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).     

Increased respiratory restriction during phosphate-limited growth in transgenic tobacco cells lacking alternative oxidase

We found that mitochondrial alternative oxidase (AOX) protein and the capacity for CN-resistant respiration are dramatically increased in wild-type tobacco (Nicotiana tabacum) suspension-cultured cells in response to growth under P limitation, and antisense (AS8) tobacco cells unable to induce AOX under these conditions have altered growth and metabolism. Specifically, we found that the respiration of AS8 cells was restricted during P-limited growth, when the potential for severe adenylate control of respiration (at the level of C supply to the mitochondrion and/or at the level of oxidative phosphorylation) is high due to the low cellular levels of ADP and/or inorganic P. As a result of this respiratory restriction, AS8 cells had altered growth, morphology, cellular composition, and patterns of respiratory C flow to amino acid synthesis compared with wild-type cells with abundant AOX protein. Also, AS8 cells under P limitation displayed high in vivo rates of generation of active oxygen species compared with wild-type cells. This difference could be abolished by an uncoupler of mitochondrial oxidative phosphorylation. Our results suggest that induction of non-phosphorylating AOX respiration (like induction of adenylate and inorganic P-independent pathways in glycolysis) is an important plant metabolic adaptation to P limitation. By preventing severe respiratory restriction, AOX acts to prevent both redirections in C metabolism and the excessive generation of harmful active oxygen species in the mitochondrion.     

氮磷营养限制影响三角褐指藻光合无机碳利用和碳酸酐酶活性

以海洋硅藻三角褐指藻为实验材料, 研究了不同氮磷比培养对其光合无机碳利用和碳酸酐酶活性的影响, 结果显示三角褐指藻生长速率在N:P=16:1时最大, 高于或低于16:1时明显下降, 表明其最适生长受到氮磷的限制。氮限制(N:P=4:1或1:1)导致叶绿素a含量分别下降30.1% 和47.6%, 磷限制(N:P=64:1或256:1)下降39.1%和52.4%, 但氮或磷限制对叶绿素c含量并没有明显影响。不同营养水平培养对光饱和光合速率具有明显的影响, 与营养充足培养相比, 在严重氮磷限制(N:P=1:1或256:1)培养下光饱和光合速率分别下降39.7%和48.0%, 光合效率与暗呼吸速率也明显下降。在氮磷限制培养下藻细胞pH补偿点明显下降; K0.5CO2值在磷限制下降低30%, 表明磷限制有助于提高细胞对CO2的亲和力, 但氮限制并没有明显影响。在氮磷限制培养的细胞反应液中Fe (CN)63-浓度下降速率较慢, 表明在氮磷限制环境中生长的细胞质膜氧化还原能力明显低于营养充足条件下生长的细胞。氮磷限制也导致胞内、外碳酸酐酶活性明显下降, 其中在氮限制下胞外碳酸酐酶活性分别下降50%和37.5%, 在磷限制下下降22.3%和42.1%。严重的氮(N:P=1:1)或磷(N:P=256:1)限制导致胞内碳酸酐酶活性下降36.5%和42.9%。研究结果表明, 三角褐指藻细胞在氮磷营养限制的环境中, 可以通过调节叶绿素含量、无机碳的利用方式和碳酸酐酶的活性以维持适度的生长。       

The Absence of Alternative Oxidase AOX1A Results in Altered Response of Photosynthetic Carbon Assimilation to Increasing CO2 in Arabidopsis thaliana

In higher plants, the mitochondrial electron transport chain has non-phosphorylating alternative pathways that include the alternative terminal oxidase (AOX). This alternative pathway has been suggested to act as a sink for dissipating excess reducing power, minimizing oxidative stress and possibly optimizing photosynthesis in response to changing conditions. The expression patterns of the AOX genes have been well characterized under different growth conditions, particularly in response to light and temperature stress. Additionally, it has been suggested that mitochondrial electron transport is important for avoiding chloroplast over-reduction and balancing energy partitioning among photosynthesis, photorespiration and respiration. Nonetheless, the role AOX plays in optimizing photosynthetic carbon metabolism is unclear. Therefore, the response of photosynthesis to the disruption of AOX was investigated in the Arabidopsis thaliana T-DNA mutant aox1a (SALK_084897). Gas exchange analysis revealed a lower net CO(2) assimilation rate (A) at high CO(2) concentrations in the aox1a mutant compared to wild type. This decrease in A was accompanied by a lower maximum electron transport rate and quantum yield of PSII, and higher excitation pressure on PSII and non-photochemical quenching. The aox1a mutant also exhibited a lower estimated rate of ribulose 1,5-bisphosphate regeneration, and the ribulose 1,5-bisphosphate content was lower at high CO(2) concentrations, suggesting an ATP limitation of the Calvin-Benson cycle. Additionally, the activity of the malate-oxaloacetate shuttle was lower in the mutant compared to wild type. These results indicate that AOX is important for optimizing rates of photosynthetic CO(2) assimilation in response to rising CO(2) concentration by balancing the NAD(P)H/ATP ratio and rates of ribulose 1,5-bisphosphate regeneration within the chloroplast.     

Transgenic plant cells lacking mitochondrial alternative oxidase have increased susceptibility to mitochondria-dependent and -independent pathways of programmed cell death

The plant mitochondrial electron transport chain is branched such that electrons at ubiquinol can be diverted to oxygen via the alternative oxidase (AOX). This pathway does not contribute to ATP synthesis but can dampen the mitochondrial generation of reactive oxygen species. Here, we establish that transgenic tobacco (Nicotiana tabacum L. cv Petit Havana SR1) cells lacking AOX (AS8 cells) show increased susceptibility to three different death-inducing compounds (H(2)O(2), salicylic acid [SA], and the protein phosphatase inhibitor cantharidin) in comparison with wild-type cells. The timing and extent of AS8 cell death are very similar among the three treatments and, in each case, are accompanied by the accumulation of oligonucleosomal fragments of DNA, indicative of programmed cell death. Death induced by H(2)O(2) or SA occurs by a mitochondria-dependent pathway characterized by cytochrome c release from the mitochondrion. Conversely, death induced by cantharidin occurs by a pathway without any obvious mitochondrial involvement. The ability of AOX to attenuate these death pathways may relate to its ability to maintain mitochondrial function after insult with a death-inducing compound or may relate to its ability to prevent chronic oxidative stress within the mitochondrion. In support of the latter, long-term treatment of AS8 cells with an antioxidant compound increased the resistance of AS8 cells to SA- or cantharidin-induced death. The results indicate that plants maintain both mitochondria-dependent and -independent pathways of programmed cell death and that AOX may act as an important mitochondrial "survival protein" against such death.     

Contrasting effects of N and P deprivation on the regulation of photosynthesis in tomato plants in relation to feedback limitation

The effects were studied of both nitrogen and phosphorus limitation and irradiance on the performance and operation of photosynthesis in tomato leaves (Lycopersicon esculentum Mill.). Plants were grown at low N, high N, low P or high P supply and at two irradiances. Using mature leaves, measurements were made of the irradiance dependencies of the relative quantum efficiencies of photosystems I and II, and of the rate of carbon dioxide fixation. Measurements were also made of foliar starch and chlorophyll concentrations. The results showed that photosynthetic light-harvesting and electron-transport activity acclimate to nutrient stress and growth irradiance such that the internal relationships between electron transport by photosystems I and II do not change; the linear relationship between PhiPSII, and PhiPSI was not affected. It was also evident that under N stress photosynthesis was reduced by a decreased light absorption and by the decreased utilization of assimilates, while P stress mainly affected the carboxylation capacity. Under N stress foliar starch levels increased and the oxygen sensitivity of CO2 fixation decreased, whereas P stress resulted in decreased starch levels and increased oxygen sensitivity of CO2 fixation. The relationship between starch accumulation and oxygen sensitivity (increased starch correlated with decreased oxygen sensitivity) was always the same across the nutrient treatments. These results are consistent with N deprivation producing an increasing limitation of photosynthesis, possibly by feedback from the leaf carbohydrate pool, whereas, although P deprivation produces a decreased rate of CO2 fixation, this is accompanied by a increase in oxygen sensitivity, suggesting that feedback limitation is decreased under P stress.     

Influence of Nutrient Stress on the Relationships between PAM Measurements and Carbon Incorporation in Four Phytoplankton Species

Two methods of measuring primary production, modulated fluorimetry (PAM) and the traditional carbon incorporation method (¹³C), were compared in four phytoplankton species, two diatoms (Pseudo-nitzschia pungens and Asterionellopsis glacialis), and two dinoflagellates (Heterocapsa sp and Karenia mikimotoï), under N (nitrogen), P (phosphorus) and Si (silicon) limited semi-continuous culture. N and Si-limited cultures showed relatively high quantum efficiency of the PSII (F_v/F_m) values, confirming that F_v/F_m is not a good proxy for nutrient stress in balanced systems, whereas P limitation had a drastic effect on many physiological parameters. In all species, the physiological capacity of phytoplankton cells to acclimate to nutrient limitations led to changes in the cellular biochemical composition and the structure of the photosynthetic apparatus. The observed physiological responses were species and nutrient specific. The values of the chlorophyll-specific absorption cross section (a*) increased with nutrient limitation due to package effect, while the carbon/Chl a ratio was higher under N and P limitations. In diatoms, Si limitation did not affect photosynthesis confirming the uncoupling between Si and carbon metabolisms. In all four species and under all treatments, significant relationships were found between photosynthetic activities, ETR^Chl (electron transport rate) and P^Chl (carbon fixation rate) estimated using PAM measurements and ¹³C incorporation, showing that the fluorescence technique can reliably be used to estimate carbon fixation by phytoplankton. The relationship between ETR^Chl and P^Chl can be described by the shape and the slope of the curve (Φ_C.e). Linear relationships were found for dinoflagellates and P. pungens under all treatments. A decrease in Φ_C.e was observed under N and P limitation probably due to structural damage to the photosynthetic apparatus. A. glacialis showed a logarithmic relationship in N and P limited conditions, due to the alternative electron flow which takes place to optimise photosynthetic performances under high light and/or nutrient stress.     

Diel variation of the cellular carbon to nitrogen ratio of Chlorella autotrophica (Chlorophyta) growing in phosphorus‐ and nitrogen‐limited continuous cultures

We investigated the relationship between daily growth rates and diel variation of carbon (C) metabolism and C to nitrogen (N) ratio under P‐ and N‐limitation in the green algae Chlorella autotrophica. To do this, continuous cultures of C. autotrophica were maintained in a cyclostat culture system under 14:10 light:dark cycle over a series of P‐ and N‐limited growth rates. Cell abundance, together with cell size, as reflected by side scatter signal from flow cytometric analysis demonstrated a synchronized diel pattern with cell division occurring at night. Under either type of nutrient limitation, the cellular C:N ratio increased through the light period and decreased through the dark period over all growth rates, indicating a higher diel variation of C metabolism than that of N. Daily average cellular C:N ratios were higher at lower dilution rates under both types of nutrient limitation but cell enlargement was only observed at lower dilution rates under P‐limitation. Carbon specific growth rates during the dark period positively correlated with cellular daily growth rates (dilution rates), with net loss of C during night at the lowest growth rates under N‐limitation. Under P‐limitation, dark C specific growth rates were close to zero at low dilution rates but also exhibited an increasing trend at high dilution rates. In general, diel variations of cellular C:N were low when dark C specific growth rates were high. This result indicated that the fast growing cells performed dark C assimilation at high rates, hence diminished the uncoupling of C and N metabolism at night.     

Modified alternative oxidase expression results in different reactive oxygen species contents in Arabidopsis cell culture but not in whole plants

Alternative oxidase (AOX) transfers electrons from ubiquinone to oxygen in the respiratory chain of plant mitochondria. It is widely accepted that AOX functions as a mechanism decreasing the formation of reactive oxygen species (ROS) produced during respiratory electron transport. However, there are no experimental data to provide unambiguous proof of this hypothesis. We have studied growth characteristics, ROS content, and stress sensitivity in Arabidopsis transgenic lines with reduced or increased levels of AOX. We demonstrated that AOX-deficient plants grown in soil had an extended reproductive phase. Changes in AOX activity did not affect ROS content or stress sensitivity in the whole plants. However in the suspension culture, cells overexpressing AOX had significantly lower ROS content, whereas the AOX-deficient cells had higher ROS contents compared to the wild-type (WT) cells. Prooxidant treatment led to the increase in ROS content and to the reduction of viability more in the cells overexpressing AOX than in WT and AOX-deficient cells. Thus, we demonstrated that differences in the metabolism of whole plants and cultured cells might affect AOX functioning.     

Soil parent material—A major driver of plant nutrient limitations in terrestrial ecosystems

Because the capability of terrestrial ecosystems to fix carbon is constrained by nutrient availability, understanding how nutrients limit plant growth is a key contemporary question. However, what drives nutrient limitations at global scale remains to be clarified. Using global data on plant growth, plant nutritive status, and soil fertility, we investigated to which extent soil parent materials explain nutrient limitations. We found that N limitation was not linked to soil parent materials, but was best explained by climate: ecosystems under harsh (i.e., cold and or dry) climates were more N‐limited than ecosystems under more favourable climates. Contrary to N limitation, P limitation was not driven by climate, but by soil parent materials. The influence of soil parent materials was the result of the tight link between actual P pools of soils and physical–chemical properties (acidity, P richness) of soil parent materials. Some other ground‐related factors (i.e., soil weathering stage, landform) had a noticeable influence on P limitation, but their role appeared to be relatively smaller than that of geology. The relative importance of N limitation versus P limitation was explained by a combination of climate and soil parent material: at global scale, N limitation became prominent with increasing climatic constraints, but this global trend was modulated at lower scales by the effect of parent materials on P limitation, particularly under climates favourable to biological activity. As compared with soil parent materials, atmospheric deposition had only a weak influence on the global distribution of actual nutrient limitation. Our work advances our understanding of the distribution of nutrient limitation at global scale. In particular, it stresses the need to take soil parent materials into account when investigating plant growth response to environment changes.     

A prokaryotic alternative oxidase present in the bacterium Novosphingobium aromaticivorans

The alternative oxidase (AOX) is a terminal oxidase present in the respiratory chain of all plants as well as some yeasts and trypanosomes, but has not previously been found in a prokaryote. We have identified an AOX homologue in Novosphingobium aromaticivorans, the first AOX found in a prokaryote. We have cloned the gene for the N. aromaticivorans AOX and showed it to have a terminal oxidase activity when heterologously expressed in Escherichia coli. We have also shown that this novel AOX is expressed in N. aromaticivorans cells, and that its expression level is greatly influenced by the oxygen level and carbon source of the growth media.     

Induction of mitochondrial alternative oxidase in response to a cell signal pathway down-regulating the cytochrome pathway prevents programmed cell death

Treatment of tobacco (Nicotiana tabacum L. cv Petit Havana SR1) cells with cysteine (Cys) triggers a signal pathway culminating in a large loss of mitochondrial cytochrome (cyt) pathway capacity. This down-regulation of the cyt path likely requires events outside the mitochondrion and is effectively blocked by cantharidin or endothall, indicating that protein dephosphorylation is one critical process involved. Generation of reactive oxygen species, cytosolic protein synthesis, and Ca(2+) flux from organelles also appear to be involved. Accompanying the loss of cyt path is a large induction of alternative oxidase (AOX) protein and capacity. Induction of AOX allows the cells to maintain high rates of respiration, indicating that the lesion triggered by Cys is in the cyt path downstream of ubiquinone. Consistent with this, transgenic (AS8) cells unable to induce AOX (due to the presence of an antisense transgene) lose all respiratory capacity upon Cys treatment. This initiates in AS8 a programmed cell death pathway, as evidenced by the accumulation of oligonucleosomal fragments of DNA as the culture dies. Alternatively, wild-type cells remain viable and eventually recover their cyt path. Induction of AOX in response to a chemical inhibition of the cyt path (by antimycin A) is also dependent upon protein dephosphorylation and the generation of reactive oxygen species. Common events required for both down-regulation of the cyt path and induction of AOX may represent a mechanism to coordinate the biogenesis of these two electron transport paths. Such coordinate regulation may be necessary, not only to satisfy metabolic demands, but also to modulate the initiation of a programmed cell death pathway responsive to mitochondrial respiratory status.     

Alterations in the mitochondrial alternative NAD(P)H Dehydrogenase NDB4 lead to changes in mitochondrial electron transport chain composition, plant growth and response to oxidative stress

The branched respiratory electron transport chain of plants contains a non-phosphorylating alternative pathway consisting of type II NAD(P)H dehydrogenases on both sides of the inner membrane linked through the ubiquinone pool to an alternative oxidase (AOX). T-DNA and RNA interference (RNAi) were used to reduce gene expression to characterize the external NAD(P)H dehydrogenase NDB4 in Arabidopsis. The ndb4 lines showed different levels of suppression of NDB4 protein, leading to increases in NBD2 and AOX1a mRNA and protein levels in all lines. These changes were associated with lower reactive oxygen species formation and an altered phenotype, including changes in growth rate, root : shoot ratios and leaf area. The general growth pattern for the ndb4 mutants was decreased leaf area early in development (6-15 d) followed by a prompt subsequent increase in leaf area that exceeded the leaf area of the wild type by maturity (the 10-12 rosette stage). This pattern was most evident for the RNAi lines that had increased mitochondrial electron transport capacity. The RNAi lines also exhibited better tolerance to salinity stress, with better growth rates and lower shoot Na? content compared with controls when grown under saline conditions. We hypothesize that these differences reflect the enhanced expression of NDB2 and AOX in the ndb4 mutant plants.     

Coping with iron limitation: a metabolomic study of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

Iron (Fe) is a key element for all living systems, especially for photosynthetic organisms because of its important role in the photosynthetic electron transport chain. Fe limitation in cyanobacteria leads to several physiological and morphological changes. However, the overall metabolic responses to Fe limitation are still poorly understood. In this study, we integrated elemental, stoichiometric, macromolecular, and metabolomic data to shed light on the responses of Synechocystis sp. PCC 6803, a non-N₂-fixing freshwater cyanobacterium, to Fe limitation. Compared to Synechocystis growing at nutrient replete conditions, Fe-limited cultures had lower growth rates and amounts of chlorophyll a, RNA, RNA:DNA, C, N, and P, and higher ratios of protein:RNA, C:N, C:P, and N:P, in accordance with the growth rate hypothesis which predicts faster growing organisms will have decreased biomass RNA contents and C:P and N:P ratios. Fe-limited Synechocystis had lower amounts Fe, Mn, and Mo, and higher amount of Cu. Several changes in amino acids of cultures growing under Fe limitation suggest nitrogen limitation. In addition, we found substantial increases in stress-related metabolites in Fe-limited cyanobacteria such antioxidants. This study represents an advance in understanding the stoichiometric, macromolecular, and metabolic strategies that cyanobacteria use to cope with Fe limitation. This information, moreover, may further understanding of changes in cyanobacterial functions under scenarios of Fe limitation in aquatic ecosystems.     

Nutrient limitation of periphyton growth in arctic lakes in south-west Greenland

Many arctic lakes are oligotrophic systems where phototrophic growth is controlled by nutrient supply. Recent anthropogenic nutrient loading is associated with biological and/or physico-chemical change in several lakes across the arctic. Shifts in nutrient limitation (nitrogen (N), phosphorus (P), or N + P) and associated effects on the growth and composition of algal communities are commonly reported. The Kangerlussuaq region of south-west Greenland forms a major lake district which is considered to receive little direct anthropogenic disturbance. However, long-range transport of pollutant N is now reaching Greenland, and it was hypothesised that a precipitation gradient from the inland ice sheet margin to the coast might also deliver increased N deposition. In situ nutrient bioassays were deployed in three lakes across the region: ice sheet margin, inland (close to Kangerlussuaq) and the coast (near Sisimiut), to determine nutrient limitation of lakes and investigate any effects of nutrients on periphyton growth and community composition. Nutrient limitation differed amongst lakes: N limitation (ice sheet margin), N and P limitation (inland) and N + P co-limitation (coast). Factors including variation in N supply, ice phenology, seasonal algal succession, community structure and physical limnology are explored as mechanisms to explain differences amongst lakes. Nutrient limitation of arctic lakes and associated ecological impacts are highly variable, even across small geographic areas. In this highly sensitive region, future environmental change scenarios carry a strong risk of significantly altering nutrient limitation; in turn, potentially severely impacting lake structure and function.     

酶化学计量揭示5年氮添加加剧毛竹林土壤微生物碳磷限制

土壤胞外酶活性和酶化学计量比能很好地反映土壤养分有效性和微生物对养分的需求变化。然而,氮(N)沉降对亚热带森林土壤微生物养分相对限制情况的影响尚不清楚。通过在亚热带毛竹林进行N添加试验来模拟N沉降,并在试验满5年时进行取样,测定不同处理下土壤养分和与碳(C)、N、磷(P)循环相关的酶活性,利用酶化学计量比及矢量分析探究微生物的养分分配情况。结果表明: N添加显著降低土壤可溶性有机碳、有效磷含量,显著提高有效氮含量。此外,N添加显著降低β-N-乙酰氨基葡糖苷酶(NAG)活性和NAG/微生物生物量碳(MBC),显著提高酸性磷酸酶(ACP)和ACP/MBC。低N和中N处理显著提高酶C/N、矢量长度和矢量角度,但显著降低酶N/P。冗余分析表明,N添加下,土壤有效磷含量的变化是影响土壤酶活性及酶化学计量比变化的主要因子。综上可知,N添加改变了微生物的养分获取策略,即通过减少分配给合成N获取酶的养分来增加合成P获取酶的养分。此外,N添加还加剧了微生物的C、P限制,未来可以施加适量P肥来提高亚热带毛竹林的土壤肥力。     

The flexibility of metabolic interactions between chloroplasts and mitochondria in Nicotiana tabacum leaf

To examine the effect of mitochondrial function on photosynthesis, wild-type and transgenic Nicotiana tabacum with varying amounts of alternative oxidase (AOX) were treated with different respiratory inhibitors. Initially, each inhibitor increased the reduction state of the chloroplast electron transport chain, most severely in AOX knockdowns and least severely in AOX overexpressors. This indicated that the mitochondrion was a necessary sink for photo-generated reductant, contributing to the ‘P700 oxidation capacity’ of photosystem I. Initially, the Complex III inhibitor myxothiazol and the mitochondrial ATP synthase inhibitor oligomycin caused an increase in photosystem II regulated non-photochemical quenching not evident with the Complex III inhibitor antimycin A (AA). This indicated that the increased quenching depended upon AA-sensitive cyclic electron transport (CET). Following 12 h with oligomycin, the reduction state of the chloroplast electron transport chain recovered in all plant lines. Recovery was associated with large increases in the protein amount of chloroplast ATP synthase and mitochondrial uncoupling protein. This increased the capacity for photophosphorylation in the absence of oxidative phosphorylation and enabled the mitochondrion to act again as a sink for photo-generated reductant. Comparing the AA and myxothiazol treatments at 12 h showed that CET optimized photosystem I quantum yield, depending upon the P700 oxidation capacity. When this capacity was too high, CET drew electrons away from other sinks, moderating the P700⁺ amount. When P700 oxidation capacity was too low, CET acted as an electron overflow, moderating the amount of reduced P700. This study reveals flexible chloroplast–mitochondrion interactions able to overcome lesions in energy metabolism.     

Bioenergetic consequences from xenotopic expression of a tunicate AOX in mouse mitochondria: Switch from RET and ROS to FET

Electron transfer from all respiratory chain dehydrogenases of the electron transport chain (ETC) converges at the level of the quinone (Q) pool. The Q redox state is thus a function of electron input (reduction) and output (oxidation) and closely reflects the mitochondrial respiratory state. Disruption of electron flux at the level of the cytochrome bc₁ complex (cIII) or cytochrome c oxidase (cIV) shifts the Q redox poise to a more reduced state which is generally sensed as respiratory stress. To cope with respiratory stress, many species, but not insects and vertebrates, express alternative oxidase (AOX) which acts as an electron sink for reduced Q and by-passes cIII and cIV. Here, we used Ciona intestinalis AOX xenotopically expressed in mouse mitochondria to study how respiratory states impact the Q poise and how AOX may be used to restore respiration. Particularly interesting is our finding that electron input through succinate dehydrogenase (cII), but not NADH:ubiquinone oxidoreductase (cI), reduces the Q pool almost entirely (>90%) irrespective of the respiratory state. AOX enhances the forward electron transport (FET) from cII thereby decreasing reverse electron transport (RET) and ROS specifically when non-phosphorylating. AOX is not engaged with cI substrates, however, unless a respiratory inhibitor is added. This sheds new light on Q poise signaling, the biological role of cII which enigmatically is the only ETC complex absent from respiratory supercomplexes but yet participates in the tricarboxylic acid (TCA) cycle. Finally, we delineate potential risks and benefits arising from therapeutic AOX transfer.     

PHOSPHATE UPTAKE UNDER NITRATE LIMITATION BY SCENEDESMUS SP. AND ITS ECOLOGICAL IMPLICATIONS1

Under nitrogen limitation the phosphate content of Scenedesmus sp. shows little variation regardless of growth rate and the N/P atomic ratio of the medium. P uptake therefore can be calculated as the product of P content and N-dependent growth rate. The maximum rate of P uptake in N limitation is lower by a factor of about 8 than the rate in P limitation. As reported earlier, P uptake by this alga under P limitation is described by the kinetics resembling non-competitive enzyme inhibition, with one or several intracellular P fractions as inhibitors. These fractions include surplus P (water extractable) and inorganic polyphosphate fractions A (acid soluble) and B, C, and D (acid insoluble). In N limitation, the ratios of fractions A, B, C, and D are quite different from the ratios of P limitation at comparable growth rates. The concentrations of polyphosphate fraction A in N-limited cells are much, higher than the levels in P-limited cells, and this fraction becomes more predominant at low growth rates in N limitation. This fraction, if introduced as the inhibitor into the noncompetitive scheme, explains the uptake kinetics in both N- and P-limited cells and the low maximum uptake rate in N limitation. This finding may have two significant ecological implications: (1) A nutrient imbalance which brings about changes in the internal, level or the metabolism, of fraction A would affect P uptake. (2) Nitrogen sufficiency would cause a competitive advantage in P uptake. This advantage would be shared by N₂ fixers and algae with low optimum N/P ratios. In Scenedesmus sp. P limitation switches to N limitation and vice versa when the cell N/P atomic ratio is about 30.