Differential Expression of Alternative Oxidase Genes in Soybean Cotyledons during Postgerminative Development

The expression of the alternative oxidase (AOX) was investigated during cotyledon development in soybean (Glycine max [L.] Merr.) seedlings. The total amount of AOX protein increased throughout development, not just in earlier stages as previously thought, and was correlated with the increase in capacity of the alternative pathway. Each AOX isoform (AOX1, AOX2, and AOX3) showed a different developmental trend in mRNA abundance, such that the increase in AOX protein and capacity appears to involve a shift in gene expression from AOX2 to AOX3. As the cotyledons aged, the size of the mitochondrial ubiquinone pool decreased. We discuss how this and other factors may affect the alternative pathway activity that results from the developmental regulation of AOX expression.     

Saline Stress Alters the Temporal Patterns of Xylem Differentiation and Alternative Oxidase Expression in Developing Soybean Roots

We conducted a coordinated biochemical and morphometric analysis of the effect of saline conditions on the differentiation zone of developing soybean (Glycine max L.) roots. Between d 3 and d 14 for seedlings grown in control or NaCl-supplemented medium, we studied (a) the temporal evolution of the respiratory alternative oxidase (AOX) capacity in correlation with the expression and localization of AOX protein analyzed by tissue-print immunoblotting; (b) the temporal evolution and tissue localization of a peroxidase activity involved in lignification; and (c) the structural changes, visualized by light microscopy and quantified by image digitization. The results revealed that saline stress retards primary xylem differentiation. There is a corresponding delay in the temporal pattern of AOX expression, which is consistent with the xylem-specific localization of AOX protein and the idea that this enzyme is linked to xylem development. An NaCl-induced acceleration of the development of secondary xylem was also observed. However, the temporal pattern of a peroxidase activity localized in the primary and secondary xylem was unaltered by NaCl treatment. Thus, the NaCl-stressed root was specifically affected in the temporal patterns of AOX expression and xylem development.     

Regulation of alternative oxidase activity during phosphate deficiency in bean roots (Phaseolus vulgaris)

Cyanide-resistant respiration was studied in mitochondria isolated from the roots of bean plants ( Phaseolus vulgaris L. cv. Złota Saxa) grown hydroponically up to 16 days on a phosphate-sufficient (+P, control) or phosphate-deficient (−P) medium. Western blotting indicated that the alternative oxidase (AOX) was present only in its reduced (active) form, both in phosphate-sufficient and phosphate-deficient roots, but in the latter, the amount of AOX protein was greater. Addition of pyruvate to the isolation, washing and reaction media made mitochondria from +P roots cyanide-insensitive, similar to mitochondria from −P roots. The doubled activity of NAD-malic enzyme (NAD-ME) in −P compared with +P root mitochondria may suggest increased pyruvate production in −P mitochondria. Lower cytochrome c oxidase (COX) activity and no uncoupler effect on respiration indicated limited cytochrome chain activity in −P mitochondria. In −P mitochondria, the oxygen uptake decreased and the level of Q reduction increased from 60 to 80%. With no pyruvate present (AOX not fully activated), inhibition of the cytochrome pathway resulted in an increased level of the ratio of reduced ubiquinone (Qr) to total ubiquinone (Qt) (Qr/Qt) in +P mitochondria, but did not change Qr/Qt in −P mitochondria. When pyruvate was present, the kinetics for AOX were similar in mitochondria from −P and +P roots. It is suggested that AOX participation in −P respiration may provide an acclimation to phosphate deficiency. Stabilization of the ubiquinone reduction level by AOX might prevent the harmful effect of an increased formation of reactive oxygen species.     

The Regulation of Electron Partitioning between the Cytochrome and Alternative Pathways in Soybean Cotyledon and Root Mitochondria

The regulation of electron partitioning between the cytochrome (Cyt) and alternative pathways in soybean (Glycine max L. cv Ransom) mitochondria in the absence of added inhibitors has been studied using the oxygen isotope fractionation technique. This regulation can depend on several factors, including the amount of alternative oxidase protein, the redox status of the alternative oxidase regulatory sulfhydryl-disulfide system, the degree of activation by [alpha]-keto acids, and the concentration and redox state of the ubiquinone pool. We studied electron partitioning onto the alternative pathway in mitochondria isolated from etiolated and light-grown cotyledons and roots to ascertain how these factors interact in different tissues. In light-grown cotyledon mitochondria there is some partitioning to the alternative pathway in state 4, which is increased dramatically by either pyruvate or dithiothreitol. In etiolated cotyledon mitochondria, the alternative pathway shows little ability to compete for electrons with the Cyt pathway under any circumstances. In root mitochondria, control of alternative pathway activity is exercised by both the ubiquinone pool and the regulatory sulfhydryl-disulfide system. In addition, oxygen isotope fractionation by the Cyt and alternative pathways in mitochondria were identical to the fractionation for the respective pathways seen in intact tissue, suggesting that residual respiration is not present in the absence of inhibitors.     

Regulation of alternative oxidase activity in six wild monocotyledonous species. An in vivo study at the whole root level

The activity of the alternative pathway is affected by a number of factors, including the level and reduction state of the alternative oxidase (AOX) protein, and the reduction state of the ubiquinone pool. To investigate the significance of these factors for the rate of alternative respiration in vivo, we studied root respiration of six wild monocotyledonous grass species that were grown under identical controlled conditions. The activity of the alternative pathway was determined using the oxygen isotope fractionation technique. In all species, the AOX protein was invariably in its reduced (high activity) state. There was no correlation between AOX activity and AOX protein concentration, ubiquinone (total, reduced, or oxidized) concentration, or the reduction state of the ubiquinone pool. However, when some of these factors are combined in a linear regression model, a good fit to AOX activity is obtained. The function of the AOX is still not fully understood. It is interesting that we found a positive correlation between the activity of the alternative pathway and relative growth rate; a possible explanation for this correlation is discussed. Inhibition of the AOX (with salicylhydroxamic acid) decreases respiration rates less than the activity present before inhibition (i.e. measured with the 18O-fractionation technique).     

The alternative oxidase in roots of poa annua after transfer from high-light to low-light conditions

The activity of the alternative pathway can be affected by a number of factors, including the amount and reduction state of the alternative oxidase protein, and the reduction state of the ubiquinone pool. To investigate the importance of these factors in vivo, we manipulated the rate of root respiration by transferring the annual grass Poa annua L. from high-light to low-light conditions, and at the same time from long-day to short-day conditions for four days. As a result of the low-light treatment, the total respiration rate of the roots decreased by 45%, in vitro cytochrome c oxidase capacity decreased by 49%, sugar concentration decreased by 90% and the ubiquinone concentration increased by 31%, relative to control values. The absolute rate of oxygen uptake via the alternative pathway, as determined using the 18O-isotope fractionation technique, did not change. Conversely, the cytochrome pathway activity decreased during the low-light treatment; its activity increased upon addition of exogenous sugars to the roots. Interestingly, no change was observed in the concentration of the alternative oxidase protein or in the reduction state of the protein. Also, there was no change in the reduction state of the ubiquinone pool. In conclusion, the concentration and activity of the alternative oxidase were not changed, even under severe light deprivation.     

Leaf respiration and alternative oxidase in field-grown alpine grasses respond to natural changes in temperature and light

? We report the first investigation of changes in electron partitioning via the alternative respiratory pathway (AP) and alternative oxidase (AOX) protein abundance in field-grown plants and their role in seasonal acclimation of respiration. ? We sampled two alpine grasses native to New Zealand, Chionochloa rubra and Chionochloa pallens, from field sites of different altitudes, over 1 yr and also intensively over a 2-wk period. ? In both species, respiration acclimated to seasonal changes in temperature through changes in basal capacity (R??) but not temperature sensitivity (E?). In C. pallens, acclimation of respiration may be associated with a higher AOX : cytochrome c oxidase (COX) protein abundance ratio. Oxygen isotope discrimination (D), which reflects relative changes in AP electron partitioning, correlated positively with daily integrated photosynthetically active radiation (PAR) in both species over seasonal timescales. Respiratory parameters, the AOX : COX protein ratio and D were stable over a 2-wk period, during which significant temperature changes were experienced in the field. ? We conclude that respiration in Chionochloa spp. acclimates strongly to seasonal, but not to short-term, temperature variation. Alternative oxidase appears to be involved in the plant response to both seasonal changes in temperature and daily changes in light, highlighting the complexity of the function of AOX in the field.     

The Responses of Cytochrome Redox State and Energy Metabolism to Dehydration Support a Role for Cytoplasmic Viscosity in Desiccation Tolerance

To characterize the depression of metabolism in anhydrobiotes, the redox state of cytochromes and energy metabolism were studied during dehydration of soaked cowpea (Vigna unguiculata) cotyledons and pollens of Typha latifolia and Impatiens glandulifera. Between water contents (WC) of 1.0 and 0.6 g H₂O/g dry weight (g/g), viscosity as measured by electron spin resonance spectroscopy increased from 0.15 to 0.27 poise. This initial water loss was accompanied by a 50% decrease in respiration rates, whereas the adenylate energy charge remained constant at 0.8, and cytochrome c oxidase (COX) remained fully oxidized. From WC of 0.6 to 0.2 g/g, viscosity increased exponentially. The adenylate energy charge declined to 0.4 in seeds and 0.2 in pollen, whereas COX became progressively reduced. At WC of less than 0.2 g/g, COX remained fully reduced, whereas respiration ceased. When dried under N₂, COX remained 63% reduced in cotyledons until WC was 0.7 g/g and was fully reduced at 0.2 g/g. During drying under pure O₂, the pattern of COX reduction was similar to that of air-dried tissues, although the maximum reduction was 70% in dried tissues. Thus, at WC of less than 0.6 g/g, the reduction of COX probably originates from a decreased O₂ availability as a result of the increased viscosity and impeded diffusion. We suggest that viscosity is a valuable parameter to characterize the relation between desiccation and decrease in metabolism. The implications for desiccation tolerance are discussed.     

Respiratory Elicitors from Rhizobium meliloti Affect Intact Alfalfa Roots

Molecules produced by Rhizobium meliloti increase respiration of alfalfa (Medicago sativa L.) roots. Maximum respiratory increases, measured either as CO₂ evolution or as O₂ uptake, were elicited in roots of 3-d-old seedlings by 16 h of exposure to living or dead R. meliloti cells at densities of 10⁷ bacteria/mL. Excising roots after exposure to bacteria and separating them into root-tip- and root-hair-containing segments showed that respiratory increases occurred only in the root-hair region. In such assays, CO₂ production by segments with root hairs increased by as much as 100% in the presence of bacteria. Two partially purified compounds from R. meliloti 1021 increased root respiration at very low, possibly picomolar, concentrations. One factor, peak B, resembled known pathogenic elicitors because it produced a rapid (15-min), transitory increase in respiration. A second factor, peak D, was quite different because root respiration increased slowly for 8 h and was maintained at the higher level. These molecules differ from lipo-chitin oligosaccharides active in root nodulation for the following reasons: (a) they do not curl alfalfa root hairs, (b) they are synthesized by bacteria in the absence of known plant inducer molecules, and (c) they are produced by a mutant R. meliloti that does not synthesize known lipo-chitin oligosaccharides. The peak-D compound(s) may benefit both symbionts by increasing CO₂, which is required for growth of R. meliloti, and possibly by increasing the energy that is available in the plant to form root nodules.     

Glyceollin I in soybean-cyst nematode interactions : spatial and temporal distribution in roots of resistant and susceptible soybeans

Accumulation of the phytoalexin glyceollin I in roots of soybean (Glycine max [L.] Merr.) following inoculation with race 1 of Heterodera glycines Ichinohe, the soybean cyst nematode (SCN), was determined in a whole-root system by high performance liquid chromatography (HPLC) and in a cross-section system by a radioimmunoassay procedure. In the whole-root system, roots were harvested from controls and nematode-inoculated seedlings immediately after inoculation and at 2-day intervals for 8 days. The roots were extracted with ethanol, and the extracts were subjected to HPLC. Glyceollin I was not detected in roots of either resistant cultivar Centennial or susceptible cultivar Ransom immediately after inoculation with SCN but steadily accumulated in large quantity in roots of Centennial. Accumulation of glyceollin I in roots of Ransom following nematode inoculation was minimal. In the cross-section system, 3-day-old soybean seedlings were inoculated with juvenile nematodes, and root segments containing a single nematode were dissected from inoculated plants at 4-hour intervals under a dissecting microscope. The root segments were embedded in ice and cut into 16-micrometer sections with a cryostat microtome. The spatial and temporal distribution of glyceollin I was determined with a radioimmunoassay procedure specific for the phytoalexin. Glyceollin I was found to accumulate in tissues immediately adjacent to the head region of the nematode in Centennial but not in Ransom. Glyceollin I was detected 8 hours after nematode penetration, and the concentration increased steadily up to 0.3 micromole per milliliter in Centennial 24 hours after penetration.     

Respiration in postharvest sugarbeet roots is not limited by respiratory capacity or adenylates

Control of respiration has largely been studied with growing and/or photosynthetic tissues or organs, but has rarely been examined in harvested and stored plant products. As nongrowing, heterotrophic organs that are reliant on respiration to provide all of their metabolic needs, harvested plant products differ dramatically in their metabolism and respiratory needs from growing and photosynthetically active plant organs, and it cannot be assumed that the same mechanism controls respiration in both actively growing and harvested plant organs. To elucidate mechanisms of respiratory control for a harvested and stored plant product, sugarbeet (Beta vulgaris L.) root respiration was characterized with respect to respiratory capacity, adenylate levels and cellular energy status in roots whose respiration was altered by wounding or cold treatment (1 degrees C) and in response to potential effectors of respiration. Respiration rate was induced by wounding in roots stored at 10 degrees C and by cold temperature in roots stored at 1 degrees C for 11-13d. Alterations in respiration rate due to wounding or storage temperature were unrelated to changes in total respiratory capacity, the capacities of the cytochrome c oxidase (COX) or alternative oxidase (AOX) pathways, adenylate concentrations or cellular energy status, measured by the ATP:ADP ratio. In root tissue, respiration was induced by exogenous NADH indicating that respiratory capacity was capable of oxidizing additional electrons fed into the electron transport chain via an external NADH dehydrogenase. Respiration was not induced by addition of ADP or a respiratory uncoupler. These results suggest that respiration rate in stored sugarbeet roots is not limited by respiratory capacity, ADP availability or cellular energy status. Since respiration in plants can be regulated by substrate availability, respiratory capacity or energy status, it is likely that a substrate, other than ADP, limits respiration in stored sugarbeet roots.     

Alternative Oxidase Dependent Respiration Leads to an Increased Mitochondrial Content in Two Long-Lived Mutants of the Ageing Model Podospora anserina

The retrograde response constitutes an important signalling pathway from mitochondria to the nucleus which induces several genes to allow compensation of mitochondrial impairments. In the filamentous ascomycete Podospora anserina, an example for such a response is the induction of a nuclear-encoded and iron-dependent alternative oxidase (AOX) occurring when cytochrome-c oxidase (COX) dependent respiration is affected. Several long-lived mutants are known which predominantly or exclusively respire via AOX. Here we show that two AOX-utilising mutants, grisea and PaCox17::ble, are able to compensate partially for lowered OXPHOS efficiency resulting from AOX-dependent respiration by increasing mitochondrial content. At the physiological level this is demonstrated by an elevated oxygen consumption and increased heat production. However, in the two mutants, ATP levels do not reach WT levels. Interestingly, mutant PaCox17::ble is characterized by a highly increased release of the reactive oxygen species (ROS) hydrogen peroxide. Both grisea and PaCox17::ble contain elevated levels of mitochondrial proteins involved in quality control, i. e. LON protease and the molecular chaperone HSP60. Taken together, our work demonstrates that AOX-dependent respiration in two mutants of the ageing model P. anserina is linked to a novel mechanism involved in the retrograde response pathway, mitochondrial biogenesis, which might also play an important role for cellular maintenance in other organisms.     

The gene for alternative oxidase-2 (AOX2) from Arabidopsis thaliana consists of five exons unlike other AOX genes and is transcribed at an early stage during germination.

We investigated the expressions of genes for alternative oxidase (AOX1a, AOX1b, AOX1c and AOX2) and genes for cytochrome c oxidase (COX5b and COX6b) during germination of Arabidopsis thaliana, and examined oxygen uptakes of the alternative respiration and the cytochrome respiration in imbibed Arabidopsis seeds. A Northern blot analysis showed that AOX2 mRNA has already accumulated in dry seeds and subsequently decreased, whereas accumulation ofAOX1a mRNA was less abundant from 0 hours to 48 hours after imbibition and then increased. The increase of the capacity of the alternative pathway appeared to be dependent on the expressions of both AOX2 and AOX1a. On the other hand, steady-state mRNA levels of COX5b and COX6b were gradually increased during germination, and the capacity of the cytochrome pathway was correlated with the increase of expressions of the COX genes. Antimycin A, the respiratory inhibitor, strongly increased the expression of AOX1a but had no effect on the expression of AOX2. A 5'RACE analysis showed that AOX2 consists of five exons, which is different from the case of most AOX genes identified so far. Analysis of subcellular localization of AOX2 using green fluorescent protein indicated that the AOX2 protein is imported into the mitochondria.     

Maintenance of growth rate at low temperature in rice and wheat cultivars with a high degree of respiratory homeostasis is associated with a high efficiency of respiratory ATP production

Some plants have the ability to maintain similar respiratory rates (measured at the growth temperature) when grown at different temperatures. This phenomenon is referred to as respiratory homeostasis. Using wheat and rice cultivars with different degrees of respiratory homeostasis (H), we previously demonstrated that high-H cultivars maintained shoot and root growth at low temperature [Kurimoto et al. (2004) Plant Cell Environ., 27: 853]. Here, we assess the relationship between respiratory homeostasis and the efficiency of respiratory ATP production, by measuring the levels of alternative oxidase (AOX) and uncoupling protein (UCP), which have the potential to decrease respiratory ATP production per unit of oxygen consumed. We also measured SHAM- and CN-resistant respiration of intact roots, and the capacity of the cytochrome pathway (CP) and AOX in isolated mitochondria. Irrespective of H, SHAM-resistant respiration of intact roots and CP capacity of isolated root mitochondria were larger when plants were grown at low temperature, and the maximal activity and relative amounts of cytochrome c oxidase showed a similar trend. In contrast, CN-resistant respiration of intact roots and relative amounts of AOX protein in mitochondria isolated from those roots, were lower in high-H plants grown at low temperature. In the roots of low-H cultivars, relative amounts of AOX protein were higher at low growth temperature. Relative amounts of UCP protein showed similar trends to AOX. We conclude that maintenance of growth rate in high-H plants grown at low temperature is associated with both respiratory homeostasis and a high efficiency of respiratory ATP production.     

Seasonal variation of leaf respiration and the alternative pathway in field-grown Populus × canadensis

The temperature response of plant respiration varies between species and can acclimate to changing temperatures. Mitochondrial respiration in plants has two terminal oxidases: the cytochrome c oxidase (COX) and the cyanide-resistant alternative oxidase (AOX). In Populus × canadensis var. italica, a deciduous tree species, we investigated the temperature response of leaf respiration via the alternative and cytochrome pathways, as well as seasonal changes in these pathways, using the oxygen isotope fractionation technique. The electron partitioning through the alternative pathway (τ(a) ) increased from 0 to 30-40% with measurement temperatures from 6 to 30°C at all times measured throughout the growing season. τ(a) at the growth temperature (the average temperature during 3 days prior to sampling) increased from 12 to 29% from spring until late summer and decreased thereafter. Total respiration declined throughout the growing season by 50%, concomitantly with decreases in both AOX (64%) and COX (32%) protein abundances. Our results provide new insight into the natural variability of AOX protein abundances and alternative respiration electron partitioning over immediate and seasonal timescales.     

Temperature-Stress-Induced Impairment of Chlorophyll Biosynthetic Reactions in Cucumber and Wheat

Chlorophyll (Chl) biosynthesis in chill (7°C)- and heat (42°C)-stressed cucumber (Cucumis sativus L. cv poinsette) seedlings was affected by 90 and 60%, respectively. Inhibition of Chl biosynthesis was partly due to impairment of 5-aminolevulinic acid biosynthesis both in chill- (78%) and heat-stress (70%) conditions. Protochlorophyllide (Pchlide) synthesis in chill- and heat-stressed seedlings was inhibited by 90 and 70%, respectively. Severe inhibition of Pchlide biosynthesis in chill-stressed seedlings was caused by inactivations of all of the enzymes involved in protoporphyrin IX (Proto IX) synthesis, Mg-chelatase, and Mg-protoporphyrin IX monoester cyclase. In heat-stressed seedlings, although 5-aminolevulinic acid dehydratase and porphobilinogen deaminase were partially inhibited, one of the porphyrinogen-oxidizing enzymes, uroporphyrinogen decarboxylase, was stimulated and coproporphyrinogen oxidase and protoporphyrinogen oxidase were not substantially affected, which demonstrated that protoporphyrin IX synthesis was relatively more resistant to heat stress. Pchlide oxidoreductase, which is responsible for phototransformation of Pchlide to chlorophyllide, increased in heat-stress conditions by 46% over that of the control seedlings, whereas it was not affected in chill-stressed seedlings. In wheat (Triticum aestivum L. cv HD2329) seedlings porphobilinogen deaminase, Pchlide synthesis, and Pchlide oxidoreductase were affected in a manner similar to that of cucumber, suggesting that temperature stress has a broadly similar effect on Chl biosynthetic enzymes in both cucumber and wheat.     

Effect of Temperature on Rates of Alternative and Cytochrome Pathway Respiration and Their Relationship with the Redox Poise of the Quinone Pool

We investigated the effect of short-term changes in temperature on alternative (Alt) and cytochrome (Cyt) pathway respiration, both in intact tissues and isolated mitochondria of 14-d-old cotyledons of soybean (Glycine max L. cv Stevens). We also established the extent to which temperature alters the interaction between the oxidizing pathways and the level of ubiquinone (UQ) reduction (UQ(r)/UQ(t)). No difference was found between the temperature coefficient of respiration (Q(10); proportional change per 10 degrees C) of Alt and Cyt pathway respiration in cotyledon slices (Q(10) = 1.92 and 1.86, respectively). In isolated mitochondria, the Q(10) of the fully activated Alt pathway (Q(10) = 2.24-2.61) was always equal to, or higher than, that of Cyt c oxidase (COX) alone (Q(10) = 2.08) and the complete Cyt pathway (Q(10) = 2.40-2.55). This was true regardless of substrate or whether ADP was present. There was little difference in the Q(10) of the Cyt pathway with or without ADP; however, the Q(10) of COX was substantially lower in the presence of an uncoupler (Q(10) = 1.61) than its absence (Q(10) = 2.08). The kinetics of Alt and Cyt pathway activity in relation to UQ(r)/UQ(t) were not affected by temperature. For a given UQ(r)/UQ(t) value, the proportion of maximum flux taking place was similar at all temperatures for both pathways (+/-ADP). However, the Q(10) of the Alt and the Cyt pathways (+ADP) increased with increasing UQ(r)/UQ(t). We conclude that the Alt pathway is not less temperature sensitive than the Cyt pathway or COX per se and that changes in the degree of control exerted by individual steps in the respiratory apparatus could result in changes in the Q(10) of mitochondrial O(2) uptake.     

Increased root oxygen uptake in pea plants responding to non-self neighbors

Recent studies have demonstrated that plants alter root growth and decrease competition with roots of the same individual (self); however, the physiological traits accompanying this response are still widely unknown. In this study, we investigated the effect of root identity on gas exchange in the model species pea (Pisum sativum L.). Split-root plants were planted so that each pot contained either two roots of the same plant (self) or of two different plants (non-self), and the responses of biomass, photosynthesis, and respiration were measured. The photosynthetic rate was not affected by the identity of the root neighbor. We found a reduction of leaf dark respiration by half, accompanied by an increase in nocturnal root respiration by 29 % in plants neighboring with non-self. The activity of the alternative oxidase (AOX) pathway increased when plants responded to non-self neighbors. The increased activity of AOX in plants responding to non-self indicates carbon imbalances in roots, possibly as a consequence of increased root exudation and communication between individuals. If such an effect occurs more widely, it may change the assumptions made for the quantity of respiration as used in carbon budget models.