Two cycad AOX genes, CrAOX1 and CrAOX2, showing different expression patterns in thermogenic male cones. Two cycad AOX genes, CrAOX1 and CrAOX2, showing different expression patterns in thermogenic male cones. A, Phylogenetic tree of the predicted amino acid sequences of CrAOX1, CrAOX2, and related proteins. Red letters indicate gymnosperm AOX proteins, and bold red letters indicate C. revoluta AOX proteins. The tree was created in MEGA7.0.14 based on the alignment produced in Clustal W and using the neighbor joining method. Bootstrap values >10 are shown and were determined from 1,000 replications. The scale bar represents one amino acid substitution per site. The AOX sequence from the green alga Ostreococcus lucimarinus CCE9901 was included as an outgroup. Accession numbers and species abbreviations are listed in Supplemental Table S2. B, Tissue-specific expression of CrAOX1, CrAOX2, and CrUCP transcripts in thermogenic male cones. Expression of CrAOX1, CrAOX2, and CrUCP mRNA in two major tissues of cycad male cones, ML and MG, was analyzed using real-time PCR. CrEF1γ was used as an internal control. Values are means from four different male cones, and bars indicate the sd. *P < 0.05 and **P < 0.005 as determined by Student’s t test. C, Quantitative analysis of CrAOX1 and CrAOX2 transcript levels. To estimate the copy number of each CrAOX transcript in plant mRNA, plasmids containing each CrAOX gene were used as the standard for real-time PCR. Each bar indicates the sd. The expression level of CrAOX1 in MG was set to 1.0. *P < 0.05 as determined by Student’s t test; ns, not significant. Yasuko Ito-Inaba et al. Plant Physiol. 2019;180:743-756 ©2019 by American Society of Plant Biologists