Thermoregulation in the appendix of the Sauromatum guttatum (Schott) inflorescence
© Skubatz; licensee Springer. 2014
Received: 10 May 2014
Accepted: 28 August 2014
Published: 26 September 2014
Three phenolic compounds are capable of activating the process that simultaneously leads to temperature rise and odor-production in the Sauromatum appendix. These compounds are salicylic acid, aspirin, and 2,6 dihydroxybenzoic acid. The objectives of the present study were to examine the effect of various concentrations of the these inducers on the temperature rise and to study the effect of mitochondrial inhibitors (KCN and SHAM) and an uncoupler (DNP) on the temperature rise.
In sections of the Sauromatum appendix two successive temperature rate maxima were detected in the presence of the three inducers. Two temperature maxima were also detected in appendices of intact inflorescences. The temperature profiles demonstrated a considerable variability within sections of one appendix in both magnitude and time of reaching a peak. When the Sauromatum temperature decreased it returned either to the same temperature baseline or to a slightly different baseline. The temperature rise was blocked by KCN (20 mM) and SHAM (40 mM) alone or when added together. DNP, an uncoupler, at 2.5 mM also blocked the rise in temperature. The thermogenic inducers also triggered a temperature rise in Arum appendix.
The presence of two rate maxima may indicate different heat-generating sources. The blockage of the temperature rise in the presence of KCN or SHAM implies that the activity of the cyanide-resistant and -sensitive pathways is required for generating heat. The variability in temperature profiles maybe related to changes in cellular control factors. This study provides the basis for investigating thermoregulation in plants.
KeywordsAppendix Aspirin Arum italicum 2,6-Dihydroxybenzoic acid Salicylic acid Sauromatum guttatum Thermoregulation
One striking example of a thermogenic plant is Sauromatum guttatum (the voodoo lily). On D-day, the day of inflorescence opening, the Sauromatum appendix (a ~20 cm-long, slender organ that comprised the upper part of the inflorescence) becomes hot, reaching 32°C above room temperature (Skubatz et al. ). The heat-loss reaches a value of 1 mWatt/mg DW (Skubatz and Meeuse ). The temperature rise correlates with a high rate of respiration (Meeuse , ). The heat facilitates the release of odorants that attract pollinators.
The current concept on the rising temperature in the Sauromatum appendix is that heat is generated by the activity of AOX, which is present in the Sauromatum mitochondria as well as in mitochondria of many non-thermogenic plants. Basically, AOX activity acts as an energy overflow of the Cyt pathway. When the Cyt pathway is restricted or saturated, AOX becomes active and since it does not generate a proton-motive force, the energy is released as heat (Elthon and McIntosh ; Wagner et al. ). On D-day, the amount of COX in the Sauromatum mitochondria is low, and the amount of AOX is high. Therefore, the electron flow to AOX is intense (Elthon et al. ). In non-thermogenic plants, AOX is involved in the protection against oxidative stress (Wagner and Moore ). In some thermogenic plants, the mitochondrial UCPs generate heat by uncoupling the electron transport from phosphorylation (Borecky et al. ; Ito-Inaba et al. ; Smith et al. ; Zhu et al. ).
Salicylic acid, ASA, and 2,6-DHBA activate the temperature rise as well as odor-production in the Sauromatum appendix (Raskin et al. ). Treatment of pre D-Day (3 to 1 d prior to heat-production) appendix sections with SA at concentrations higher than 1 μM induced significant increases in temperature (Skubatz et al. ). Salicylic acid effects are not confined only to the spadix of arum lilies. The acid has many roles in non-thermogenic plants and is considered a plant hormone (Vlot et al. ).
In the present study the temperature rise in sections of the Sauromatum and Arum appendices in the presence of the three inducers was recorded. The temperature profiles revealed two successive temperature rates that fluctuated in regard to their maximum values and the time it took to reach a maximum. Two temperature maxima were also detected in appendices of intact Sauromatum inflorescences. Surprisingly, application of KCN alone blocked the rise in temperature of the Sauromatum appendix whereas SHAM significantly but not completely blocked the rise in temperature. Application of KCN and SHAM together blocked completely the temperature rise. DNP, an uncoupler, also unexpectedly blocked the rise in temperature. These results provide evidence that the temperature rise may involve AOX and Cyt pathways. The blockage of the temperature rise by DNP, an uncoupler that dissipates the proton gradient and increases the electron flux via the Cyt pathway suggests that in vivo the Cyt pathway is required for heat-production and that UCPs may not be involved in this process in the Sauromatum appendix.
Dinitrophenol, KCN, SHAM, 2,6-DHBA, and ASA were purchased from Sigma-Aldrich, St. Louis, MO. SA was purchased from Thermo Fisher Scientific Inc., Pittsburgh, PA. Salicylic acid, ASA, and 2,6-DHBA were dissolved in distilled water. Salicylhydroxamic acid was dissolved in 100% DMSO to produce 1 M stock solution.
Corms of Sauromatum guttatum were kept at 4°C and the inflorescences were allowed to develop under a 15/9 day/night cycle at room temperature. When an inflorescence matures the base of its spathe becomes swollen and its color changes to burgundy. These characteristics appear 3 d prior to heat-production (pre D-day stage). At this stage, application of an inducer to sections of the appendix triggers heat-production.
Arum itallicum was grown outdoors at the University of Washington. Arum inflorescences were also cut at pre D-day stage, prior to inflorescence opening and heat-production. The thermogenic response of sections of the Arum appendix in the presence of inducers was recorded.
The Sauromatum appendix is ~5 times longer than the Arum appendix and therefore more experiments were conducted with the Sauromatum appendix. Another advantage of the Sauromatum appendix is that the inflorescence can develop from a corm without any soil.
Induction of a thermogenic response
Pre D-day appendices of the Sauromatum and Arum inflorescences were cut transversely into equal length sections and immediately placed either in inducer solutions with different final concentration or in a control solution (without the inducer) in a 96-well plate in an environmental chamber (SANYO) under constant light and temperature. Each set of experiments was performed with sections from one appendix. The Sauromatum appendix was cut into sections of 2–3 cm in length and Arum appendix was cut into sections of ~0.5 cm in length. The sections were placed in an appropriate solution containing SA, ASA, or 2,6-DHBA in 2.5 mM HEPES buffer, pH 7.0. The appendix sections were in the solution throughout the experimental period. Control appendix sections were treated in the same manner but without the inducers. The sectioning and placing of the sections in various solutions were completed in ~1 min for the Sauromatum appendix and ~1 h for the Arum appendix. Data collection started as soon as the appendix sections were placed in the appropriate solution.
T-type thermocouple probes 0.5 mm in diameter (copper/constantan, TMTSS-020G, Omega Engineering, Stamford, CT) were inserted into the appendix sections and the temperature was recorded every 2 min with a data logger (Omega Engineering). The temperature sensitivity of T-type thermocouples is liner at physiological temperatures. There were 720 consecutive readings per 24 h. The effect of a treatment on the temperature rise was calculated as the difference in temperature between treated and untreated sections. Temperature rate was defined as Δ°C/min and was calculated by subtracting consecutive readings and dividing by time (2 min). The temperature rate data was smoothed using Excel’s Moving Average function.
Temperature wave induced by ASA and 2,6-DHBA in the Sauromatum appendix
The variations in temperature induction by ASA are further illustrated in Figure 1G. When sections of one appendix were treated with ASA in duplicate or triplicate only the peak at ~19 h was detected, and the temperature did not rise at all in two out of three sections at 60 μM ASA. The variability in temperature did not seem to be a result of heat loss because the thermocouples were inserted into the tissue and they recorded the section temperature where heat loss to the environment is small. These variations were observed in two D-3 appendices treated with SA, one D-2 appendix treated with ASA, and one D-1 appendix treated with 2,6-DHBA. Additionally, variations in temperature were observed in six appendices treated with SA or ASA and one appendix treated with 2,6-DHBA at pre D-day stage. Consequently, each set of treatments described in the results was carried out with one appendix.
In order to account for the temperature differences seen among sections of the appendix, the temperature of an appendix of an intact inflorescence was also recorded (Figure 3G). It seems that the rise in temperature was lower at the tip of the appendix (18 cm from the base) than in the middle part (6–12 cm from the base) and the base. The appearance of two successive temperature maxima was also observed with the intact appendix. The two temperature maxima were seen at the base and the middle part of the appendix (1–6 cm). The temperature was also recorded at two parallel points along the vertical axis of the appendix at 6 and 12 cm from the base. No difference in temperature was observed at these vertical points indicating that the heat is distributed uniformly over the vertical axis. The moderate temperature gradient along the horizontal axis of the appendix can be explained by the difference in the appendix diameter. The base of the appendix is wider than its tip and therefore, it may better retain heat than its tip. Consequently, the tip temperature can be lower than the base temperature.
Temperature wave induced by SA, ASA and 2,6-DHBA in the Arum appendix
Two successive temperature rate maxima
Another characteristic of the the temperature maxima was that it always followed the rate maxima by ~1–3 h. When the temperature rate reached its maximum it slowed down and crossed the x-axis at the temperature maximum. The rate values became negative when the temperature decreased and when the rate values returned to zero the temperature returned to baseline. The time lag between the temperature peak and rate maximum is only shown for 1 μM ASA treatment (Figure 5A). This lag in time was observed at all concentrations with the three inducers. These results suggest the presence of a control mechanism over the temperature maximum. The temperature rate of the control and environmental chamber had a slope of zero that did not change over time (C). It also appears that when the rate peak at the ~23 h was high (D and E) the rate peak at ~17 h was low, and when the peak at ~17 was high (A) the peak at ~23 was relatively low. In (F) a rate maximum at 13 h was detected and it could be associated with the maximum at 17 h.
In Arum appendix one rate maximum was detected when appendix sections were treated with 1-70 μM ASA and SA. The maximum rate was 0.07 Δ°C/min with ASA treatment and 0.03 Δ°C/min with SA treatment, and it occurred at ~7 h from the beginning of the treatment.
The appearance of two rat maxima is not a result of the variations within an appendix. In many concentrations the two maxima appeared together at a single concentration of the inducer. For example, in Figure 4A-C two sequential maxima were detected at the same concentration of ASA. The two rate maxima were also detected when sections were treated with 2,6-DHBA (Figure 6A and E) and with SA (Figure 7A-B).
Effect of KCN and SHAM on temperature rise
This finding is hard to reconcile with the concept that heat is generated by AOX that is resistance to KCN. One explanation for this effect is that the operation of the Cyt pathway prior to the temperature rise is required for some unknown functions. Another explanation is the activity of UCPs to generate heat is decreased because of the inhibition of COX by KCN.
Effect of DNP on temperature rise
Two temperature waves
Two temperature maxima were observed in sections and intact appendices of the S. guttatum inflorescence. The temperature maxima seem to be coupled and the coupling is partially controlled by the concentrations of the thermogenic inducers. These data raise the question whether the two temperature waves represent one heat source, the mitochondria or, two different sources of heat. One source can be AOX that is blocked by SHAM and the other source can be a cyanide-sensitive activity, presumably, the Cyt pathway and/or another activity that is affected by KCN. It is conceivable that in the presence of an inducer coupling between the two sources occurs and heat is generated. The coupling strength between the two sources can vary and each source can also operate individually. For example, 2,6-DHBA may trigger the two sources separately (Figure 3C versus E) whereas ASA (Figure 1) and SA may trigger them simultaneously. If this hypothesis is correct than the time between the two temperature waves represents the time of the feedback between the two temperature waves.
In our previous publications, we have described the isolation of a novel 34.1 kDa NAD(P) reductase like protein (RL) from the thermogenic appendix of the Sauromatum inflorescence (Skubatz and Howald ). We also have shown that RL is present in two global conformations. An extended conformation (state A) that is only present at pre D-day when SA is undetectable, and a compact conformation (state B) that is present on D-day, when SA level is high. The protein was present in state B during the rise in temperature in thermogenic organs of several other members of the Araceae family and in thermogenic plants of other plant families. In Arabidopsis leaves, a non-thermogenic plant, it was present in state A. Furthermore, addition of SA at pM concentration to the purified protein solution induced a reversible volume phase transition every 4–5 min for at least 30 min (Skubatz et al. ). These changes in the protein conformation may result in variation in the response to the inducers. It is also conceivable that this protein induces two systems to generate heat by conformational changes.
Inhibition of temperature rise
The fact that KCN blocked the rise in temperature is surprising. The 24-h incubation of the appendix sections with KCN should not affect AOX activity. In Cocklebur seeds (Xanthium strumarium) pretreatment with 30 mM KCN for 16 h did not abolish AOX activity (Esashi et al. ). It is plausible that KCN binds to COX and to another protein(s) involved in the temperature rise. A number of enzymes are inhibited by KCN, for example, catalase (Wolfe et al. ) and malic dehydrogenase (Zołnierowicz et al. ).
The effect of DNP on the rise in temperature is also puzzling. If UCPs were active on D-day then DNP should not block the temperature rise because the mitochondrial proton gradient had been partially dissipated. Uncoupling of mitochondrial oxidative phosphorylation could have a negative impact when ATP production is needed prior to D-day. On D-day, the ATP/ADP ratio is low ~0.9 maybe because of a high ATP turnover rate (Skubatz et al. ). In a previous study, when DNP was applied to sections of the Sauromatum appendix at a micromolar range no significant effect on the respiration rate on D-1 and D-Day was detected, but the respiration rate increased when it was applied post D-Day (Hess and Meeuse ).
By reexamination of temperature data, this study has demonstrated the presence of two successive temperature rates and the capability of the appendix tissue to maintain and change its initial temperature. These in vivo results do not correspond to the cyanide-insensitive respiration of isolated mitochondria. They may indicate the involvement of cellular components not present in isolated mitochondria.
Aspirin, acetylsalicylic acid
The day of heat-production
6-DHBA: 2,6 Dihydroxybenzoic acid
- Pre D-day:
Three to 1 days prior to D-day
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