Mitochondria in microsporophylls had higher AOX capacity and protein expression levels than those in microsporangia. Mitochondria in microsporophylls had higher AOX capacity and protein expression levels than those in microsporangia. A, Isolation of mitochondria from microsporophylls (ML) and microsporangia (MG) using Percoll density gradient centrifugation. Photographs (left) show the band patterns obtained following centrifugation. Arrows indicate mitochondrial fractions. The graph (right) shows the protein content in the mitochondria of microsporophylls and microsporangia. The averages were calculated from at least four different thermogenic male cones (ML: n = 4; MG: n = 6). Error bars indicate the sd. **P < 0.005 as determined by Student’s t test. B, Representative traces (left) of oxygen uptake by intact mitochondria in microsporophylls (ML) and microsporangia (MG). Changes in oxygen uptake rate by the sequential addition of succinate, ADP, KCN, and n- propyl gallate (nPG) in both mitochondria are shown. Averages (right) were calculated from at least three independent experiments from different thermogenic male cones (n = 3). Error bars indicate the sd. *P < 0.05 and **P < 0.005 as determined by Student’s t test; ns, not significant. C, Expression of AOX, UCP, and the mitochondrial marker heat shock protein 60 (HSP60) in mitochondria of microsporophylls (MLs) and microsporangia (MGs). In total, 10 µg of mitochondrial proteins were separated by SDS-PAGE and analyzed by immunoblotting using the indicated antibodies. Mitochondrial proteins were prepared from three thermogenic male cones (n = 3). Yasuko Ito-Inaba et al. Plant Physiol. 2019;180:743-756 ©2019 by American Society of Plant Biologists