This study has three key findings. First, despite apparently using different fuels, heat production in both fertile and sterile male florets of P. bipinnatifidum occurs predominantly via the alternative pathway. Second, both male floret types can maintain their thermoregulatory activity ex planta for up to 30 h. Finally, with the exception of the sacred lotus (Watling et al., 2006; Grant et al., 2008), measurements of respiratory fluxes and discrimination using isotope techniques have not been possible in other thermogenic tissues to date because of the high diffusional resistances (Guy et al., 1989). Our third key finding, that diffusional effects on O2 isotope discrimination in dense tissues can be largely overcome by using elevated O2 partial pressures, provides an important advance in stable isotope measurements of respiration.
Thermogenesis and thermoregulation by fertile male florets
We demonstrated that fertile male (FM) florets heat in a pattern similar to that characterized for sterile male (SM) florets except that FM florets typically commenced heating earlier than SM florets, and had a less pronounced peak and dip than SM florets. Furthermore, measurements of dissected inflorescences in the laboratory demonstrated that both floret types heat independently.
In the present study, heating in both SM and FM florets lasted for at least 30 h following excision from the plant, and was similar to that recorded on intact inflorescences. This contrasts with previous studies reporting that excision of spadices from P. bipinnatifidum stimulates a respiratory burst lasting only 1–2 h, with respiration dropping to very low rates 2 h after removal from the plant (Seymour et al., 1983; Seymour, 1991), but is similar to P. melinonii where isolated FM and SM florets heated for at least 14 h once cut from the plant (Seymour & Gibernau, 2008). The duration and magnitude of heating in isolated FM and SM florets suggests that all that is required for heat generation (e.g. fuel) and for temperature regulation (e.g. signalling) is contained within the detached inflorescence. Consistent with this, our data indicated that thermogenesis is unlikely to be limited by substrate (lipid or carbohydrate) supply. Calorimetric studies of P. bipinnatifidum spadices also concluded that there was no substrate import into the inflorescence during thermogenesis (Seymour, 1991). By contrast, thermogenesis in other aroids, for example Symplocarpus foetidus (skunk cabbage) relies on carbohydrate import, and inflorescence heating ceases upon removal from the plant (Knutson, 1974; Ito et al., 2003b).
Mechanisms of heating in P. bipinnatifidum
We identified a clear relationship between in vivo alternative pathway (AOX) flux and heating in both FM and SM florets of P. bipinnatifidum. Based on our oxygen isotope measurements, the AOX pathway accounts for the bulk of respiratory activity in both of these thermogenic tissues, and indeed the proportions of flux via AOX in SM florets (96%) are the highest measured to date (Ribas-Carbo et al., 2005; Watling et al., 2006; Grant et al., 2008). The high proportions of AOX flux in both FM (up to 92%) and SM florets are similar to those reported in the thermoregulatory receptacles of N. nucifera where up to 93% of respiration was via AOX in the most strongly heating flowers, and where AOX flux was strongly correlated with heating (Watling et al., 2006; Grant et al., 2008). Similarly, 78% of total respiratory flux was via the AOX in isolated mitochondria of thermogenic S. foetidus (Guy et al., 1989). In our study, SM florets, which reach the highest peak temperatures (Table 1), also had the highest mean total respiration rate (0.15 μmol O2 g−1 FW s−1; stage C), although peak respiration rates may not have been captured in FM florets (Fig 2a). Given the high proportional engagement of the alternative pathway in P. bipinnatifidum thermogenic tissues, fluxes via the AOX are substantial – up to 0.094 μmol O2 g−1 FW s−1 and 0.15 μmol O2 g−1 FW s−1 in FM and SM florets, respectively.
Our finding that discrimination was essentially the same in FM florets in air or elevated O2 suggests that diffusional limitations were not an issue with FM florets. By contrast, diffusional limitations to discrimination were observed in SM florets but were essentially overcome by increasing the O2 concentration, which confirmed that the majority of the respiratory flux in stage C and E florets is via the AOX pathway. The use of higher O2 partial pressures to largely mitigate the effects of diffusional limitations to discrimination in these dense tissues opens up the possibility of using stable isotope methodologies not only to measure alternative pathway flux in thermogenic plants, but also in other diffusionally limited tissues. That SM florets displayed O2 diffusional limitations but FM florets did not could be a result of the higher total respiration rates in SM florets, and/or because of differences in floret morphology. For example, FM florets have a higher surface area to volume ratio and thinner cuticle than SM florets (Grant, 2010).
The strong relationship between AOX flux and heating in FM florets, and the substantial proportions of total flux via AOX in both FM and SM florets, suggest there is little room for contribution by pUCPs, except alongside AOX to totally uncouple respiration via concurrent operation of pUCPs and AOX (Onda et al., 2008; Wagner et al., 2008). If pUCPs alone were responsible for heat generation in P. bipinnatifidum, then we would expect an increase in flux through the cytochrome pathway during thermogenesis; however, we detected no change in COX flux during heating by FM florets across all thermogenic stages, and comparatively low proportions of total flux via COX in peak heating SM florets when measured under increased O2 supply. Our protein data further support the substantial role for AOX in thermogenesis in P. bipinnatifidum; whereas AOX increases in thermogenic tissues and stages, pUCP does not. Synchronicity between onset of thermogenic activity and the increase in AOX protein in both floret types is similar to the pattern found in sacred lotus (Grant et al., 2008), but contrasts with other Araceae (e.g. S. guttatum and Arum maculatum) where significant increases in AOX protein precede the onset of thermogenesis by several days (Rhoads & McIntosh, 1992; Chivasa et al., 1999).
Our data provide evidence for developmental regulation of thermogenesis at the level of protein synthesis in P. bipinnatifidum; however, no significant relationship between AOX protein content and AOX flux was detected during the thermogenic stages. This indicates that fine scale post-translational regulation of AOX activity most likely occurs and is responsible for regulating heat production. Activation of AOX is controlled, in part, by the redox status of the protein, which is regulated via the formation of disulfide bonds between conserved cysteine residues (Rhoads et al., 1998). At least one isoform of AOX from P. bipinnatifidum contains the regulatory cysteines (Ito & Seymour, 2005; Grant et al., 2009); however, c. 40% of the protein resists oxidation by diamide (Grant, 2010), suggesting it may lack this redox control. The activity of the reduced protein can be further moderated by effectors such as α-keto acids (e.g. pyruvate, succinate) (Rhoads et al., 1998), the specific effector varying depending on the AOX isoform. For example, AOX from thermogenic N. nucifera also shows significant redox insensitivity, and stimulation of AOX occurs via succinate rather than pyruvate (Grant et al., 2009). An AOX that is not redox regulated (Onda et al., 2007; Grant et al., 2009) but is controlled by effectors could provide greater control of AOX flux for the precise temperature control these plants achieve over a prolonged period. By contrast, AOX from S. guttatum, which does not thermoregulate but rather heats in a single burst (Meeuse, 1966; Meeuse & Raskin, 1988), is constitutively active (Crichton et al., 2005).
The co-occurrence of AOX and pUCP in thermogenic tissues, such as P. bipinnatifudum, has raised speculation that both may contribute to heating, but to date there is little evidence that pUCPs function in heat generation in plants (Grant et al., 2008; Wagner et al., 2008). Based on pUCP and AOX transcript abundances, the mechanism of thermogenesis in P. bipinnatifidum was assumed to be pUCPs (Ito & Seymour, 2005); however, our data clearly demonstrate a predominant role for AOX in heating in this species. Between 70% and 96% of total flux was via the alternative pathway in heating FM and SM florets, AOX protein increased specifically in thermogenic male tissues, and no significant difference in amounts of pUCP was found between non-thermogenic and thermogenic stages. If pUCP operated alongside AOX in these tissues we would expect concurrent increases in both proteins throughout thermogenesis. Intriguingly, we did observe an increase in COXII protein with the onset of thermogenesis in both FM and SM florets. Relative amounts, however, were very similar to those observed in non-thermogenic female florets unlike AOX protein, which was several-fold higher in male florets than in female florets.
Studies indicating that lipids were used as respiratory substrates in thermogenic P. bipinnatifidum florets have been used to support a role for pUCPs in thermogenesis in this species (Ito & Seymour, 2005). The assumption derives from the fact that lipids are the substrate for animal UCPs (Argyropoulos & Harper, 2002), and that fatty acids (e.g. linoleic acid) which stimulate pUCP inhibit AOX activity (Sluse et al., 1998). Calorimetric studies yielding a respiratory quotient of 0.83, and C isotope analyses suggest that spadices switch from carbohydrate to direct lipid oxidation once the spathe opens and thermogenesis commences (Nagy et al., 1972; Walker et al., 1983; Seymour et al., 1984). We found significant declines in lipid content (total TAGs) towards the end of the thermogenic phase and post-thermogenesis in both SM and FM florets, consistent with lipid oxidation during thermogenesis. In addition, in FM florets, concurrent with the decline in TAGs post-thermogenesis, total starch content also decreased significantly. It is difficult to draw definitive conclusions about the specific substrate for thermogenesis in FM florets because changes in starch and lipids during anthesis may also be associated with maturation of male florets. Nevertheless, the significant decline in starch in FM florets is similar to that recorded in other thermogenic species, including the sacred lotus receptacle (Grant et al., 2008), S. foetidus and A. maculatum (ap Rees et al., 1976, 1977). By contrast, other Araceae may use both lipids and carbohydrates (e.g. S. guttatum; Wilson & Smith, 1971). Our flux and protein data strongly support a role for AOX and demonstrate that AOX and pUCP activity cannot be inferred based on substrate type alone. It does seem, however, that lipids are the major substrate for thermogenesis in SM florets of P. bipinnatifidum. If so, this suggests that AOX activity may not be as sensitive to fatty acids in these tissues as has been observed in non-thermogenic plants such as tomato (Sluse et al., 1998).
In summary, we have shown that both sterile and fertile male florets of P. bipinnatifidum have independent thermoregulatory phases that persist ex planta. Thermogenic activity is driven predominantly via increased flux through the alternative respiratory pathway in both floret types. While increased expression of AOX protein during the thermogenic phase provides the capacity for the increased AOX flux, fine-scale regulation of AOX activity must also occur. Although both floret types primarily use the alternative pathway to produce heat, the respiratory fuel appears to differ, with lipids and carbohydrates more predominant in SM and FM florets, respectively. A further important finding of this study is that diffusional limitations, that have to date prevented measurements of oxygen fractionation in most thermogenic species, can be mostly overcome, or potentially estimated, as a result of measurement at elevated partial pressures of oxygen. This latter finding provides an important advance to studies aimed at understanding the mechanisms that regulate heating in thermogenic plants, and roles of AOX in dense tissues of non-thermogenic plants. This study clearly demonstrates the importance of functional measurements of respiratory pathways to compliment molecular studies.