Pyruvate Activation)

Transgenic Nicotiana tabacum (cv Petit Havana SR1) containing high levels of mitochondrial alternative oxidase (AOX) protein due to the introduction of a sense transgene(s) of Aox1, the nuclear gene encoding AOX, were used to investigate mechanisms regulating AOX activity. After purification of leaf mitochondria, a large proportion of the AOX protein was present as the oxidized (covalently associated and less active) dimer. High AOX activity in these mitochondria was dependent on both reduction of the protein by DTT (to the noncovalently associated and more active dimer) and its subsequent activation by certain [alpha]-keto acids, particularly pyruvate. Reduction of AOX to its more active form could also be mediated by intramitochondrial reducing power generated by the oxidation of certain tricarboxylic acid cycle substrates, most notably isocitrate and malate. Our evidence suggests that NADPH may be specifically required for AOX reduction. All of the above regulatory mechanisms applied to AOX in wild-type mitochondria as well. Transgenic leaves lacking AOX due to the introduction of an Aox1 antisense transgene or multiple sense transgenes were used to investigate the potential physiological significance of the AOX-regulatory mechanisms. Under conditions in which respiratory carbon metabolism is restricted by the capacity of mitochondrial electron transport, feed-forward activation of AOX by mitochondrial reducing power and pyruvate may act to prevent redirection of carbon metabolism, such as to fermentative pathways.     

Evidence for Activation of the Oxidative Pentose Phosphate Pathway during Photosynthetic Assimilation of NO(3) but Not NH(4) by a Green Alga

Addition of NO₃⁻ to N-limited Selenastrum minutum during photosynthesis resulted in an immediate drop in the NADPH/NADP ratio and a slower increase of the NADH/NAD ratio. These changes were accompanied by a rapid decrease in glucose-6-phosphate and increase in 6-phosphogluconate, indicating activation of glucose-6-phosphate dehydrogenase and a role for the oxidation pentose phosphate pathway during photosynthetic NO₃⁻ assimilation. In contrast, the short-term changes in pyridine nucleotides and metabolites during photosynthetic assimilation of NH₄⁺ were not consistent with a stimulation of the oxidative pentose phosphate pathway.     

Evolution and diversity of Arsenophonus endosymbionts in aphids

Endosymbiotic bacteria are important drivers of insect evolutionary ecology, acting both as partners that contribute to host adaptation and as subtle parasites that manipulate host reproduction. Among them, the genus Arsenophonus is emerging as one of the most widespread lineages. Its biology is, however, entirely unknown in most cases, and it is therefore unclear how infections spread through insect populations. Here we examine the incidence and evolutionary history of Arsenophonus in aphid populations from 86 species, characterizing the processes that shape their diversity. We identify aphids as harbouring an important diversity of Arsenophonus strains. Present in 7% of the sampled species, incidence was especially high in the Aphis genus with more than 31% of the infected species. Phylogenetic investigations revealed that these Arseno‐phonus strains do not cluster within an aphid‐specific clade but rather exhibit distinct evolutionary origins showing that they undergo repeated horizontal transfers (HT) between distantly related host species. Their diversity pattern strongly suggests that ecological interactions, such as plant mediation and parasitism, are major drivers for Arsenophonus dispersal, dictating global incidence across insect communities. Notably, plants hosting aphids may be important ecological arenas for global exchange of Arsenophonus, serving as reservoirs for HT.     

Mitochondrial alternative oxidase acts to dampen the generation of active oxygen species during a period of rapid respiration induced to support a high rate of nutrient uptake

When wild type (wt) tobacco ( Nicotiana tabacum L. cv. Petit Havana SR1) suspension cells were grown under phosphate (P) limitation, they contained large amounts of mitochondrial alternative oxidase (AOX). When these cells were resupplied with P, there was a large, immediate and sustained stimulation of respiration to support a period of rapid P uptake. Two lines of evidence suggest that the abundant level of AOX present in wt cells contributed to this stimulated rate of respiration. First, when P-limited transgenic antisense tobacco cells (AS8) lacking AOX were resupplied with P, the stimulation of respiration was much less dramatic even though these cells displayed similar rates of P uptake. Second, while the stimulated rate of respiration in AS8 cells was insensitive (as expected) to the AOX inhibitor n -propyl gallate (nPG), much of the stimulated rate of respiration in wt cells could be inhibited by nPG. Given the non-phosphorylating nature of AOX respiration, wt cells required higher rates of electron transport to O₂ than AS8 cells to support similar rates of P uptake. The utilization of AOX by wt cells during P uptake was apparently not occurring because the cytochrome (Cyt) pathway alone could not fully support the rate of P uptake, as the respiration of cells lacking AOX (either untreated AS8 cells or wt cells treated with nPG) supported similar rates of P uptake as wt cells with abundant AOX. Rather, we provide in vivo evidence that the utilization of AOX during the period of high respiration supporting P uptake was to dampen the mitochondrial generation of active oxygen species (AOS).     

In Organello and in Vivo Evidence of the Importance of the Regulatory Sulfhydryl/Disulfide System and Pyruvate for Alternative Oxidase Activity in Tobacco

After isolation of tobacco (Nicotiana tabacum) leaf mitochondria, alternative oxidase (AOX) is predominantly present as the disulfide-linked, less-active “oxidized” form. In an in organello assay, significant AOX activity was dependent upon both the reduction of the regulatory disulfide bond (such as occurs by dithiothreitol) and upon the presence of the activator pyruvate. However, AOX activity in these assays was substantially affected when mitochondria were isolated in the presence of pyruvate. First, pyruvate protects against the oxidation of the regulatory sulfhydryl during isolation, such that subsequent in organello AOX activity is not dependent upon dithiothreitol. Second, pyruvate stabilizes AOX activity, such that mitochondria kept in the presence of pyruvate have higher maximum rates of AOX activity than mitochondria kept for some time in the absence of pyruvate. The ability of pyruvate to protect against AOX oxidation was exploited to assess the in vivo status of the regulatory sulfhydryl/disulfide system. In both tobacco suspension cells and tobacco leaves with high levels of AOX protein, the protein is predominantly present as the “reduced” active form in vivo under a range of respiratory conditions. Experiments also indicate that, while the presence of reduced protein may be a necessary prerequisite for significant AOX activity, it is not sufficient for activity and other factors must also be critical.