Mechanisms of thermoregulation in plants

Endothermic heating of floral tissues and even thermoregulation is known to occur in a number of plant species across a wide taxonomic range. The mechanisms by which flowers heat, however, are only just beginning to be understood, and even less is known about how heating is regulated in response to changes in ambient temperature. We have recently demonstrated that the alternative pathway of respiration, in which the alternative oxidase (AOX) rather than cytochrome C (COX) acts as terminal electron acceptor, is responsible for heat generation in one thermoregulating species, the sacred lotus (Nelumbo nucifera). In the March issue of the Journal of Experimental Botany we further demonstrated that AOX-mediated heat production in this species is regulated at both the level of gene expression and also post-translationally. Similarly, AOX has also been implicated in heat production in other thermogenic species. In this addendum we discuss the central role of AOX in heat production and how post-translational mechanisms may provide the fine control necessary for thermoregulation.Key words: alternative oxidase, Nelumbo nucifera, thermogenic plants, uncoupling proteins     

Contribution of the alternative pathway to respiration during thermogenesis in flowers of the sacred lotus

We report results from in vivo measurements, using oxygen isotope discrimination techniques, of fluxes through the alternative and cytochrome respiratory pathways in thermogenic plant tissue, the floral receptacle of the sacred lotus (Nelumbo nucifera). Fluxes through both pathways were measured in thermoregulating flowers undergoing varying degrees of thermogenesis in response to ambient temperature. Significant increases in alternative pathway flux were found in lotus receptacles with temperatures 16 degrees C to 20 degrees C above ambient, but not in those with lesser amounts of heating. Alternative pathway flux in the hottest receptacles was 75% of the total respiratory flux. In contrast, fluxes through the cytochrome pathway did not change significantly during thermogenesis. These data support the hypothesis that increased flux through the alternative pathway is responsible for heating in the lotus and that it is unlikely that uncoupling proteins, which would have produced increased fluxes through the cytochrome pathway, contribute significantly to heating in this tissue. Comparisons of actual flux, with capacity determined using inhibitors, suggested that the alternative pathway was operating at close to maximum capacity in heating tissues of lotus. However, in nonheating tissues the inhibitor data significantly overestimated the alternative pathway flux. This confirms that isotopic measurements are necessary for accurate determination of fluxes through the two pathways.     

In the heat of the night--alternative pathway respiration drives thermogenesis in Philodendron bipinnatifidum

? Philodendron bipinnatifidum inflorescences heat up to 42 °C and thermoregulate. We investigated whether they generate heat via the cytochrome oxidase pathway uncoupled by uncoupling proteins (pUCPs), or the alternative oxidase (AOX). ? Contribution of AOX and pUCPs to heating in fertile (FM) and sterile (SM) male florets was determined using a combination of oxygen isotope discrimination, protein and substrate analyses. ? Both FM and SM florets thermoregulated independently for up to 30 h ex planta. In both floret types, AOX contributed > 90% of respiratory flux during peak heating. The AOX protein increased fivefold with the onset of thermogenesis in both floret types, whereas pUCP remained low throughout development. These data indicate that AOX is primarily responsible for heating, despite FM and SM florets potentially using different substrates, carbohydrates or lipids, respectively. Measurements of discrimination between O? isotopes in strongly respiring SM florets were affected by diffusion; however, this diffusional limitation was largely overcome using elevated O?. ? The first in vivo respiratory flux measurements in an arum show AOX contributes the bulk of heating in P. bipinnatifidum. Fine-scale regulation of AOX activity is post-translational. We also demonstrate that elevated O? can aid measurement of respiratory pathway fluxes in dense tissues.     

Two Cys or Not Two Cys? That Is the Question; Alternative Oxidase in the Thermogenic Plant Sacred Lotus

Sacred lotus (Nelumbo nucifera) regulates temperature in its floral chamber to 32°C to 35°C across ambient temperatures of 8°C to 40°C with heating achieved through high alternative pathway fluxes. In most alternative oxidase (AOX) isoforms, two cysteine residues, Cys₁ and Cys₂, are highly conserved and play a role in posttranslational regulation of AOX. Further control occurs via interaction of reduced Cys₁ with α-keto acids, such as pyruvate. Here, we report on the in vitro regulation of AOX isolated from thermogenic receptacle tissues of sacred lotus. AOX protein was mostly present in the reduced form, and only a small fraction could be oxidized with diamide. Cyanide-resistant respiration in isolated mitochondria was stimulated 4-fold by succinate but not pyruvate or glyoxylate. Insensitivity of the alternative pathway of respiration to pyruvate and the inability of AOX protein to be oxidized by diamide suggested that AOX in these tissues may lack Cys₁. Subsequently, we isolated two novel cDNAs for AOX from thermogenic tissues of sacred lotus, designated as NnAOX1a and NnAOX1b. Deduced amino acid sequences of both confirmed that Cys₁ had been replaced by serine; however, Cys₂ was present. This contrasts with AOXs from thermogenic Aroids, which contain both Cys₁ and Cys₂. An additional cysteine was present at position 193 in NnAOX1b. The significance of the sequence data for regulation of the AOX protein in thermogenic sacred lotus is discussed and compared with AOXs from other thermogenic and nonthermogenic species.

Thermogenesis in Sacred Lotus

Sacred lotus (Nelumbo nucifera) is a thermogenic plant that regulates the temperature of its floral chamber between 32°C and 35°C for up to 4 d (Seymour and Schultze-Motel, 1996). Heating of plant tissues has been described as an adaptation to attract insect pollinators either by volatilization of scent compounds (Meeuse, 1975) or by providing a heat reward (Seymour et al., 1983), protect floral parts from low temperatures (Knutson, 1974), or provide the optimum temperature for floral development (Ervik and Barfod, 1999; Seymour et al., 2009). In sacred lotus, heat is produced by high rates of alternative pathway respiration (Watling et al., 2006; Grant et al., 2008); however, the mechanisms of heat regulation, which likely occur at a cellular level, remain unclear.

Alternative Oxidase

Alternative pathway respiration is catalyzed by the alternative oxidase protein (AOX), which acts as a terminal oxidase in the electron transport chain but, unlike the energy conserving cytochrome pathway (COX), complexes III and IV are bypassed and energy is released as heat. Traditionally, AOX activity was measured using oxygen consumption of tissue, cells, or isolated mitochondria in the presence or absence of AOX and COX inhibitors. However, this method does not accurately measure activity in vivo but does indicate the capacity of the alternative pathway (Ribas-Carbo et al., 1995; Day et al., 1996). The only method to date to accurately determine AOX activity, that is, flux of electrons through the AOX pathway in vivo, is to use oxygen isotope discrimination techniques (for review, see Robinson et al., 1995). Determining AOX activity in vivo is important because heat production in plants could be due to activity of either the AOX and/or plant uncoupling proteins. Using oxygen fractionation techniques, we have shown that flux through the AOX pathway is responsible for heating in sacred lotus (Watling et al., 2006; Grant et al., 2008). Furthermore, we were unable to detect any uncoupling protein in these tissues (Grant et al., 2008). AOX protein content within the sacred lotus receptacle increases markedly prior to thermogenesis, but it remains constant during heating (Grant et al., 2008), suggesting that regulation of heating occurs through posttranslational modification of the protein.

Posttranslational Regulation of AOX Protein

The plant AOX is a cyanide-insensitive dimeric protein located in the inner mitochondrial membrane (Day and Wiskich, 1995). The dimer subunits (monomers) can be linked via a noncovalent association (reduced protein) or covalently through the formation of a disulfide bridge (oxidized protein; Umbach and Siedow, 1993). The reduced protein when run on SDS-PAGE has a molecular mass of approximately 30 to 35 kD and the oxidized protein 60 to 71 kD; this holds true for AOX from a number of species, including soybean (Glycine max) roots and cotyledons (Umbach and Siedow, 1993), tobacco (Nicotiana tabacum) leaf (Day and Wiskich, 1995), and the thermogenic spadix of Arum maculatum (Hoefnagel and Wiskich, 1998).Regulation of AOX has been well studied in nonthermogenic plant species, and two mechanisms have been identified. Most AOX isoforms have two highly conserved Cys residues, Cys₁ and Cys₂ (defined in Berthold et al., 2000 and Holtzapffel et al., 2003), located near the N-terminal hydrophilic domain of the protein. In these isoforms, Cys₁ can either be reduced on both subunits of the AOX dimer, or the Cys₁ sulfhydryl groups can be oxidized to form a disulfide bridge (Rhoads et al., 1998). Reduction/oxidation modulation of AOX in vitro can be achieved using the sulfhydryl reductant dithiothreitol (DTT) to reduce the protein or diamide to oxidize the Cys residues. The reduced dimer can be further activated via the interaction of Cys₁ with α-keto acids, principally pyruvate (Rhoads et al., 1998; see McDonald [2008] for a model of posttranslational regulation of AOX). In addition, Cys₂ may also be involved in regulating AOX activity through interaction with the α-keto acid glyoxylate (which can also stimulate activity at Cys₁; Umbach et al., 2002).Recently, however, AOX proteins with different regulatory properties have been reported. Naturally occurring AOX proteins without the two regulatory Cys residues have been identified and, along with site-directed mutagenesis studies, used to further elucidate the specific roles of Cys₁ and Cys₂. The LeAOX1b isoform from tomato (Lycopersicon esculentum), which has a Ser residue at the position of Cys₁ and thus does not form disulfide linked dimers, is also activated by succinate rather than pyruvate when expressed in Saccharomyces cerevisiae (Holtzapffel et al., 2003). In Arabidopsis (Arabidopsis thaliana), uncharged or hydrophobic amino acid substitutions of either Cys result in an inactive enzyme, while positively charged substitutions produce an enzyme with higher than wild type basal activity but that is insensitive to pyruvate or succinate (Umbach et al., 2002). Single substitutions at Cys₁ or Cys₂ have revealed that glyoxylate can activate AOX via both Cys residues, but only one is needed for glyoxylate stimulation (Umbach et al., 2002, 2006). Double substitution mutants were not stimulated by either pyruvate or glyoxylate (Umbach et al., 2006).Previously, we determined that thermogenesis via the AOX pathway in the sacred lotus receptacle is precisely regulated through changes in AOX flux rather than changes to protein content (Grant et al., 2008). In this study, we investigated the nature of this regulation in mitochondria isolated from heating receptacles. Our aim was to elucidate the reduction/oxidation behavior of the AOX protein and the mechanisms of activation of cyanide-resistant respiration in sacred lotus receptacles to provide insights into the mechanism(s) of heat regulation in this species. We further investigated AOX regulation by determining the amino acid sequence of two novel AOX genes isolated from thermogenic receptacle tissue of sacred lotus.     

Regulation of thermogenesis in flowering Araceae: the role of the alternative oxidase

The inflorescences of several members of the Arum lily family warm up during flowering and are able to maintain their temperature at a constant level, relatively independent of the ambient temperature. The heat is generated via a mitochondrial respiratory pathway that is distinct from the cytochrome chain and involves a cyanide-resistant alternative oxidase (AOX). In this paper we have used flux control analysis to investigate the influence of temperature on the rate of respiration through both cytochrome and alternative oxidases in mitochondria isolated from the appendices of intact thermogenic Arum maculatum inflorescences. Results are presented which indicate that at low temperatures, the dehydrogenases are almost in full control of respiration but as the temperature increases flux control shifts to the AOX. On the basis of these results a simple model of thermoregulation is presented that is applicable to all species of thermogenic plants. The model takes into account the temperature characteristics of the separate components of the plant mitochondrial respiratory chain and the control of each process. We propose that 1) in all aroid flowers AOX assumes almost complete control over respiration, 2) the temperature profile of AOX explains the reversed relationship between ambient temperature and respiration in thermoregulating Arum flowers, 3) the thermoregulation process is the same in all species and 4) variations in inflorescence temperatures can easily be explained by variations in AOX protein concentrations.     

高等植物的交替氧化酶研究进展

交替氧化酶(Alternative Oxidase,AOX)广泛存在于高等植物、藻类和原生生物线粒体内膜。从主呼吸链的辅酶Q分岔,是氧化辅酶Q、还原氧分子生成水的另一终端氧化酶。氧化过程没有质子穿膜运动、热量以产热方式散发。产热植物中交替氧化产生的热量使花粉发出芳香味吸引虫传粉。推测植物AOX使植物在环境胁迫下维持呼吸,调节能量平衡,抵抗氧化胁迫,保持三羧酸循环的运行。AOX是首次发现的双铁羧酸蛋白质成员中的膜蛋白质,AOX与膜分离后容易失活,至今尚未有三级结构的报导,只有二级结构的2种假设模式,最新的模式AOX为膜界面蛋白质而不是跨膜蛋白。最近我们的研究表明有2个途径可获得适量有活性的AOX:建立优化的pFLAG-1-AOX大肠杆菌超量表达系统;从产热植物如斑叶阿若母(Arum maculatum)花序组织线粒体分离纯化有活性的AOX。     

Metabolic activities in germinated ancient lotus seeds

Seeds of Taizi lotus (Nelumbo nucifera Gaertn cv. Taizi) wereunearthed in a western suburb of Beijing in 1984, and determinedto be 58070-years-old by radiocarbon dating. Pretreatment withconcentrated sulphuric acid for 6 h promoted 75% of the seedstested to germinate. Over 3 weeks of incubation after the additionof water, storage starch, total soluble sugar and globulin inthe cotyledons decreased whereas albumin and soluble -aminonitrogen increased. These changes in cotyledonary componentswere similar to the changes seen in four present-day Indianlotus varieties, although the content of reducing sugar in Taizilotus was significantly lower. The polypeptide compositionsin extracts from the cotyledons and embryonic axis of Taiziseeds, as analysed by SDS-PAGE, were similar to those of present-dayJiangxi seeds. Taizi lotus also showed the ability to incorporateradioactive leucine into the trichloroacetic acid (TCA)-insolublefraction of a cotyledonary extract during the 24 h post-imbibitionperiod. The incorporation was inhibited when seeds of Taizilotus were allowed to rehydrate for 24 h in the presence of-amanitin or cycloheximide. Key words: Ancient lotus, Nelumbo nucifera, protein synthesis, seed germination, seed viability     

Expression of uncoupling protein and alternative oxidase depends on lipid or carbohydrate substrates in thermogenic plants

Thermogenesis, in which cellular respiratory activity is considerably stimulated, requires mitochondrial uncoupling protein (UCP) in mammals and an alternative oxidase (AOX) in plants. Here, we show that the genes for both proteins are expressed in thermogenic plants, but the type correlates with the respiratory substrate. A novel gene termed PsUCPa encoding a variant of UCP was specifically expressed in thermogenic flowers of Philodendron selloum, which uses lipids as substrates. However, a gene termed DvAOX encoding for AOX protein was expressed in thermogenic flowers of Dracunculus vulgaris, which presumably uses carbohydrates as substrates. These findings suggest that cellular metabolism is a major determinant in selective expression of appropriate thermogenic genes in plants.     

Analysis of Respiratory Chain Regulation in Roots of Soybean Seedlings

Changes in the respiratory rate and the contribution of the cytochrome (Cyt) c oxidase and alternative oxidase (COX and AOX, respectively) were investigated in soybean (Glycine max L. cv Stevens) root seedlings using the ¹⁸O-discrimination method. In 4-d-old roots respiration proceeded almost entirely via COX, but by d 17 more than 50% of the flux occurred via AOX. During this period the capacity of COX, the theoretical yield of ATP synthesis, and the root relative growth rate all decreased substantially. In extracts from whole roots of different ages, the ubiquinone pool was maintained at 50% to 60% reduction, whereas pyruvate content fluctuated without a consistent trend. In whole-root immunoblots, AOX protein was largely in the reduced, active form at 7 and 17 d but was partially oxidized at 4 d. In isolated mitochondria, Cyt pathway and succinate dehydrogenase capacities and COX I protein abundance decreased with root age, whereas both AOX capacity and protein abundance remained unchanged. The amount of mitochondrial protein on a dry-mass basis did not vary significantly with root age. It is concluded that decreases in whole-root respiration during growth of soybean seedlings can be largely explained by decreases in maximal rates of electron transport via COX. Flux via AOX is increased so that the ubiquinone pool is maintained in a moderately reduced state.     

Decreased expression of cytosolic pyruvate kinase in potato tubers leads to a decline in pyruvate resulting in an in vivo repression of the alternative oxidase

The aim of this work was to investigate the effect of decreased cytosolic pyruvate kinase (PKc) on potato (Solanum tuberosum) tuber metabolism. Transgenic potato plants with strongly reduced levels of PKc were generated by RNA interference gene silencing under the control of a tuber-specific promoter. Metabolite profiling showed that decreased PKc activity led to a decrease in the levels of pyruvate and some other organic acids involved in the tricarboxylic acid cycle. Flux analysis showed that this was accompanied by changes in carbon partitioning, with carbon flux being diverted from glycolysis toward starch synthesis. However, this metabolic shift was relatively small and hence did not result in enhanced starch levels in the tubers. Although total respiration rates and the ATP to ADP ratio were largely unchanged, transgenic tubers showed a strong decrease in the levels of alternative oxidase (AOX) protein and a corresponding decrease in the capacity of the alternative pathway of respiration. External feeding of pyruvate to tuber tissue or isolated mitochondria resulted in activation of the AOX pathway, both in the wild type and the PKc transgenic lines, providing direct evidence for the regulation of AOX by changes in pyruvate levels. Overall, these results provide evidence for a crucial role of PKc in the regulation of pyruvate levels as well as the level of the AOX in heterotrophic plant tissue, and furthermore reveal that these parameters are interlinked in vivo.     

Functional expression of plant alternative oxidase decreases antimycin A-induced reactive oxygen species production in human cells

Alternative oxidase (AOX) plays a pivotal role in cyanide-resistance respiration in the mitochondria of plants, fungi and some protists. Here we show that AOX from thermogenic skunk cabbage successfully conferred cyanide resistance to human cells. In galactose medium, HeLa cells with mitochondria-targeted AOX proteins were found to have significantly less reactive oxygen species production in response to antimycin-A exposure, a specific inhibitor of respiratory complex III. These results suggest that skunk cabbage AOX can be used to create an alternative respiration pathway, which might be important for therapy against various mitochondrial diseases.     

Possible involvement of the mitochondrial alternative pathway in ethylene-induced apoptosis in tomato protoplasts

In this study, a monoclonal antibody to the terminal oxidase of the alternative pathway from Sauromatum guttatum was used to detect the expression of alternative oxidase (AOX) protein in tomato mitochondria. The results show that there was an obvious correlation between the ethylene-induced apoptosis and the levels of AOX protein in tomato cells undergoing ethylene-induced apoptosis. In addition, when tomato protoplasts were preincubated with 2 mM salicylhydroxamic acid (SHAM), an inhibitor of the alternative pathway, before their exposure to ethylene, the TUNEL positive reaction and DNA fragmentation were obviously accelerated. We suggest that AOX may play an important role in protecting tomato protoplasts against ethylene-induced apoptosis.     

Regulation of alternative oxidase 1 in Chlamydomonas reinhardtii during sulfur starvation

The mitochondrial respiratory chain in plants, some protists and many fungi consists of the ATP-coupling cyanide-sensitive cytochrome pathway and the cyanide-resistant alternative respiratory pathway. The alternative pathway is mediated by alternative oxidase (AOX). Although AOX has been proposed to play essential roles in nutrient stress tolerance of plants and protists, the effects of sulfur (S) deprivation, on AOX are largely unknown. The unicellular green alga Chlamydomonas reinhardtii reacts to S limitation conditions with the induced expression of many genes. In this work, we demonstrated that exposure of C. reinhardtii to S deprivation results in the up-regulation of AOX1 expression and an increased AOX1 protein. Furthermore, S-deprived C. reinhardtii cells display the enhanced AOX1 capacity. Moreover, nitrate assimilation regulatory protein (NIT2) is involved in the control of the AOX1 gene expression in the absence of S. Together, the results clearly indicate that AOX1 relates to S limitation stress responses and is regulated in a NIT2-dependent manner, probably together with yet-unknown regulatory factor(s).     

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.     

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).     

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.