Research Report

Alternative Oxidase Capacity of Mitochondria inMicrosporophylls May Function inCycad Thermogenesis1[OPEN]

Yasuko Ito-Inaba,a,2,3 Mayuko Sato,b Mitsuhiko P. Sato,c Yuya Kurayama,a Haruna Yamamoto,a

Mizuki Ohata,a Yoshitoshi Ogura,c Tetsuya Hayashi,c Kiminori Toyooka,b and Takehito Inabaa

aDepartment of Agricultural and Environmental Sciences, Faculty of Agriculture, University of Miyazaki,1-1 Gakuenkibanadai-nishi, Miyazaki 889-2192, JapanbRIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa230-0045, JapancDepartment of Bacteriology, Faculty of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan

ORCID IDs: 0000-0001-9598-5196 (Y.I.-I.); 0000-0002-9876-5612 (M.S.); 0000-0001-5383-5499 (M.P.S.); 0000-0002-4509-2653 (Y.O.);0000-0001-6366-7177 (T.H.); 0000-0002-6414-5191 (K.T.); 0000-0003-1750-454X (T.I.).

Cone thermogenesis is a widespread phenomenon in cycads and may function to promote volatile emissions that affectpollinator behavior. Given their large population size and intense and durable heat-producing effects, cycads are importantorganisms for comprehensive studies of plant thermogenesis. However, knowledge of mitochondrial morphology and functionin cone thermogenesis is limited. Therefore, we investigated these mitochondrial properties in the thermogenic cycad speciesCycas revoluta. Male cones generated heat even in cool weather conditions. Female cones produced heat, but to a lesser extentthan male cones. Ultrastructural analyses of the two major tissues of male cones, microsporophylls and microsporangia, revealedthe existence of a population of mitochondria with a distinct morphology in the microsporophylls. In these cells, we observedlarge mitochondria (cross-sectional area of 2 mm2 or more) with a uniform matrix density that occupied .10% of the totalmitochondrial volume. Despite the size difference, many nonlarge mitochondria (cross-sectional area ,2 mm2) also exhibited ashape and a matrix density similar to those of large mitochondria. Alternative oxidase (AOX) capacity and expression levels inmicrosporophylls were much higher than those in microsporangia. The AOX genes expressed in male cones revealed twodifferent AOX complementary DNA sequences: CrAOX1 and CrAOX2. The expression level of CrAOX1 mRNA in themicrosporophylls was 100 times greater than that of CrAOX2 mRNA. Collectively, these results suggest that distinctivemitochondrial morphology and CrAOX1-mediated respiration in microsporophylls might play a role in cycad conethermogenesis.

Thermogenesis has been observed in the reproduc-tive organs (inflorescences, flowers, and cones) ofseveral primitive seed plants, from gymnospermsto angiosperms (Tang, 1987; Gibernau et al., 2005;Seymour, 2010). Many roles have been proposed forfloral or cone thermogenesis, such as attracting polli-nators by spreading odor (Meeuse and Raskin, 1988),

providing an energetic benefit to adult insects (Seymouret al., 2003b), enhancing pollinator larval develop-ment (Terry et al., 2004), stimulating mating (Terryet al., 2004), facilitating the arrival and departure ofadult insects (Terry et al., 2007), facilitating fertili-zation (Li and Huang, 2009), and preventing freezedamage in plants (Knutson, 1974). Of these multipleroles, the most commonly proposed role for plantthermogenesis is to promote the emission of vola-tiles that may serve to attract pollinators such asbeetles (Kumano and Yamaoka, 2006; Gottsbergeret al., 2013), weevils (Terry et al., 2004; Suinyuyet al., 2013), thrips (Terry et al., 2007), and niti-dulids (Kono and Tobe, 2007).Cone thermogenesis is a widespread phenomenon in

two families of cycads (gymnosperms): Zamiaceae andCycadaceae (Tang, 1987). In most cycad species, themale cones are thermogenic, whereas female cones areonly slightly thermogenic or nonthermogenic (Table 1;Tang, 1987; Seymour et al., 2004). Thermogenic cycadsare defined as those species that can increase their malecone temperature up to at least 0.5°C above ambienttemperature. So far, 43 cycad species have been recog-nized as thermogenic (Table 1), possibly accounting for

1This work was supported by a Grant-in-Aid for Scientific Re-search (grant no.17K07762 to Y.I.-I., and grant nos. 15K07843 and15K07843 to T.I.), and by the Japan Society for the Promotion ofScience KAKENHI (grant no. 16H06279 to Y.I.-I.).

2Author for contact: ykoina@cc.miyazaki-u.ac.jp.3Senior author.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) isYasuko Ito-Inaba (ykoina@cc.miyazaki-u.ac.jp).

Y.I.-I. contributed to the experimental design and wrote the article.Y.I.-I., M.P.S., Y.O., T.H., and T.I. analyzed the data. Y.I.-I., Y.K., H.Y.,and M.O. conducted the biochemical and physiological experiments.M.S. and K.T. carried out experiments with electron microscopy. Allauthors have read and approved the article.

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more than half of all known thermogenic gymnospermand angiosperm plants. The levels of temperature ele-vation of cones vary across cycad species, but severalspecies are able to increase their cone temperature to.10°C above ambient (Table 1). Another prominent

feature of cycad thermogenesis is that cone thermo-genesis can occur for long periods of time. In severalMacrozamia species, thermogenic temperature peaksoccur daily for up to two weeks (Terry et al., 2004;Roemer et al., 2005, 2008), and inCycas micronesica, male

Table 1. Gymnosperms with known thermogenic ability in their cones

–, no data; CR, critically endangered; EN, endangered; VU, vulnerable; NT, near threatened; LC, least concern.

Family Genus Botanical Name Conservation StatusaTcone 2 Tair (°C)b

ReferenceMale Cone Female Cone

Zamiaceae Bowenia B. spectabilis LC 1.8 — Tang (1987)Ceratozamia C. hildae EN 1.7 0.1 Tang (1987)

C. matudae EN 5.4 — Tang (1987)C. microstrobila VU 2.1 — Tang (1987)C. miqueliana CR 4.9 — Tang (1987), Skubatz et al. (1993)C. robusta EN 3.2 1.7 Tang (1987)C. spp — 7.0 1.0 Tang (1987)

Dioon D. edule NT 7.5 3.0 Tang (1987)D. mejiae LC 8.3 — Tang (1987)D. merolae VU 6.3 — Tang (1987)D. spinulosum EN 7.1 4.8 Tang (1987)

Encephalartos E. altensteinii VU 10.7 — Tang (1987)E. barteri VU 4.7 — Tang (1987)E. bubalinus NT 12.3 — Tang (1987)E. ferox NT 6.6 2.8 Tang (1987)E. gratus VU 11.2 6.7 Tang (1987)E. hildebrandtii NT 6.3 5.8 Tang (1987)E. longifolius NT 7.0 — Tang (1987)E. manikensis VU 4.8 — Tang (1987)E. villosus LC 9.3 2.0 Tang (1987), Suinyuy et al. (2013)

Lepidozamia L. peroffskyana LC 7.4 — Tang (1987)Macrozamia M. fawcettii NT 0.5 — Tang (1987)

M. lucida LC 12.0 5.4 Tang (1987), Terry et al. (2004),Roemer et al. (2012), Roemer et al. (2005),Terry et al. (2007)

M. macleayi LC 12.0 — Terry et al. (2004), Roemer et al. (2012),Roemer et al. (2005)

M. machinii VU 12.0 4.6 Terry et al. (2004), Roemer et al. (2005),Seymour et al. (2004)

M. miquellii LC 1.9 — Tang (1987)M. moorei NT 3.7 1.0 Tang (1987)M. pauli-guilielmi EN 1.8 — Tang (1987)

Microcycas M. calocoma CR 2.6 — Tang (1987)Zamia Z. boliviana NT 7.4 — Tang (1987)

Z. fischeri EN 1.2 — Tang (1987)Z. furfuracea EN 2.5 0.8 Tang (1987), Skubatz et al. (1993)Z. loddigesii NT 0.7 0.2 Tang (1987)Z. manicata NT 0.8 — Tang (1987)Z. pumila NT 1.2 — Tang (1987)Z. skinneri EN 7.4 — Tang (1987)

Cycadaceae Cycas C. cairnsiana VU 2.4 — Tang (1987)C. media LC 1.9 — Tang (1987)C. micronesica EN 5.1 — Roemer et al. (2013)C. pectinata VU 0.8 — Tang (1987)C. revoluta LC 11.5 8.3 Tang (1987), Seymour (2010),

this studyC. rumphii NT 1.5 — Tang (1987), Skubatz et al. (1993)C. spp — 5.1 — Tang (1987)

aConservation status was obtained from the International Union for Conservation of Nature Red List of Threatened Species Version2016.1. bMean of the greatest difference between the internal temperature of the cones (Tcone) and ambient air temperature (Tair). The valuewas recorded or calculated in previous reports, except for C. revoluta, where the value was recorded in this study. If there were more than tworeferences for the same species, the highest value was incorporated in this list.

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cones exhibit a continuous thermogenesis for multipleweeks (Roemer et al., 2013). In contrast, floral thermo-genesis in most angiosperms only occurs for one day ora few days at a time (Seymour et al., 1983, 2003a; Raskinet al., 1989; Seymour and Schultze-Motel, 1998, 1999;Miller et al., 2011). The large number of thermogeniccycad species, as well as the intense and durableheat-producing abilities of these species, makes cycadsvery important in comprehensive studies on plantthermogenesis.Alternative oxidase (AOX), which is a terminal oxi-

dase found in all plants, has been proposed to functionto prevent reactive oxygen species production in therespiratory chain (Maxwell et al., 1999), dissipateexcess cellular reducing equivalents (Vanlerbergheand McIntosh, 1997; Yoshida et al., 2008), and regu-late floral thermogenesis in angiosperms (Elthon andMcIntosh, 1987). In thermogenic gymnosperms, thepresence of cyanide-resistant respiration was initiallydemonstrated in a respiratory assay using sporophylls(microsporophylls) of the male cones of Zamia pumila(Zamiaceae; Skubatz et al., 1993). However, AOXrespiration was also confirmed in nonthermogenicgymnosperms, such as white spruce (Picea glauca;Johnson-Flanagan and Owens, 1986; Weger and Guy,1991) and Araucaria angustifolia (Mariano et al., 2008;Valente et al., 2012). In addition, since the first report ofmolecular evidence for the existence of AOX in Pinuspinea (Frederico et al., 2009), high-throughput se-quencing has revealed several deduced AOX proteinsequences in gymnosperms (Neimanis et al., 2013).There is, therefore, no clear evidence that AOX-mediated respiration in sporophyll tissues is relatedto cone thermogenesis in gymnosperms, and if it is,it has not yet been determined which AOX genes areinvolved in cone thermogenesis.The positive correlation between the energy metab-

olism of a tissue and the morphological features of itsmitochondria, such as the number, size, and cristaedensities (Ghadially, 1988), is well known. Inmammals,mitochondrial morphology varies with sex (Rodriguez-Cuenca et al., 2002), type of tissue (Benard et al., 2006),and environmental conditions (Loncar et al., 1988;Cousin et al., 1992; Morroni et al., 1995). The mor-phology of plant mitochondria is also markedly alteredduring development and by cellular and environmentalsignals. Recent studies on mitochondrial dynamics inmodel plants have revealed the existence of elongatedor networked mitochondria with typical small mito-chondria in the chondriome (the collective mitochon-dria in a cell) in meristem-like cells such as shoot apicalmeristems (Seguí-Simarro et al., 2008), germinatingcells (Paszkiewicz et al., 2017), and culture-startingprotoplasts (Sheahan et al., 2005). Elevated CO2 leadsto changes in the mitochondrial morphology of severalplants (Griffin et al., 2001, 2004; Tissue et al., 2002;Gomez-Casanovas et al., 2007). Plant mitochondriatend to be longer in the dark in the absence of sucrose(Jaipargas et al., 2015) or under low-oxygen conditions(Ramonell et al., 2001; Van Gestel and Verbelen, 2002).

Thermogenic skunk cabbage (Symplocarpus renifolius)contains abundant mitochondria in its heat-producingorgans, the spadices (Ito-Inaba et al., 2009b; Ito-Inaba,2014). The correlation between mitochondrial mor-phology and energy metabolism in plants is, however,largely unknown. It is therefore of great interest toelucidate whether mitochondrial morphology is relatedto energy demands in plants.In this study, we investigated cycad cone thermo-

genesis, its underlying mechanisms, and mitochondrialfunction/morphology in a thermogenic cycad species,Cycas revoluta (family Cycadaceae). The categorizationof the species as a least-concern species by the Inter-national Union for Conservation of Nature (Table 1)made it a suitable species for investigation. Findings ofour study suggest that CrAOX1-mediated cyanide-resistant respiration in thermogenic microsporophyllsand the distinctive mitochondrial population that theycontain play important roles in cycad thermogenesis.

RESULTS

Heat-Producing Abilities in C. revoluta Male ConesAre Significant

Cycad thermogenesis has mostly been studied inZamiaceae (Seymour et al., 2004; Terry et al., 2004;Roemer et al., 2005, 2008, 2012), and seldom inCycadaceae(Roemer et al., 2013). However, even in Zamiaceae,thermal images have not been obtained. As cone ther-mogenesis by C. revoluta occurs during the rainy sea-son, in which cool temperatures often last for severaldays, we expected that the prevalent cool-weatherconditions during this season would assist in thecapturing of thermal images of the cycad cones. Asexpected, on the day when daytime ambient tempera-tures did not exceed 25°C, thermal images of C. revolutamale cones (but not female cones) were successfullytaken using an infrared thermal camera (Fig. 1A). Theincrease in surface temperature was more prominent inearly- and middle-stage male cones than in late-stagemale and female cones. When ambient temperaturewas ;22°C, the difference between the air temperature(Tair) and surface temperature of the cones was largerin early- and middle-stage male cones (DT = ;6.3°C)than in late-stage male and female cones (DT =;1.4°C;Fig. 1A). We also assessed the progressive temperaturechanges in both male and female cones over 10 suc-cessive days using thermal recorders (Fig. 1B; Table 2).The internal temperatures (Tcone) of both male andfemale cones were consistently higher than Tair, nota-bly even when daytime temperatures did not exceed25°C (Fig. 1B, blue background). Because male conesmature earlier than female cones, i.e. in early summer, itis more likely that they will be exposed to coolerweather conditions (as shown by the blue backgroundin Fig. 1B). It should also be noted that the maximumtemperature differences between night-time recordingsof Tair and Tcone inmale and female cones were 11.5°C

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and 8.3°C, respectively (Table 2). Collectively, the in-creased body temperatures observed in both male andfemale cones suggest that both have a heat-producingability, although this ability is significantly intensifiedin male versus female cones.

Ultrastructural Analyses of Individual Tissues inThermogenic Male Cones

We analyzed the intracellular structures of twomajor tissues of male cones, microsporophylls andmicrosporangia, using transmission electron micros-copy (Fig. 2) to explore how these individual tissues orintracellular structures are involved in male cone ther-mogenesis. The male cone consists of a central axis withmany spirally attached microsporophylls, which carry

dense microsporangia in which numerous microsporesdevelop into pollen grains (Fig. 2A). Ultrastructuralanalyses using three male cones revealed that the mi-crosporophylls, especially the epidermal cells, have alarge number of large, round-shaped mitochondriawith a cross-sectional area of 2 mm2 or more and arelatively uniform matrix density (Fig. 2B). Suchenormous mitochondria, which were labeled large mi-tochondria (LMt), were never observed in the micro-sporangia. It is noteworthy that in the microsporophyllepidermal cells, many mitochondria with area,2 mm2,i.e. not categorized as LMt, also exhibited round shapeand uniform matrix density similar to those of LMt(Fig. 2C). In addition, similarly shaped and nonlargemitochondria were also observed in the microsporo-phyll parenchyma cells (Fig. 2D). A large number ofsmall mitochondria of various shapes (round, oval, or a

Figure 1. Thermogenesis in cycad cones.A, Thermal images (right) of Cycas revolutamale and female cones obtained using anFLIR SC620 thermal imager (FLIR Systems)at night at ;22°C. Photographs acquiredduring daytime (left). B, Internal tempera-ture of male (left) and female (right) cones(Tcone) and ambient temperature (Tair)recorded over 10 successive days usinga thermal recorder. Tcone and Tair areshown in red and gray, respectively. Theblue background indicates the day whenTair did not exceed 25°C due to cloudy orrainy conditions.

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dumbbell- or rod-like configuration) were observed inthe microsporangia (Fig. 2E). Quantitative analysis toestimate the ratio of LMt in each tissue revealed that inthe microsporophyll cells, the appearance frequency ofLMt with an area $2 mm2 and ,4 mm2 was 8.98 60.78% and that of LMt with an area of $4 mm2 was4.07 6 0.27%; however, such large mitochondria werenot observed in the microsporangia (Fig. 2F, left).Similarly, mitochondria with perimeters of 6 mm ormore were found in most microsporophylls but only ina few microsporangia (Fig. 2F, right). The above find-ings suggest that a certain proportion of mitochondriafound in microsporophyll cells, especially in theepidermis, are made up of LMt. Given the structuralsimilarities of both nonlarge Mt and large Mt in mi-crosporophyll cells, they might exhibit similar func-tioning, e.g. in mitochondrial respiration. Interestingly,in microsporophylls, mitochondrial numbers in theparenchyma cells were significantly larger than those inthe epidermal cells, suggesting that the density of LMtin the epidermal cells might contribute to the lowernumber of mitochondria in this tissue compared to theparenchymal cells (Fig. 2G). These data indicate that thechondriomes observed in both epidermis and paren-chyma cells of microsporophylls are clearly differentfrom those observed in microsporangia. The differencein the chondriome of microsporophylls and micro-sporangia might be correlated with their functionaldifferences. No typical features of the central-axis mi-tochondrial structure were identified, because very fewmitochondria were present in this tissue.

Cyanide-Resistant Respiration in Intact MitochondriaIsolated from Thermogenic Male Cones

Cyanide-resistant respiration via AOX seems to playamajor role in angiosperm floral thermogenesis, but therole of AOX in gymnosperm cone thermogenesis islargely unknown. If cycad AOX plays a role in conethermogenesis, it is reasonable to expect that AOX

capacity would be larger in microsporophylls than inother tissues, such as in microsporangia, because mi-crosporophylls are proposed to be the thermogenictissues in the male cones (Skubatz et al., 1993). To de-termine AOX capacity in both microsporophylls andmicrosporangia, we first isolated intact mitochondriafrom both tissues. Many more mitochondria were re-covered from microsporophylls than from micro-sporangia in a male cone (Fig. 3A). The intactness ofthe outer membrane of isolated mitochondria wassimilar in both tissues, showing that intact mitochon-dria were successfully isolated from the two tissues(Table 3). Using these isolated mitochondria, we con-ducted an oxygen consumption assay (Fig. 3B; Ta-ble 4). Under succinate oxidation conditions (complexII substrate), the addition of ADP stimulated the oxy-gen uptake to an average of 67.87 6 6.19 and67.15 6 21.72 nmol O2 min21 mg21 of protein in mi-crosporophylls andmicrosporangia, respectively. Thisindicated that state 3 respiration rates in microsporo-phylls and microsporangia were almost identical.However, AOX activity was affected differently inmicrosporophylls and microsporangia by the additionof potassium cyanide (KCN). This compound reducedstate 3 respiration in microsporophylls by half, to anaverage of 30.846 6.04 nmol O2min21 mg21 of protein,whereas in microsporangia it significantly reducedAOX activity to one-tenth of the state 3 respiration, toan average of 7.20 6 1.38 nmol O2 min21 mg21 ofprotein. In both tissues, cyanide-resistant oxygen up-take was almost completely inhibited by the AOX in-hibitor n-propyl gallate. The mitochondria isolatedfrom both tissues revealed that the cytochrome oxidase(COX) activity in microsporophylls was not signifi-cantly different from that in microsporangia (Table 3;Supplemental Fig. S1). These data indicated that thealternative respiration capacity in microsporophyllswas much higher than that in microsporangia, sug-gesting that mitochondrial respiration via the AOXpathway in microsporophylls may play a role in thecone thermogenesis of C. revoluta.

Table 2. Average and maximum temperature differences between internal temperature of intact C. revoluta cones (Tcone) and ambient air tem-perature (Tair)

Averages 6 SD are presented for Tcone, Tair, and DT (Tcone 2 Tair). Maximum temperature differences are shown as DTmax, and the times atwhich differences were measured are shown as tTmax.

Sex of ConesAverage Temperature (°C) Average Difference (°C) Maximum Difference (°C) Times

Tcone Tair DT DTmax tTmax

Malea 29.69 6 2.25 23.58 6 3.40 6.19 6 1.09 11.5 2:18Femalea 31.48 6 3.52 28.28 6 3.67 3.35 6 0.58* 8.3 19:01Maleb 27.72 6 1.89 21.39 6 1.51 5.80 6 1.46 9.5 2:32Femaleb 27.39 6 0.81 23.31 6 0.68 4.05 5.4 19:46

aTemperature data for intact male cones (n = 3), intact female cones (n = 4), and ambient air were recorded over 10 successive days and used tocalculate the average and maximum differences between Tcone and Tair. The asterisk indicates a significant difference (Student’s t test; P , 0.05)between male and female DT values. bData corresponding to the day that temperature did not exceed 25°C were extracted from the 10-day dataand used to calculate the average and maximum differences between Tcone and Tair on that particular day. Since only one female cone experiencedair temperature that did not exceed 25°C, as shown in Fig. 1B right (blue background), the statistical analysis between male DT and female DT couldnot be executed. Male cones bloom earlier than female cones, and therefore, it is more likely that male cones will be exposed to cooler weatherconditions.

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Figure 2. Ultrastructural analyses of microsporophylls and microsporangia in thermogenic male cones of C. revoluta. A, Pho-tographs of a male cone with both microsporophylls and microsporangia. Cross section of a male cone (left); a large number ofmicrosporophylls are attached to a central axis. Numerous microsporangia were attached to the surface of a microsporophyll(middle), as evidenced in its cross section (right). B, Electron photographs showing typical LMt observed in the epidermal cells ofmicrosporophylls. Nuc, nucleus. LMt were defined as mitochondria with a cross-sectional area of 2 mm2 or more. The size of thisLMt is 5.62 mm2. C, Common structure of LMt and nonlarge mitochondria (Mt) in the microsporophyll epidermis cells. Eachmitochondrial size is as follows: LMt1 = 3.41mm2;Mt1 = 1.44mm2;Mt2 = 1.54mm2. Nonlargemitochondria exhibited a uniformmatrix density and round shape similar to those of the LMt. D, Mitochondria, which exhibited a roundish configuration and auniformmatrix density, in microsporophyll parenchyma cells. Although LMtswere not observed in these cells, mitochondriawithuniform matrix density similar to that of LMt were observed. Pt, plastid. E, Electron photographs showing typical mitochondriaobserved in microsporangia cells. F, The frequency of the area and perimeter of mitochondria in microsporophyll (ML) andmicrosporangia cells (MG). In total, 494 mitochondria in microsporophyll cells and 436 mitochondria in microsporangia cells inthree male cones were analyzed. The frequency of the area and perimeter were calculated from three independent male cones(n = 3). Error bars indicate the SE; *P, 0.05, as determined by Student’s t test; **P, 0.005; ns, not significant. G, Mitochondrialnumber in the microsporophylls and microsporangia of thermogenic male cones. Mitochondrial number in microsporophyll

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Instead of succinate, NAD(P)H dehydrogenase sub-strate, NADH, and a combination of tricarboxylic acidcycle intermediates, pyruvate and malate or glutamateand malate (Pyr + Mal and Glu + Mal, respectively;Supplemental Fig. S2), were used as respiratory sub-strates. However, neither NADH, pyruvate + malate,nor Glu + malate initiated oxygen uptake by mito-chondria, and ADP did not stimulate their respiratoryactivities in either microsporophylls or microsporangia.Therefore, these substrates may not be effectively uti-lized for mitochondrial respiration in cycadmale cones.Protein expression was analyzed in purified mito-

chondria from both tissues to determine whether AOXcapacities in microsporophylls and microsporangiadepend on the levels of AOX proteins (Fig. 3C;Supplemental Fig. S3). An equal amount of mitochon-drial protein was separated by SDS-PAGE and ana-lyzed by Coomassie Brilliant Blue staining andimmunoblotting. This staining revealed slightly differ-ent protein band profiles between the two tissues(Supplemental Fig. S3). Immunoblots detected AOX inmicrosporophylls but not in microsporangia. Uncou-pling proteins (UCPs), which are proposed to be an-other type of thermogenic protein, weremore abundantin microsporophylls than in microsporangia. The levelsof the mitochondrial marker heat shock protein60 (HSP60) were almost identical in the two tissues.Collectively and in combination with the results fromthe AOX capacity assay (Fig. 3B; Table 4), these findingsindicate that the high AOX capacity may be attributableto the abundance of AOX proteins in microsporophyllmitochondria. As UCPs were also abundant in micro-sporophylls, we do not exclude the possibility that theyare involved in thermogenesis.

Isolation and Identification of Partial CrAOX1 and CrAOX2Genes in Male Cones

To determine the molecular characteristics of theAOX proteins involved in cyanide-resistant respirationin C. revoluta male cones, partial AOX gene sequenceswere obtained using two degenerate primer sets: P1and P2 (Saisho et al., 1997) and 42AOX-F (forward) and42AOX-R (reverse; Frederico et al., 2009). The 440 bpcomplementary DNA (cDNA) fragments that wereamplified using each primer set were cloned indepen-dently in a pZErO vector, and the resulting nucleotidesequences of 69 independent clones (44 clones from P1and P2 and 25 clones from 42AOX-F and 42AOX-R)enabled the identification of two different sequences,indicating that C. revoluta had at least two AOXisoforms.

The two AOX cDNA sequences, designated CrAOX1and CrAOX2, exhibited 84% sequence identity in theC-terminal region containing ;224 amino acid resi-dues. The putative structural features of CrAOX1 andCrAOX2, and their amino acid sequence alignmentwith that of AOX from trypanosome, Arabidopsis(Arabidopsis thaliana), and other gymnosperms areshown in Supplemental Figure S4. CrAOX1 was ho-mologous to the AOX1 of gymnosperms (89% sequencesimilarity to PgAOX_subtype2 and PsAOX_subtype1,and 85% sequence identity to PgAOX_subtype1), andCrAOX2 was homologous to the AOX2 of gymno-sperms (81% sequence identity to PgAOX2). Althoughthe Cys residues CysI and CysII, which correspond toCys-127 and Cys-177, respectively, in ArabidopsisAOX1 (AtAOX1), are highly conserved in most AOXisoforms, CysI is replaced by Ser in the gymnospermAOX1, which includes CrAOX1 (Supplemental Fig. S4).This type of AOX has been known to be stimulated bysuccinate, but not by pyruvate (Holtzapffel et al., 2003;Umbach et al., 2006; Grant et al., 2009; Selinski et al.,2017). Indeed, under succinate oxidation conditions,pyruvate did not stimulate the AOX capacity in mi-crosporophyll mitochondria (Supplemental Fig. S5).The sequence of the N-terminal region of all gymno-sperm AOX2 proteins containing the CysI position hasnot been determined yet.The phylogenic tree based on several amino acid se-

quences of AOX homologs from gymnosperms andangiosperms (Fig. 4A) presented three major clades:angiosperm AOX1, gymnosperm AOX1, and AOX2.CrAOX1 was clustered with gymnosperm AOX1 andwas close to Picea sp. AOX proteins (PgAOX_subtype 1,PgAOX_subtype 2, and PsAOX_subtype 2). CrAOX2was clustered with gymnosperm and angiospermAOX2, and it was closer to Malus domestica AOX2proteins (MdAOX2d) than to the gymnosperm P. pineaAOX2 proteins (PpAOX2). These data indicate thatCrAOX1 and CrAOX2 clearly belong to two distinctAOX subfamilies, AOX1 and AOX2, respectively.A full-length UCP was isolated from cycad male

cones and the cDNA sequence was determined.C. revoluta UCP, designated as CrUCP, was homolo-gous to isoforms in gymnosperm UCP (85% sequenceidentity to Picea sitchensis UCP; Supplemental Fig. S6).A phylogenic tree for the UCP group was constructedusing several amino acid sequences of other homologsfrom gymnosperms and angiosperms (SupplementalFig. S7). The tree was divided into three majorclades: PUMP1 and PUMP2; PUMP 3; and PUMP 4,PUMP5 and PUMP6. CrUCP was located in the gym-nosperm group of the PUMP 1 subfamily, and was alsolocated close to PsUCP. Collectively, these results show

Figure 2. (Continued.)epidermis (ML-E) and parenchyma (ML-P) cells and in microsporangia (MG) were recorded on each ultrathin section andexpressed as the mean 6 SE for 25 cells within the epidermis in microsporophylls, 25 cells within the parenchyma in micro-sporophylls, and 20 cells in MG. Two male cones were analyzed. Different letters indicate statistically significant differencesbetween samples within each genotype by the Tukey-Kramer multiple comparison test (P , 0.05).

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that CrUCPs are typical UCP proteins found ingymnosperms.

Tissue-Specific Expression of CrAOX1 and CrAOX2mRNAs in Male Cones

The AOX proteins accumulated differently in themitochondria of microsporophylls and microsporangiain C. revoluta male cones (Fig. 3C; SupplementalFig. S3B). Therefore, it was of interest to deter-mine whether this difference was regulated at thetranscriptional level and to analyze which AOX pro-teins, CrAOX1 or CrAOX2, were responsible for AOXcapacity and expression in microsporophyll mito-chondria. Real-time PCR using primers that specificallyamplified CrAOX1 and CrAOX2 (Fig. 4B) revealedthat the CrAOX1 transcript was more abundant inmicrosporophylls, whereas CrAOX2 was significantlyupregulated in microsporangia. The transcript level ofCrUCP (Fig. 4B) was increased in microsporophyllscompared with that in microsporangia, although thedifference between these two tissues was less signifi-cant than that observed for CrAOX1. We also analyzedthe copy number of CrAOX1 and CrAOX2 transcriptsusing the plasmids pZAOX1 and pZAOX2, each ofwhich contained one of two CrAOX genes, as the stan-dard for real-time PCR (Fig. 4C). The transcript level ofCrAOX1 was significantly higher (.100-fold) than thatof CrAOX2 in microsporophylls, but the difference be-tween these two CrAOX genes was not statistically sig-nificant in microsporangia. These results, together withthe abundance of AOX proteins in microsporophyllmitochondria (Fig. 3C), suggest that the AOX proteinsdetected in microsporophyll mitochondria were mostlikely the products of CrAOX1 transcripts.

Collectively, these data suggest that CrAOX1-mediated cyanid-resistant respiration in microsporo-phylls with a distinctive chondriome may play a role incycad male cone thermogenesis.

DISCUSSION

Heat-Producing Ability of C. revoluta Cones IsComparable to That of Some Species in Zamiaceae

Tang (1987) reported that cone thermogenesis was awidespread phenomenon in Zamiaceae and Cycada-ceae, and that the heat-producing abilities of thermo-genic cycads in Cycadaceae were likely to be weakerthan those in Zamiaceae. Several species, which belongto the genera Encephalartos and Macrozamia in Zamia-ceae, are able to increase their cone temperature to.10°C above the ambient temperature, whereas inCycadaceae, the increase in cone temperature above theambient was at most 5°C (Table 1). Weak thermogen-esis was also observed in C. revoluta male cones, andthe maximum increase of the cone temperature wasonly 1.6°C above air temperature (Tang, 1987). Another

Figure 3. Mitochondria in microsporophylls had higher AOX ca-pacity and protein expression levels than those in microsporangia.A, Isolation of mitochondria from microsporophylls (ML) andmicrosporangia (MG) using Percoll density gradient centrifugation.Photographs (left) show the band patterns obtained following cen-trifugation. Arrows indicate mitochondrial fractions. The graph(right) shows the protein content in the mitochondria of microspo-rophylls and microsporangia. The averages were calculated from atleast four different thermogenic male cones (ML: n = 4; MG: n = 6).Error bars indicate the SD. **P , 0.005 as determined by Student’st test. B, Representative traces (left) of oxygen uptake by intact mi-tochondria in microsporophylls (ML) and microsporangia (MG).Changes in oxygen uptake rate by the sequential addition of succi-nate, ADP, KCN, and n- propyl gallate (nPG) in both mitochondriaare shown. Averages (right) were calculated from at least three in-dependent experiments from different thermogenic male cones(n = 3). Error bars indicate the SD. *P , 0.05 and **P , 0.005 asdetermined by Student’s t test; ns, not significant. C, Expression ofAOX, UCP, and the mitochondrial marker heat shock protein 60(HSP60) in mitochondria of microsporophylls (MLs) and micro-sporangia (MGs). In total, 10 mg of mitochondrial proteins wereseparated by SDS-PAGE and analyzed by immunoblotting usingthe indicated antibodies. Mitochondrial proteins were preparedfrom three thermogenic male cones (n = 3).

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study also reported that the temperature differencebetween the C. revoluta male cones and air was 5.4°C(Seymour, 2010). However, in marked contrast to theseprevious reports, the Tcone of male cones in our studyreached 11.5°C above Tair at midnight (Tables 1 and 2),and the thermal images of the male cones were suc-cessfully taken (Fig. 1A). In addition, Tcone was con-sistently higher than Tair even when Tair did notexceed 25°C during the day (Fig. 1B, blue background).The paradoxical difference between the previous re-ports and our study may be attributable to differencesin experimental conditions; for example, Tang (1987)used cut cones removed from intact plants, whereaswe used intact cones attached to plants. As male conescut from C. revoluta exhibit a gradual decline in tem-perature and die before pollen is released from themicrosporangia, the weak thermogenesis reported inthe previous study may be attributed to cone detach-ment. Our findings showed that the heat-producingability of the male cones in C. revoluta may be muchhigher than previously believed, and it may occur at anintensity similar to that of several other Zamiaceaerepresentatives that possess a high capacity to produceheat in their cones.Heat productionwas not detected in the female cones

of several cycad species and, when present, the maxi-mum temperature was consistently lower than thatdetected in male cones (Tang, 1987; Seymour et al.,2004). Our findings concur with those previouslyreported, as both the surface temperature and the Tconeof C. revoluta female cones were lower than those ofearly- and middle-stage male cones (Fig. 1, A and B).However, the Tcone of female cones, even under lowTair conditions and weak sunlight, was consistentlyhigher than Tair (Fig. 1B, blue background), and atnight, female cones raised their Tcone to 8.3°C aboveTair (Tables 1 and 2). Unexpectedly, 8.3°C was the

highest recorded value in all cycad female cones stud-ied to date (Table 1), and therefore, it is possible that thethermogenic abilities of female cones in other generahave been underestimated.

The Correlation between Mitochondrial Morphology andEnergy Metabolism in Thermogenic Tissues

It is well known that the energy metabolism ofmammalian tissue is positively correlated with themorphological features of its mitochondria (Ghadially,1988). For example, significant differences are likelyto exist between thermogenic brown adipose tissues(BATs) and nonthermogenic white adipose tissues(WATs; Cinti, 2001; Sell et al., 2004), wherein manymitochondria with tightly packed cristae are observedin BATs and very few mitochondria with sparse cristaeare observed in WATs (Cinti, 2001; Sell et al., 2004).Several studies have also demonstrated that BAT mi-tochondria become larger in cold-acclimated rats thanin control rats (Loncar et al., 1988; Morroni et al., 1995).However, in plants, only a few studies have suggested apositive correlation between heat production and mi-tochondrial morphology, such as size, number, andmatrix density, in tissues or cells of heat-producingplants. In thermogenic skunk cabbages, a larger num-ber of mitochondria were seen to accumulate in thetissues of both petals and pistils during the thermogenicstage than during the postthermogenic stage (Ito-Inabaet al., 2009b; Ito-Inaba, 2014), and mitochondrial pro-tein content was much higher in thermogenic skunkcabbage than in a nonthermogenic skunk cabbage(Lysichiton camtschatcensis; Ito-Inaba et al., 2009a). In thecurrent study, we observed large mitochondria withround shape and uniform matrix densities in the mi-crosporophylls, especially in the epidermis, but not in

Table 3. COX activity and outer membrane integrity of the mitochondria isolated from MLs and MGs of heat-producing C. revoluta male cones

Data are the means 6 SD of at least three measurements. There were no significant differences between MLs and MGs.

COX Activity and Outer Membrane Integrity Microsporophylls Microsporangia

COX activity (nmol O2 min21 mg21 protein)a 24.48 6 9.04 33.31 6 5.13COX activity (units mg21 of protein)b 0.456 6 0.295 0.288 6 0.127Outer membrane integrity of mitochondria (%)c 85.7 84.4

aCOX activity was analyzed using the ascorbate-TMPD assay. bCOX activity was analyzed using CYTOCOX1 (Sigma-Aldrich). cOutermembrane integrity was measured as described previously (Ito-Inaba et al., 2008a).

Table 4. Respiratory rate in mitochondria isolated from MLs and MGs of heat-producing C. revoluta male cones

Oxygen uptake was measured as described in “Materials and Methods.” Data are means 6 SD of at least five measurements. *P , 0.05 and **P ,0.005 as determined by Student’s t test.

Sequential AdditionsOxygen Uptake (nmol O2 min21 mg21 protein)

ML MG

Succinate 34.77 6 2.90 27.35 6 9.33*ADP 67.87 6 6.19 67.15 6 21.72KCN 30.84 6 6.04 7.20 6 1.38**n-Propyl gallate 5.50 6 3.39 1.99 6 1.36

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the microsporangia of C. revoluta male cones. In addi-tion, even smaller mitochondria, i.e. those not catego-rized as large mitochondria, in microsporophyllsexhibited morphological (matrix) features similar tothose of the large mitochondria (Fig. 2, C and D). Given

that mitochondrial dynamics have been observed invarious plants (Arimura, 2018), the morphologicalsimilarities observed in the chondriomes of both largeand nonlarge mitochondria in microsporophyll cellsimplies that theymight be derived from the consecutivefission and fusion of mitochondria, and thus, theirfunction, including cellular respiration, might be simi-lar. Interestingly, the shape of the mitochondria inC. revoluta microsporophylls was very similar to thatobserved in the appendix of Sauromatum guttatum in-florescences during its thermogenic stage (Skubatz andKunkel, 2000). Although the large mitochondria inC. revoluta microsporophylls were not reported inS. guttatum appendices, the similarities in mitochon-drial morphology, i.e. the almost spherical shape with auniform matrix density, between these two thermo-genic tissues may be correlated with the thermogeniccapacity or activity, or both, in plant tissues.

Mitochondria with Distinctive Morphology in MoreGeneral Plants and Green Algae

There has been a long-standing interest in mito-chondrial pleomorphy, and giant or enlarged mito-chondria have been observed in various plants andgreen algae. There are a few reports addressing giantmitochondria in egg cells of Pelargonium zonale(Kuroiwa and Kuroiwa, 1992) and in synchronized cellsof Chlamydomonas reinhardtii (Ehara et al., 1995) andEuglena gracilis (Osafune et al., 1975). Many of thesegiant mitochondria contain a large electron-lucent areawith a few peripherally localized cristae. The structureof these previously reported giant mitochondria is sig-nificantly different from that of the large mitochondriaseen in the cycad microsporophylls in the current study(Fig. 2B), as the latter have a uniform matrix density.The notable difference in the mitochondrial structurebetween the three previously reported noncycad spe-cies and the cycads suggests that the respiratory func-tion of the large mitochondria in cycads likely differsfrom that of the giant or enlarged mitochondriaobserved in the three noncycad species. Actually, theformation of giant mitochondria in C. reinhardtii in-duced a temporary reduction in respiratory function(Ehara et al., 1995). Enlarged mitochondria have alsobeen observed in cultured cells of tobacco (Van Gesteland Verbelen, 2002) and Arabidopsis leaves (Ramonellet al., 2001). However, the formation of those mito-chondria was induced by low oxygen pressure. Despitethe inward parenchyma cells in cycad microsporo-phylls likely being under lower oxygen partial pressurecompared with the epidermal cells, they contained fewlarge mitochondria, and it is therefore improbablethat the formation of large mitochondria in cycads wasinduced by hypoxia. In addition, although elongatedor networked mitochondria have also been observedin germinating seeds, shoot apical meristems, andculture-starting protoplasts in several angiospermplants (Sheahan et al., 2005; Seguí-Simarro et al., 2008;

Figure 4. Two cycad AOX genes, CrAOX1 and CrAOX2, showing dif-ferent expression patterns in thermogenic male cones. A, Phylogenetictree of the predicted amino acid sequences of CrAOX1, CrAOX2, andrelated proteins. Red letters indicate gymnosperm AOX proteins, andbold red letters indicate C. revoluta AOX proteins. The tree wascreated in MEGA7.0.14 based on the alignment produced in Clustal Wand using the neighbor joining method. Bootstrap values .10 areshown and were determined from 1,000 replications. The scale barrepresents one amino acid substitution per site. The AOX sequence fromthe green algaOstreococcus lucimarinus CCE9901 was included as anoutgroup. Accession numbers and species abbreviations are listed inSupplemental Table S2. B, Tissue-specific expression of CrAOX1,CrAOX2, andCrUCP transcripts in thermogenicmale cones. Expressionof CrAOX1, CrAOX2, and CrUCPmRNA in two major tissues of cycadmale cones, ML and MG, was analyzed using real-time PCR. CrEF1gwas used as an internal control. Values are means from four differentmale cones, and bars indicate the SD. *P , 0.05 and **P , 0.005 asdetermined by Student’s t test. C, Quantitative analysis of CrAOX1 andCrAOX2 transcript levels. To estimate the copy number of each CrAOXtranscript in plant mRNA, plasmids containing each CrAOX gene wereused as the standard for real-time PCR. Each bar indicates the SD. Theexpression level of CrAOX1 in MG was set to 1.0. *P , 0.05 as deter-mined by Student’s t test; ns, not significant.

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Paszkiewicz et al., 2017), these mitochondria markedlydiffer in shape from the spherical mitochondria ob-served in cycad microsporophylls.In nonthermogenic plants or tissues, energy metab-

olism and mitochondrial morphology seem to be notalways correlated each other. The number of mito-chondria increases in the leaves of several plants underconditions of elevated CO2 levels, but in many cases,irrespective of the effects on mitochondrial number, theelevated CO2 decreases the rate of mass-based darkrespiration (Griffin et al., 2001, 2004; Tissue et al., 2002;Gomez-Casanovas et al., 2007). In the shoot apicalmeristem of Arabidopsis, very large and tentaculate/cage-like mitochondria have been located in closeproximity to the nucleus, but such a distinctive mito-chondrial structure has not been observed in the rootapical meristem cells, suggesting that such a uniquetype of mitochondrion may not be required to satisfythe energy needs of meristematic cells (Seguí-Simarroet al., 2008). Such an elongated or enlarged mitochon-drial structure in those plants might be related to mi-tochondrial DNA replication/recombination ratherthan energy demand, to provide a kind of qualitycontrol for mitochondrial populations in the new gen-eration (Rose and McCurdy, 2017; Arimura, 2018).

AOX Respiration, via CrAOX1, in Microsporophylls Playsa Role in Male Cone Thermogenesis

Although the presence of cyanide-resistant respira-tion in the microsporophylls of male cones of thethermogenic cycad Z. pumila (Zamiaceae) was firstdemonstrated in a respiratory assay using the tissueitself (Skubatz et al., 1993), it had not been exploredwhether such AOX respiration is involved in cycadthermogenesis. Thus, the current study examined theAOX capacities using intact mitochondria isolatedfrom microsporophylls (proposed to be thermogenic)and microsporangia (possibly nonthermogenic) of C.revolutamale cones. The findings clearly indicated thatthe capacity of cyanide-resistant respiration was sig-nificantly higher in microsporophylls than in micro-sporangia (Fig. 3B). The AOX capacities in mitochondriaisolated from cell cultures of the possibly nonthermogenicgymnosperm A. angustifolia (Mariano et al., 2008; Valenteet al., 2012) were similar to those in C. revoluta micro-sporangia obtained in the current study (Fig. 3B; Table 4).The tissue-specific AOX capacity in the C. revoluta mi-crosporophylls, therefore, suggested thatAOX respirationin microsporophylls and that in microsporangia mayplay a major and a minor role, respectively, in cycadthermogenesis. In a male cone, the total weight of mi-crosporophylls was about 10-fold greater than that ofmicrosporangia, and thus, intact mitochondria recoveredfrom microsporophylls were .10-fold larger than thosefrom microsporangia (Fig. 3A, right). The higher AOXcapacities and greater weight of microsporophyllssuggests that most of the heat in a male cone is producedin microsporophylls rather than in microsporangia.

Recent increases in gymnosperm genomic and Ex-pression sequence tag data revealed that the gymno-sperm AOX is encoded by a multigene family(Frederico et al., 2009; Neimanis et al., 2013). However,in gymnosperms, the AOX2 subtypes are limited to twospecies, P. pinea (Frederico et al., 2009) and Cryptomeriajaponica (Neimanis et al., 2013), both belonging toConiferophyta. In the current study, two AOX genes,CrAOX1 and CrAOX2, were identified in the gymno-sperm C. revoluta (Cycadophyta). CrAOX1 mRNA washighly expressed in the microsporophylls, whereasCrAOX2 mRNA was highly expressed in the micro-sporangia. The transcript level of CrAOX1was 100-foldgreater than that ofCrAOX2 in themicrosporophylls. Inaddition, because the mitochondria in microsporo-phylls had higher AOX capacity and protein expressionlevels than those in microsporangia (Fig. 3, B and C),CrAOX1 is proposed to play a major role in C. revolutamale cone thermogenesis. This study provides firmexperimental evidence that cycad CrAOX1, but notCrAOX2, is involved in cone thermogenesis.

MATERIALS AND METHODS

Plant Materials

Cycads (Cycas revoluta) used in the experiments were grown at theUniversity of Miyazaki and along prefectural road no. 367 in Tsunehisa,Miyazaki-city, Japan.

Surface and Internal Temperature Measurements

The surface temperature of cycad cones was analyzed using images from athermal imager, FLIR SC620 (FLIR Systems). To eliminate the influence of solarradiation during daytime, thermal images were taken at night when the heatacquired from sun exposure is negligible. The internal temperatures of cycadcones were measured using a thermal recorder (TR-52, T&D).

Ultrastructural Analyses

Ultrastructural analyses were conducted as previously described (Toyookaet al., 2000; Ito-Inaba et al., 2009b), with modifications. Briefly, the microspo-rophylls, microsporangia, and central axis were separated from each otherusing a knife and clamping forceps and cut into 1- to 3-mm cubes. Each tissuetype was fixed with 4% (w/v) paraformaldehyde and 2% (v/v) glutaraldehydein 50 mM sodium cacodylate buffer (pH 7.4) overnight at 4°C. Tissues werepostfixed with 1% (v/v) osmium tetroxide in 50 mM cacodylate buffer (pH 7.4)for 3 h at room temperature. After dehydration in a graded methanol series,tissue cubes were further substituted in methanol:propylene oxide (1:1), 100%(v/v) propylene oxide, and propylene oxide:Epon812 resin (Taab; 3:1, 1:1,1:3), and finally embedded in 100% (v/v) Epon812 resin. Ultrathin sections(60–80 nm) were obtained using a diamond knife on ultramicrotome (EM UC7;Leica) and transferred to Formvar-coated grids to be stained with 4% (w/v)uranyl acetate for 12 min and with lead citrate solution for 2 min. Stainedsections were examined under a transmission electron microscope (JEM-1400;JEOL). Images were acquired using a charge-coupled device camera. Mito-chondria and plastids were distinguished based on the membrane structureinside each organelle. Mitochondrial size, perimeter, and number were quan-tified using Photoshop CS3 extended software (Adobe).

Mitochondrial Isolation from C. revoluta Male Cones

Mitochondria were isolated using differential centrifugation and Percolldensity gradient centrifugation as described by Ito-Inaba et al. (2008a))),with minor modifications of the cell disruption procedures. Briefly, the

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microsporophylls, microsporangia, and central axis of male cones were sepa-rated with a knife and a microspatula and immediately soaked in grindingmedium (0.4 M mannitol, 25 mM MOPS-KOH, 2 mM EDTA, 10 mM KH2PO4,1% [w/v] PVP-40, 20 mM ascorbic acid, 4 mM Cys, 2 mM pyruvate, 1% [w/v]bovine serum albumin [fatty acid free], 2% [w/v] poly[vinylpolypyrrolidone];pH 7.2). Microsporophylls and central axis tissues were then disrupted in aMixer HR-9391 (EÜPA) for 10 s. Microsporangia were ground in a PolytronPT 2500E (EYELA) for 5 s. These processes were performed at 4°C. Mitochon-dria were successfully isolated frommicrosporophylls andmicrosporangia, butnot from the central axes.

Oxygen Consumption Assay

Oxygen consumptionwasmonitored using anOxytherm-1 oxygen electrode(Hansatech) at 25°C. Mitochondria (300 mg) were added to 1.2 mL assay buffer(0.3 M sucrose, 20 mMMOPS, 1 mMMgCl2/6H2O, 10mMKCl, 5 mMKH2PO4,0.1% (w/v) bovine serum albumin; pH 7.2). Respiratory substrates NADH(1 mM), succinate (5 mM), or a mixture of pyruvate (10 mM) and malate (10 mM)were then added to the assay buffer. When amixture of Glu (10mM) andmalate(10 mM) was used as the respiratory substrate, the assay buffer was prepared topH 6.8 (Jacoby et al., 2015). After the addition of the substrate, ADP (0.5 mM),KCN (1 mM), and n-propyl gallate (0.1 mM) were added to the assay buffer, inthis order, as previously described (Ito-Inaba et al., 2009a). When succinate wasused as the respiratory substrate, ATP (0.1 mM) was included in the assay bufferto activate succinate dehydrogenase. Prior to the addition of pyruvate + malateor Glu + malate, a cofactor mix that included NAD+ (2 mM), CoA (12 mM), andthiamine pyrophosphate (200 mM) was added. A mixture of pyruvate (10 mM)and dithiothreitol (5 mM) was added after the addition of KCN (1 mM) to ana-lyze the cycad AOX sensitivity to pyruvate.

Measurements of COX Activity and OuterMembrane Integrity

Cytochrome c oxidase (COX) activity was measured using two methods:ascorbate-N,N,N9,N9-tetramethyl-p-phenylenediamine (TMPD) assay (Jacobyet al., 2015) and CYTOCOX1 assay kit (Sigma-Aldrich). In the ascorbate-TMPD assay, mitochondria (300 mg) were added to 1.2 mL assay buffer (thesame buffer used in the oxygen consumption assay; pH 7.2), and ascorbate andTMPD were sequentially added. The CYTOCOX1 assay was carried outaccording to the manufacturer’s instructions. Outer membrane integrity wasmeasured as previously described (Ito-Inaba et al., 2008a).

SDS-PAGE and Immunoblotting

The SDS-PAGE and immunoblotting procedures were conducted as de-scribed by Ito-Inaba et al. (2008a).

Antibodies

Polyclonal antibodies against AOX (Ito-Inaba et al., 2009a) and against UCP(Ito-Inaba et al., 2008b) were prepared as previously described. Anti-HSP60(Stressgen) antibodies were purchased.

Isolation and Sequencing of CrAOX1 and CrAOX2 cDNAs

To isolate CrAOX transcripts, total RNAwas first extracted frommale conesusing the RNeasyMini Kit (QIAGEN). First-strand cDNAswere generatedwitha PrimeScript RT Reagent Kit (Takara) using oligo(dT) primers and randomhexamers. Reverse transcription PCR was successively performed using thedegenerate primer pairs, P1 and P2, which were used to amplify Arabidopsis(Arabidopsis thaliana) AOX genes (Saisho et al., 1997), and 42AOX-F and42AOX-R, which were used to amplify Pinus pinea AOX genes (Fredericoet al., 2009). The obtained fragments were cloned into the EcoRV site of pZErO-2 vector (Invitrogen) and sequenced. The two partial cDNA fragments obtainedhad high levels of sequence similarity to plant AOX1 and AOX2, respectively.

To isolate CrAOX1 cDNA, primers for 39-RACE reactions were designedbased on the partial sequences derived from fragments amplified using P1 andP2 primers; 39-RACE reactions were performed using the RNA Ligase-Mediated kit (Applied Biosystems) and the primers CrAOX-39RACE-OP1 forthe first PCR and CrAOX-39RACE-IP1 for the second PCR. Obtained products

were cloned into the EcoRV site of pZErO-2 vector (Invitrogen) and sequenced.Unfortunately, 59-RACE reactions were unsuccessful. To extend the 59-cDNAends of the obtained partial fragments, conserved nucleotide sequences wereidentified by aligning the following AOX1 sequences: AtAOX1a (ArabidopsisAOX1a; NM_113135); PgAOX_s1 (Picea glauca AOX subtype 1; BT119347);PgAOX_s2 (P. glauca AOX subtype 2; BT104039); and PsAOX_s2 (Picea sitchensisAOX subtype 2; EF084004). Based on conserved portions of these sequences, aforward primer (CrAOX-degF3) was designed and used to amplify cDNA frag-ments together with primer CrAOX-R1N. The obtained fragments were cloned intotheEcoRV site of pZErO-2 vector (Invitrogen), resulting in pZAOX1, and sequenced.

To isolate CrAOX2 cDNA, primers for 39-RACE reactions were designedasdescribed above and reactionswereperformedusing theRNALigase-MediatedRACE kit (Applied Biosystems) and the primers CrAOX2-39RACE-OP1 forthe first PCR and CrAOX2-39RACE-IP1 for the second PCR. The productsobtained were treated by ExoSAP (Thermo Fisher Scientific) and directlysequenced. The 59-RACE reactions were unsuccessful. The 59-cDNA endsof the partial fragments obtained were, therefore, also extended basedon the alignment of AOX2 sequences from several plants: AtAOX2(Arabidopsis AOX2; NP_201226); NnAOX2d (Nelumbo nucifera AOX2d; gi|719971362); GmAOX2a and GmAOX2d (Glycine max AOX2a [U87906] andAOX2d [U87907], respectively); ClAOX2 (Citrullus lanatus AOX2; ADD84880);VuAOX2a and VuAOX2d (Vigna unguiculata AOX2a [AJ319899] and AOX2d[AJ421015], respectively); and DcAOX2a and DcAOX2b (Daucus carota AOX2a[EU286575] and AOX2b [EU286576], respectively). Based on the conservedportions of these sequences, the forward primer CrAOX2-degF2 was designedand used to amplify cDNA fragments together with the primer CrAOX2-SP-R2.The obtained fragments were cloned into the EcoRV site of pZErO-2vector (Invitrogen), resulting in pZAOX2, and sequenced. The primersdesigned in the current study are listed in Supplemental Table S1.

Molecular Phylogenetic Analysis of AOX Proteins

Amino acid sequences of the AOX proteins were retrieved from the DNAData Bank of Japan/EMBL/GenBank and RefSeq databases. In addition, short-read data from the Sequence Read Archive (SRA; available at the NationalCenter for Biotechnology Information; https://www.ncbi.nlm.nih.gov/sra)were used to retrieve AOX genes from magnoliids and basal Magnoliophyta.The SRA data were assembled into contigs using the CAP3 Sequence AssemblyProgram (http://doua.prabi.fr/software/cap3). Deduced cDNAs from theSRA data were translated into amino acid sequences using the translate toolat the ExPASy web server (http://web.expasy.org/translate/). All deducedproteins were checked against the AOX protein sequences using the BLASTBLASTp, available at the National Center for Biotechnology Information. Thephylogenetic analysis was performed in MEGA version 7.0.14 (Kumar et al.,2016) using 146 residues within the conserved regions of AOX proteins, whichcorresponded to positions 181 to 326 of SrAOX (AB183695). Sixty-five AOXprotein sequences were aligned using CLUSTALW (Larkin et al., 2007), and thephylogenetic tree was constructed using the neighbor-joining method; boot-strap values presented on the consensus tree were based on 1,000 replications.

Expression Analyses of CrAOX1 and CrAOX2 Transcriptsby Real-Time PCR

Total RNAwas extracted frommicrosporangia and microsporophylls usingthe RNeasy Mini Kit (QIAGEN). First-strand cDNAs were generated with aPrimeScript RT Reagent Kit (Takara) using oligo(dT) primers and randomhexamers. Real-time PCR was performed on a Thermal Cycler Dice Real-TimeSystem (Takara) using TB Green Premix ExTaq II (Takara). All the real-timePCR analyses were performed using biological quadruple samples. Primersused for real-time PCR are listed in Supplemental Table S1. Transcript levels ofeach gene were normalized to that of CrEF1g.

Statistical Analysis

Data were statistically analyzed by Student’s t test or Turkey-Kramer mul-tiple comparison test, as described in the figure legends for each experiment.

Accession Numbers

The nucleotide sequences reported in this paper were submitted to the DNAData Bank of Japan under accession numbers LC081345 for CrAOX1, LC229699for CrAOX2, and LC081346 for CrUCP.

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Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Assay of cytochrome pathway respiration andCOX (Complex IV) rate in microsporophyll (ML) and microsporangia(MG) mitochondria.

Supplemental Figure S2. Oxygen uptake measured in microsporophylls(left) and microsporangia (right) mitochondria using various respiratorysubstrates.

Supplemental Figure S3. Expression of AOX proteins in the microsporo-phylls and microsporangia mitochondria.

Supplemental Figure S4. Deduced amino acid sequences of CrAOX1 andCrAOX2 aligned with trypanosomal AOX (XP_822944) and AOX iso-forms in Arabidopsis and other gymnosperms.

Supplemental Figure S5. Oxygen uptake rate in intact mitochondria iso-lated from microsporophylls.

Supplemental Figure S6. Deduced amino acid sequences of CrUCPaligned with UCP isoforms in other gymnosperms and thermogenicskunk cabbage.

Supplemental Figure S7. Phylogenic relationships of CrUCP with UCPisoforms in other plants.

Supplemental Table S1. Gene-specific primers used in PCR.

Supplemental Table S2. List of AOX genes used in phylogenetic analysis.

Supplemental Table S3. List of UCP genes used in phylogenetic analysis.

ACKNOWLEDGMENTS

We thank Mayumi Wakazaki and Kei Hashimoto (RIKEN Center forSustainable Resource Science) for their assistance in experiments with electronmicroscopy. We also thank Yoko Katayama and Saori Hamada (University ofMiyazaki) for their assistance in molecular biological experiments. We aregrateful to Takehiko Ito (Tokyo Institute of Technology) for valuable advice.

Received February 4, 2019; accepted March 12, 2019; published March 27, 2019.

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