Essay 12.6 Respiration by Thermogenic Flowers
Roger S. Seymour, Ecology and Evolutionary Biology, Adelaide University, Australia, and Kikukatsu Ito, Cryobiofrontier Research Center, Iwate University, Japan
Over 200 years ago, the French biologist Jean-Baptiste de Lamarck wrote that the blossom of a European arum lily warmed up during the sequence of blooming (de Lamarck 1803–1815). Since then, botanists have recorded significant self-heating in flowers, inflorescences or cones in about 14 families of seed plants. These plants usually have rather large, fleshy floral structures, are often pollinated by beetles, bees or flies, and are always protogynous (female parts are receptive to pollination before male parts release pollen, thus preventing self-pollination).
Some species, such as the arum lilies, are so intensely thermogenic that the temperature of their flowers can increase up to 35°C above the surroundings. For example, in Brazil, the inflorescence of Philodendron selloum can warm to over 40°C at air temperatures close to freezing (Figure 1) (Nagy et al. 1972). In North America and Japan, skunk cabbage, Symplocarpus, can maintain temperatures above 15°C when the air temperature drops to –15°C, and it often melts the snow around it (Onda et al. 2008; Seymour 2004).
Figure 1 Thermal image of the inflorescence of Philodendron selloum during thermogenesis. The warm spadix is visible, because the spathe (V-shaped structure) has been cut away. Sterile male florets in the center of the spadix are warmest, but the fertile male florets also produce heat. Female florets at the base of the spadix do not produce significant heat. (From Ito and Seymour 2005.)
To generate this heat, the respiratory rates of some thermogenic flowers are the highest among plants, reaching over 900 nmol CO2 s–1 g–1 in the male florets of Arum concinnatum (Figure 2). This is comparable to the highest mass-specific respiration rate of any animal tissue, that of the flight muscle of a bee. Considering the whole blossom, a 125 g inflorescence of P. selloum produces about five times the amount of heat produced by a 125 g rat at an air temperature of 10°C.
Figure 2 Maximal mass-specific respiration rates of selected tissues from thermogenic flowers. (From Seymour 2010.)
Such high rates of heat production demand a good supply of oxygen. In the florets of P. selloum, this is achieved by diffusion through a network of tiny intercellular gas spaces that permeate the tissue to the center. Oxygen demand is so high that the oxygen partial pressure at the center of the floret drops to about one-quarter of atmospheric, but remains just above the critical level where oxygen uptake becomes diffusion-limited (Seymour 2001). Oxygen delivery through individual cells apparently limits the mass-specific respiration rate, and higher rates of floral heat production are possible only by increasing the floral size (Seymour 2010).
Heat production occurs by rapid respiration in the cells of the flowers. In most thermogenic species studied so far, the substrate for respiration is carbohydrate, often imported from other parts of the plant. In P. selloum, however, the substrate is lipid that is stored in the florets prior to blooming (Seymour et al. 1984). Analysis of heat production by direct calorimetry and respirometry show that all of the energy in the substrates ends up as heat in P. selloum (Seymour et al. 1983) and in the lotus, Nelumbo nucifera (Lamprecht et al. 1998). Although there is the possibility of some energy going into synthesis of floral structures, this appears to be negligible.
Physiological Temperature Regulation
A few species with the most powerfully thermogenic flowers also exhibit temperature regulation, which is the maintenance of a relatively constant temperature in the flower, regardless of external air temperature. Rather precise thermoregulation has been discovered in P. selloum (Nagy et al. 1972), Symplocarpus sp. (Knutson 1974; Ito et al. 2004) and N. nucifera (Seymour and Schultze-Motel 1996). In these cases, the respiratory rate increases linearly as the ambient temperature drops below 30°C, and the mean temperature of the flower is almost independent of ambient temperature. In N. nucifera, flower temperature varies only 6°C while ambient temperature varies 35°C (Figure 3). At low ambient temperatures during the night, heat production by the flower rises to about 1 Watt. On hot days, flower temperature is reduced to as much as 10°C below effective ambient temperature by evaporative cooling. Respiration clearly responds to ambient temperature, not time of day (Seymour and Schultze-Motel 1998). However, it is apparent that the flowers do not detect ambient temperature directly, but instead respond to changes in the temperature of their own cells. Close examination of the data reveals that there is a small decrease in flower temperature that occurs with falling ambient temperature (see Figure 3). Therefore is it clear that respiration in these species is negatively related to tissue temperature, opposite to the usual positive relationship between temperature and rates of biochemical reactions.
Recent results from three thermoregulatory species show steeply decreasing respiration as tissue temperature increases (Figure 4). What causes this inhibition is still unknown, but it is entirely reversible, and temperature regulation occurs within a relatively narrow range of tissue temperature. As environmental temperature decreases, the rate of heat loss increases, causing a decrease in tissue temperature. But this in turn causes heat production to rise to a level that matches the rate of heat loss, and tissue temperature stabilizes. Further decreases in environmental temperature cause further decreases in tissue temperature and rises in respiration rate. Eventually the flower reaches its maximum respiration rate, which occurs at a tissue temperature called the ‘switching temperature.’ Temperatures below this reduce respiration and heat production (see Figure 4), which causes the rate of heat production to always be less than the rate of heat loss, until the flower cools to the environmental temperature. Thus below the switching temperature, thermoregulation is impossible.
Figure 3 Rate of oxygen consumption and heat production (top) and temperature of the central receptacle (bottom) in the sacred lotus Nelumbo nucifera, in relation to ambient temperature. The dashed line is isothermal, showing that evaporative heat loss predominates at high ambient temperatures, but metabolic heat production prevails at low ambient temperature. The means were derived from intact flowers in the field, during the thermoregulatory period associated with female receptivity. The inset shows a flower cut in half, revealing the yellow receptacle that is obviously warm in the thermal image. (From Seymour and Schultze-Motel 1998.)
Figure 4 Respiration rates of three species of thermoregulatory flowers in which tissue temperature was experimentally clamped at 5°C intervals, revealing the reversible decrease in thermogenesis that occurs at higher temperatures. The highest respiration occurs at the ‘switching temperature,’ above which thermoregulation occurs and below which regulation is impossible. The curves fitted to the data indicate the precision of temperature regulation, with Nelumbo being the most precise, followed by Symplocarpus and Dracunculus. (From Seymour et al. 2010.)
Molecular Pathways for Thermogenesis
Heat production in thermogenic plants has been generally considered to be associated with an increase in activity of the cyanide-resistant electron transport pathway in mitochondria (Vanlerberghe and McIntosh 1994). This pathway is mediated by the “alternative oxidase” (AOX), which accepts electrons from the ubiquinone pool and uses them to reduce oxygen to water. The free energy created by the flow of electrons from ubiquinol to AOX does not generate ATP; instead, it is lost as heat. The AOX pathway seems to be present in all plants in varying capacities, although it is particularly active in thermogenic species (see Web Topic 12.3).
Plant AOX activity is generally regulated not only through transcriptional control but also by two interrelated posttranslational mechanisms. Most plant AOXs have two highly conserved Cys residues, CysI and CysII (Berthold et al. 2000; Holtzapffel et al. 2003), and reduction of an intermolecular disulfide bond formed by the CysI residues results in a more active, noncovalently linked dimer (Umbach and Siedow 1993). Moreover, α-keto acids, such as pyruvate, stimulate the AOX activity by forming a thiohemiacetal with CysI (Millar et al. 1993), and CysII regulates the AOX activity by interacting with glyoxylate (Umbach et al. 2002) (Figure 5A). To date, AOX activities have been analyzed at the molecular level in four thermogenic species: Sauromatum guttatum, Symplocarpus renifolius, Nelumbo nucifera, and Arum maculatum. N. nucifera and S. renifolius are capable of regulating thermogenesis to achieve relatively constant flower temperatures, whereas A. maculatum and S. guttatum show transient and rather uncontrolled heat production. Among these plants, only N. nucifera expresses the genes that encode AOXs without the regulatory CysI (termed NnAOX1a and NnAOX1b), and these AOXs have been suggested to be stimulated by succinate and not pyruvate (Grant et al. 2009). The other three thermogenic plants express AOXs with conserved CysI and CysII; however, their responsiveness to α-keto acids, especially pyruvate, seems to vary. S. renifolius expresses SrAOX, which is pyruvate-sensitive (Onda et al. 2007). In contrast, A. maculatum and S. guttatum abundantly express AOXs that are pyruvate-insensitive and constitutively active, termed AmAOX1e and SgAOX, respectively (Crichton et al. 2005; Ito et al. 2011). Recent bioinformatic and functional analyses have suggested that an element consisting of three adjacent residues, termed ENV (Glu-Asn-Val), is critical for the regulation (Albury et al. 2009). The ENV motif is present in thermogenic SrAOX, similar to pyruvate-sensitive AOXs found in non-thermogenic plants, such as Arabidopsis AOX1a (Onda et al. 2007) (Figure 5B). It appears that the ENV motif is replaced by a QDC motif in SgAOX and by a QNT motif in AmAOX1e (Crichton et al. 2005; Ito et al. 2011) (Figure 5B).
Figure 5 Structural elements proposed to influence alternative oxidase (AOX) regulatory activity. (A) Schematic representation of the AOX structure as predicted previously (Andersson and Nordlund 1999; Crichton et al. 2005). The red stars indicate positions of conserved CysI and CysII. Filled red circles show the di-iron center. The black star marks the position of the ENV motif. Predicted α-helices are shown as yellow cylinders. (B) Amino acid sequence alignment of AOXs from Arabidopsis thaliana (AtAOX1a), Symplocarpus renifolius (SrAOX), Nelumbo nucifera (NnAOX1a and NnAOX1b), Sauromatum guttatum (SgAOX), and Arum maculatum (AmAOX1e). Pyruvate-sensitive, succinate-sensitive, and pyruvate-insensitive and constitutively active AOXs are boxed in red, blue, and green, respectively.
Ubiquinone (UQ) is a redox intermediate of the mitochondrial respiratory chain that translocates electrons from dehydrogenases to the cytochrome pathway and AOX. The UQ pool is controlled to be approximately 60% reduced in non-thermogenic plants (Wagner and Wagner 1995). In contrast, the UQ pool in the thermogenic appendices of A. maculatum has been shown to be approximately 90% reduced during heating (Wagner et al. 1998; Wagner et al. 2008) (Figure 6A). As mentioned before, plant AOXs are more active in their reduced forms, so a highly reduced UQ pool facilitates maximal AOX activity for thermogenesis. However, analysis of the redox state of the UQ pool in vivo has shown that the reduction level in the thermogenic and post-thermogenic spadices of S. renifolius remains in the range of 40–75% (Figure 6B).
Figure 6 Relationship between thermogenic tissue temperatures and ubiquinone reduction (UQred) levels as a percentage of total UQ (UQtotal) in Arum maculatum (A) and Symplocarpus renifolius (B). Data from Wagner et al. (1998, 2008) and Kamata et al. (2009) were re-plotted to provide a comparison between short-term, non-thermoregulatory A. maculatum and long-term, thermoregulatory S. renifolius. UQ reduction levels in thermogenic (Hot) and post-thermogenic (Post) tissues are represented by white circles and black triangles, respectively.
The reduction state of UQ is a reflection of the metabolic balance between the rate at which the upstream dehydrogenases reduce oxidized UQ and the rate at which reduced UQ is oxidized by the AOX and cytochrome c oxidase pathways. The regulatory mechanisms that control the redox status of the UQ pool in thermoregulating S. renifolius and transiently thermogenic A. maculatum seem to differ. A recent comparative study of respiratory component expression in mitochondria showed abundant expression of a type II NAD(P)H dehydrogenase (NDB) found on the external face of the mitochondrial inner membrane in A. maculatum and of uncoupling protein (UCP) in S. renifolius (Kakizaki et al. 2012). Like AOX, neither of these mitochondrial enzymes (NDB and UCP) pumps protons, and therefore they do not contribute to energy conservation via ATP synthesis (Rasmusson et al. 2004). Therefore, it is possible that such factors, which control electron flow in the mitochondrial respiratory chain, are involved in the molecular mechanisms of thermoregulation in plants. Moreover, because the type of respiratory substrate has been suggested to be a major determinant in selective expression of the UCP gene in the inflorescence of P. selloum (Ito and Seymour 2005), further analyses are needed to clarify the relationship between cellular metabolism and respiration control in thermogenic plants.
The physiological thermoregulatory control mechanism that decreases respiration at higher temperatures in certain species is not yet known. Unlike thermoregulation in birds and mammals, which is mediated by complex neural integration, thermoregulation in plants must occur at a strictly biochemical or molecular level. One clue is that the rate of AOX-mediated respiration of mitochondria isolated from Arum maculatum increases with temperature to a peak at about 32°C, above which it falls steeply (Wagner et al. 2008). Control of AOX activity has been shown to respond to changes in the disulfide bridge that binds the two halves of the AOX dimer (Umbach and Siedow 1993) or activation by α-keto acids (Millar et al. 1993). However neither of these mechanisms seems to occur in A. maculatum, and respiration rate has been proposed to result from an interaction between the thermal profiles of the AOX and dehydrogenases earlier in the metabolic pathway (Wagner et al. 2008).
The Role of Thermogenesis in Nature
Thermogenesis is usually associated with an increase in scent production that makes the flowers more attractive to insects. Attraction, thermogenesis, and scent production are indeed tightly coordinated during the initial floral opening in most species that do not display temperature regulation (Meeuse and Raskin 1988). However, there is now evidence that thermoregulation in some species is a service offered as a direct reward to insect visitors. Thermoregulatory flowers are often pollinated by large flying insects, chiefly beetles, which remain within the warm floral chamber for about 24 h. During this period, they mate avidly, while consuming and digesting floral parts. These activities apparently require high body temperatures, because the beetles raise their temperature by a kind of “shivering” that involves heat-generating contractions of their flight muscles. This is potentially very energy costly, but the warmth from the flower reduces the cost substantially. For example, large scarab beetles inside the floral chamber of Philodendron solimoesense in lowland French Guiana experience temperatures only 4°C warmer than the outside air during the night, but reduce their energy cost of activity by 50–75% (Seymour et al. 2003). Instead of providing energy solely as food, as most flowers do, thermogenic flowers offer an energy savings. Even if the flower does not thermoregulate, the warm environment is an energy-saving reward. Thermogenesis can have mixed roles, even in the same inflorescence. For example, the exposed appendix of Dracunculus vulgaris inflorescences has highly thermogenic, but non-thermoregulatory, tissue that emits a powerful, obnoxious scent during the day to attract carrion beetles. However, the male florets in the floral chamber are thermoregulatory during the night, when the insects tend to reside there (Seymour and Schultze-Motel 1999).
Temperature regulation is also important for proper floral development and pollination success. Artificially enforced low temperatures reduce fertilization and seed set in thermoregulatory N. nucifera (Li and Huang 2009). In vitro pollen germination rate and pollen tube growth rate in S. renifolius are optimal at 23°C, which is the regulated temperature, and decrease so steeply at higher and lower temperatures that pollination would be impossible at natural ambient temperature (Seymour et al. 2009). It is likely that the temperature sensitivities of developmental features have evolved in response to the primary thermal stability of the flowers, rather than vice versa.
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