Functional coexpression of the mitochondrial alternative oxidase and uncoupling protein underlies thermoregulation in the thermogenic florets of skunk cabbage

Abstract

Two distinct mitochondrial energy dissipating systems, alternative oxidase (AOX) and uncoupling protein (UCP), have been implicated as crucial components of thermogenesis in plants and animals, respectively. To further clarify the physiological roles of AOX and UCP during homeothermic heat production in the thermogenic skunk cabbage (Symplocarpus renifolius), we identified the thermogenic cells and performed expression and functional analyses of these genes in this organism. Thermographic analysis combined with in situ hybridization revealed that the putative thermogenic cells surround the stamens in the florets of skunk cabbage and coexpress transcripts for SrAOX, encoding Symplocarpus AOX, and SrUCPb, encoding a novel UCP that lacks a fifth transmembrane segment. Mitochondria isolated from the thermogenic florets exhibited substantial linoleic acid (LA)-inducible uncoupling activities. Moreover, our results demonstrate that LA is capable of inhibiting the mitochondrial AOX pathway, whereas the proportion of pyruvate-stimulated AOX capacity was not significantly affected by LA. Intriguingly, the protein expression levels for SrAOX and SrUCPb were unaffected even when the ambient air temperatures increased from 10.3 degrees C to 23.1 degrees C or from 8.3 degrees C to 24.9 degrees C. Thus, our results suggest that functional coexpression of AOX and UCP underlies the molecular basis of heat production, and that posttranslational modifications of these proteins play a crucial role in regulating homeothermic heat production under conditions of natural ambient temperature fluctuations in skunk cabbage.

Figure 1.

Stigma-stage-specific homeothermic control in the spadix of skunk cabbage. Changes in both the spadix (Ts) and air temperatures (Ta) during the stigma, bisexual, and male stages are shown.

Figure 2.

Thermographic analysis of longitudinal sections of skunk cabbage spadices. A to C, Structural features of skunk cabbage used for thermographic analysis. The cylindrical organ is the thermogenic spadix. A, An intact skunk cabbage. B, The spathe has been cut to reveal the developmental stage and level of thermogenesis. C, Longitudinal sectioning of the spadix. D, Thermal imaging of the plant shown in C using a high-resolution infrared thermal camera. E to G, High-magnification images of B to D, respectively. The temperature scale of the thermographic analysis is shown on the bottom right. Bars, 1 cm.

Figure 3.

Expression analysis of SrAOX, SrUCPa, and SrUCPb transcripts among various tissues from stigma-stage (thermogenic) skunk cabbage samples. Ten-microgram aliquots of total RNA extracted from the leaf, spathe, spadix, and root were resolved on formaldehyde gels, blotted onto a nylon filter, and hybridized with a labeled cDNA probe for SrAOX, SrUCPa, or SrUCPb. The rRNA bands in the ethidium bromide-stained gels are shown in the bottom panel as a loading control.

Figure 4.

In situ hybridization analysis of SrAOX and SrUCPb transcripts in the spadix of skunk cabbage. A, Transverse sections of the stigma-stage (thermogenic) spadix were hybridized with digoxigenin-labeled antisense (left) and sense (right) RNA probes for SrAOX (top) and SrUCPb (bottom). B, Magnification of the boxed area in A. C, Transverse sections of a male-stage (postthermogenic) spadix were hybridized with digoxigenin-labeled antisense (left) and sense (right) RNA probes for SrAOX (top) and SrUCPb (bottom). D, Magnification of the boxed area in C. an, Anther; pe, petal; pi, pistil; fi, filament; v, vascular bundle. Bars, 2 mm in A and C; 500 μm in B and D.

Figure 5.

Western-blot analysis of mitochondria purified from the thermogenic florets and nonthermogenic roots of a skunk cabbage. Ten micrograms of purified mitochondrial proteins were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and incubated with antibodies directed against the proteins indicated below the panels. The predicted position of each protein species is indicated by an arrow on the right. Molecular mass standards are shown on the left of each panel.

Figure 6.

Stimulation of proton conductance by LA in isolated mitochondria from stigma-stage thermogenic florets. Purified mitochondria were incubated with 1 mm NADH substrate in assay medium containing 1 μm oligomycin (to inhibit ATP synthase), 0.1 μm nigericin (to clamp the ΔpH), 6 μm carboxyatractyloside (to inhibit adenine nucleotide translocase), and 100 μm n-propyl gallate (to inhibit SrAOX). The kinetics of proton conductance were then examined by simultaneous measurement of respiration and membrane potential. A, LA-induced dose-dependent increase in proton conductance. Increasing concentrations of LA (0–15 μm) were obtained by successive additions when the steady-state respiration rate and membrane potential were attained. B, The effects of 5 μm LA on the kinetics of proton conductance were examined with a KCN titration. The results shown are the mean ± sd of three measurements using mitochondria from the same preparation.

Figure 7.

The effects of LA on both the alternative and cytochrome pathway respirations in mitochondria purified from thermogenic florets of skunk cabbage. Purified mitochondria were incubated in the reaction medium without bovine serum albumin (containing 16.5 μm pyruvate) in the presence of 100 μm n-propyl gallate or 0.5 mm KCN for measurement of the cytochrome or alternative pathway respirations, respectively. Increasing concentrations of LA (0–15 μm) were obtained by successive additions when the steady-state respiration rate was attained. The results shown are the mean ± sd of three measurements using mitochondria from the same preparation.

Figure 8.

Expression profiles of the SrAOX and SrUCPb proteins under different ambient temperatures. Mitochondria were purified immediately after sampling of the spadices (see Table I). Ten micrograms of purified mitochondrial proteins, prepared without reducing agents, were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and incubated with AOA, UCP-cab, or Hsp60 antibodies. The ambient temperatures are indicated above the panels. The position of each protein species is indicated by an arrow on the right. Molecular mass standards are shown on the left.