Genetic defects in the CDP-choline pathway for phosphatidylcholine biosynthesis cannot be transmitted to offspring via male gametophytes owing to interruption of autophagy-like processes required for pollen germination in Arabidopsis thaliana

Genetic defects in the CDP-choline pathway for phosphatidylcholine biosynthesis cannot be transmitted to offspring via male gametophytes owing to interruption of autophagy-like processes required for pollen germination in Arabidopsis thaliana | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genetic defects in the CDP-choline pathway for phosphatidylcholine biosynthesis cannot be transmitted to offspring via male gametophytes owing to interruption of autophagy-like processes required for pollen germination in Arabidopsis thaliana Momoka Wada, Chiaki Kuga, Kimie Atsuzawa, Atsuko Miyagi, Toshiki Ishikawa, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7822087/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Feb, 2026 Read the published version in Journal of Plant Research → Version 1 posted 5 You are reading this latest preprint version Abstract Phosphatidylcholine is a major plant membrane phospholipid that contributes to the biogenesis and desaturation of membrane lipids and storage lipids. Thus, to ensure reproductive capacity, any genetic defect that affects phosphatidylcholine biosynthesis must be eliminated before fertilization. In Arabidopsis thaliana , phosphatidylcholine biosynthesis depends on CCT1 and CCT2 , both encoding CTP:phosphorylcholine cytidylyltransferase. Using A. thaliana T-DNA-tagged mutants, we demonstrate that neither cct1-3 cct2-3 nor cct1-3 cct2-5 seedlings are viable. Reciprocal crosses of cct2-3/CCT2 or cct2-5/CCT2 plants in the cct1-3 background revealed that neither cct2-3 nor cct2-5 was transmitted via cct1-3 male gametophytes, although each allele was transmitted via cct1-3 female gametophytes. Although all pollen grains on a pollen quartet from qrt1-1 cct1-3 cct2-5/CCT2 plants were viable, none of cct1-3 cct2-5 pollen grains from cct2-5 cct1-3/CCT1 and cct1-3 cct2-5/CCT2 plants were able to germinate in vitro. Transmission electron microscopy analysis of pollen grains subjected to pollen germination revealed that cct1-3 cct2-5 pollen grains developed unusual ultrafine structures, such as lipid bodies of disproportionate size including extremely enlarged ones, swelling of small granular structures, inhibition of vacuole development, and accumulation of incomplete autophagosome-like bodies enclosing various intracellular compartments. Thus, transmission of cct1-3 cct2-5 to offspring via male gametophytes appears to be strictly prohibited by interruption of autophagic processes required for pollen germination, thereby preventing the widespread dispersal of deleterious mutations among the progeny. By contrast, cct1-3 cct2-5 was partly transmissible via female gametophytes, so the background genome could be rescued by fertilization. Autophagy Essential genes Phosphatidylcholine Pollen germination Reciprocal cross Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Recent studies on gametogenesis have focused on how gametophytic selection has contributed to diverse evolutionary processes (Beaudry et al. 2020 ). In mammals, gametogenesis refers to a process that produces a haploid cell (n) from a diploid cell (2n) through meiosis. In plants, however, meiosis is not sufficient for the completion of gametogenesis: for maturation, both microspores and megaspores generally require mitosis and differentiation, which depend on the successful progression of certain fundamental processes such as biosynthesis of cellular components, polarized vacuolation, catabolism of storage materials, vesicular trafficking and membrane biogenesis (McCormick 1993 ; Skinner and Sundaresan 2018 ). Furthermore, autophagy is required for pollen germination (Fujiki et al. 2007 ; Qin et al. 2007 ). Some of these processes may be essential for sporophyte development. In this context, any mutation that is unfavorable for sporophytes must be eliminated during gametogenesis and/or before fertilization. Mutations in essential genes may have similar consequences such as the lethality of male and female gametophytes, which ensures that unfavorable mutant genes will not be transmitted to offspring. However, certain mutations are transmitted at different rates via male or female gametophytes (Bolaños‑Villegas et al. 2015; El-Kasmi 2011; Liu et al. 2021 ). For example, functional loss of the canonical α-SNAP in Arabidopsis results in gametophytic lethality by arresting first mitosis during gametogenesis, but reciprocal crosses revealed that transmission of the mutation via female gametophytes is leaky whereas that via male gametophytes is strictly prohibited (Liu et al. 2021 ). Lipids are essential components of biomembranes, and some of genes responsible for lipid biosynthesis are among the essential genes (Meinke et al. 2008 ). However, it is not well understood how mutations in lipid genes are eliminated before fertilization or if there is any difference in the rate of transmission of mutant lipid-associated genes via male or female gametophytes. In plant cells, phosphatidylcholine (PC) is a major membrane phospholipid that serves as not only a biosynthetic precursor to galactolipids (Kobayashi el al. 2013) that are abundant in photosynthetic membranes but also a substrate for fatty-acid desaturases that regulate membrane fluidity (Ohlrogge and Browse 1995 ). Thus, any failure of PC biosynthesis is anticipated to negatively impact sporophyte growth and development. In this context, any mutation that affects PC biosynthesis must be eliminated from the genome before fertilization or during the early stages of embryogenesis. However, no critical studies have been reported to evaluate the importance of PC biosynthesis in plants or to examine if and how mutations in PC biosynthesis could be eliminated before fertilization. In Arabidopsis thaliana , PC biosynthesis occurs via the CDP-choline pathway, which is regulated by the genes CCT1 (AT2G32260) and CCT2 (AT4G15130), each of which encodes CTP:phosphorylcholine cytidylyltransferase (CCT, E.C. 2.7.7.15) (Inatsugi et al. 2002 , 2009 ). CCT synthesizes CDP-choline, a precursor to the polar-head group of PC, from CTP and phosphorylcholine; amino alcohol phosphotransferases (AAPT; E.C. 2.7.8.2) then transfer the phosphorylcholine residue of CDP-choline to the sn -3 position of sn -1,2-diacylglycerol to produce PC. Apart from the CDP-choline pathway, other eukaryotes like yeast can synthesize PC via triple methylation of phosphatidylethanolamine (PE) (Vance and Ridgeway 1988 ; Carman and Zeimets 1996 ). However, the Arabidopsis genome does not have a gene responsible for the first methylation step (Ohlrogge and Browse 1995 ; Keogh et al. 2009 ) and, hence, the PE methylation pathway towards PC does not complement the deficiency of the CDP-choline pathway towards PC in Arabidopsis. Several studies have revealed the roles of phospholipids in reproductive development of Arabidopsis. For S -adenosyl- l -methionine:phosphoethanolamine N -methyltransferase (EC 2.1.1.103), which is required for phosphorylcholine biosynthesis, the temperature-sensitive mutant t365 exhibits male sterility (Mou et al. 2002 ). For CTP:phosphorylethanolamine cytidylyltransferase (PECT1; EC 2.7.7.14), which is required for PE biosynthesis, the null mutant pect1-6 is unable to proceed to embryo development beyond the early globular stage (Mizoi et al. 2006 ), whereas the pect1-4 mutant that retains 26% of the PECT1 activity causes a delay in anther development. Thus, downregulation of the CDP-ethanolamine pathway towards PE biosynthesis causes a delay in anther development, but disruption of the CDP-ethanolamine pathway does not completely abolish the development of male and female gametophytes. AAPT1 and AAPT2 are required for the final step in PC and PE biosynthesis. Disruption of both AAPT1 and AAPT2 abolishes the establishment of aapt1 aapt2 seeds, and 50% of pollen grains from aapt1 aapt2 / AAPT2 or aapt1/AAPT1 aapt2 plants having the qrt1 background die, suggesting that disruption of both PC and PE biosynthesis induces the lethality of male gametophytes (Liu et al. 2015 ). Phosphatidic acid is the common precursor to glycerolipids, and glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.2.15) catalyzes the first step of phosphatidic acid biosynthesis. GPAT9 is responsible for the production of phosphatidic acid that is necessary for cytoplasmic glycerolipids, and disruption of GPAT9 abolishes the establishment of gpat9 seeds (Shockey et al. 2016 ). Reciprocal crosses of gpat9-2/GPAT9 plants demonstrated that gpat9-2 is not transmissible via male gametophytes, and gpat9-2 pollen grains are unable to germinate in vitro in the qrt1 background. In the qrt1 background, gpat9-2 pollen grains are smaller than GPAT9 pollen grains (Shockey et al. 2016 ). Because disruption of phosphatidic acid biosynthesis inhibits triacylglycerol (TAG) biosynthesis, the inhibition of pollen germination is thought to be caused by TAG shortage (Shockey et al. 2016 ). Choline kinase is required for the biosynthesis of phosphorylcholine, the substrate of CCT, and the choline/ethanolamine kinase CEK4 is involved in embryo development (Lin et al. 2015 ). Finally, phosphatidylserine is involved in vesicular trafficking and required for normal progression of cell plate formation (Yamaoka et al. 2021 ). Disruption of phosphatidylserine biosynthesis affects embryo development and causes pollen lethality, but even the null mutant pss1 can escape pollen lethality and establish pss1 seedlings (Yamaoka et al. 2011 ). Using T-DNA-tagged mutants of A. thaliana , designated cct1-3 , cct2-3 and cct2-5 , we investigated whether the double mutation cct1-3 cct2-3 or cct1-3 cct2-5 can be carried over to the F3 progeny. Our results show that neither cct1-3 cct2-3 nor cct1-3 cct2-5 seedlings are present in F3 progeny. We then investigated the mechanism by which the cct1-3 cct2-3 or cct1-3 cct2-5 mutation can be eliminated from the F3 seed population by conducting reciprocal crossing, Alexander’s pollen viability test, in vitro pollen germination test, and observation of ultrathin sections of germinating pollen grains by transmission electron microscopy (TEM). We conclude that cct1-3 cct2-3 and cct1-3 cct2-5 are strictly eliminated during pollen germination owing to PC shortage; this elimination inhibits the progression of autophagy required for pollen germination. We also showed that cct1-3 cct2-3 and cct1-3 cct2-5 are partly transmissible via female gametophytes. Our results suggest that plants utilize distinct transmission strategies between male and female gametophytes, at least in regards to mutant genes in PC biosynthesis: transmission of the mutant genes via male gametophytes is strictly prohibited by inhibition of pollen germination so that a wide dispersal of deleterious mutation among the progeny is prevented, whereas transmission of the same mutant genes via female gametophytes is partly permissive so that the background genome of the escaped mutant ovules can be rescued by fertilization with normal pollen. Materials and methods Plant materials Arabidopsis thaliana (L.) Heynh. ecotype Columbia was obtained from Lehle seeds (Round Rock, TX, U.S.A., http://www.arabidopsis.com/ ). cct1-3 (GK-349C03-016244) was obtained from GABI-Kat ( G enom a nalyse im bi ologischen System Pflanze - K ölner A rabidopsis T -DNA lines) via the Arabidopsis Biological Resource Center (ABRC). cct2-3 (SK34804, CS1012739) was obtained from ABRC, whereas cct2-5 (SALK_200207, originally distributed by ABRC) was a gift from Peter Moffett (Fig. 1 ). Plant growth Arabidopsis seeds were sown on soil (Supermix A, Sakata, Kanagawa, Japan) packed in a stainless steel pan (185 mm W × 140 mm L × 30 mm D) or a plastic pot (60 mmφ × 60 mm H). After a 2-day incubation at 4 ℃ under 100% relative humidity, pans (or pots) were incubated in a growth room regulated at 23 ℃ under a 16-h light/8-h dark photo regime at a photon flux density of 110 µmol m –2 s –1 . Arabidopsis transformation and selection for the transformants A. thaliana was transformed by the floral dip method (Clough and Bent 1998 ) using Agrobacterium tumefaciens strain GV3101. Seeds were sterilized by immersion in 70% ethanol for 1 min then in a sterilizing mixture containing 5% (v/v) sodium hypochlorite and 0.02% (w/v) Triton X-100 for 5 min twice. After washing with sterilized water, seeds were aseptically sown on 1/2 MS plates containing a half-strength Murashige-Skoog salts (Company, City, Japan), 1× Gamborg B5 vitamins (Gamborg et al. 1968 ), 0.5% MES (pH 5.7), and 0.7% agar for plant growth (Company, City, Japan). Genotyping by PCR One cotyledon or an equivalent size of leaf was homogenized in 400 µl DNA extraction buffer containing 0.2 M Tris-HCl (pH 9.0), 0.4 M LiCl, 25 mM EDTAཥ2Na and 1% SDS in a round-bottomed 1.5-ml microtube (As one, Osaka, Japan) using a homogenizer pestle (CT1.5 3-325-0268, Kenis, Osaka, Japan). After centrifugation at 16,000 × g for 1 min, a 300-µl portion of the supernatant was recovered for DNA precipitation with an equivalent volume of 2-propanol. Each DNA pellet was recovered by centrifugation twice at 16,000 × g for 5 min, carefully eliminating the residual supernatant. After a 15-min evacuation under vacuum, DNA was dissolved in 100 µl of buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTAཥ2Na) to make a template DNA solution. PCR for genotyping was conducted using a Thermal cycler (2720, Applied Biosystems, Tokyo, Japan) under reaction conditions summarized in Table S1 . Each reaction mixture contained 5 µl 2×Quick Taq® HS DyeMix (TOYOBO, Tokyo, Japan), 1 µl template DNA solution, and 1 µl each of 10 pmol µl –1 primer solutions (Table S1 ). Construction of cct mutants in the qrt1-1 background qrt1-1 plants form inseparable pollen tetrads and hence are useful for meiotic segregation analysis of mutant phenotypes. To construct qrt1-1 cct1-3/CCT1 cct2-3/CCT2 and qrt1-1 cct1-3/CCT1 cct2-5/CCT2 plants, qrt1-1 plants (♂) were first crossed with cct1-3 cct2-3/CCT2 and cct1-3 cct2-5/CCT2 plants (♀) to obtain qrt1-1/QRT1 cct1-3/CCT1 cct2-3/CCT2 and qrt1-1/QRT1 cct1-3/CCT1 cct2-5/CCT2 plants, respectively, from which seedlings with the cct1-3/CCT1 cct2-3/CCT2 and cct1-3/CCT1 cct2-5/CCT2 genotypes were identified in the respective offspring by PCR. Finally, seedlings with qrt1-1 were identified under a scanning electron microscope (TM-1000, HITACHI, Tokyo, Japan). Reciprocal crosses Reciprocal crosses of cct2-3/CCT and cct2-5/CCT2 in the cct1-3 background were conducted by crossing cct1-3 vs. cct1-3 cct2-3/CCT2 and cct1-3 vs. cct1-3 cct2-5/CCT2 , respectively. Segregation ratios of mutant alleles in the F1 offspring were determined by PCR. Alexander’s staining to assess pollen survival Pollen viability was determined by Alexander staining (Alexander 1969 , Atlagić et al. 2012 ). On the day of flowering, open flowers were sampled in a 1.5-ml microtube and immediately submerged in Alexander’s solution. After 15 min, samples were washed with water, and anther and pollen grains were observed under a fluorescence microscope (LEICA DMR, Leica Microsystems, Tokyo, Japan). Pollen germination in vitro Pollen collected on the day of flowering was spread over a 1.5% agar plate made up with pollen germination medium containing 0.01% boric acid, 5 mM CaCl 2 , 5 mM KCl, 1 mM MgSO 4 , 10% sucrose, 10 µM brassinosteroid (a gift from Dr. Miho Ikeda at Saitama University) and 0.5% ethanol (Vogler et al. 2014) and incubated in a growth cabinet (BiOTRON LPH-240, NK Systems Limited, Tokyo, Japan) regulated at 23℃ in darkness. Pollen germination and pollen-tube elongation were observed under a fluorescence microscope (LEICA DMR, Leica Microsystems, Tokyo, Japan). Observation of pollen morphology by scanning electron microscopy On the day of anthesis, anthers were harvested, and pollen was spread over a glass slide. The morphology of the pollen surface was observed under a scanning electron microscope. DAPI staining for evaluation of mitosis in pollen Four to five opened flowers sampled on the day of flowering were immersed in DAPI staining medium containing 0.4 µg ml –1 DAPI, 0.1% (w/v) Triton X-100, 1 mM EDTAཥ2Na and 100 mM sodium phosphate pH 7 (Schnedl et al. 1977 ). Pollen grains were collected by centrifugation and observed under a fluorescence microscope (LEICA DMR, Leica Microsystems, Tokyo, Japan). Transmission electron microscopy Samples for transmission electron microscopy were made according to Kaneko ( 2007 ). Pollen collected on the day of anthesis was spread over an agar plate made up with pollen germination medium (Vogler et al. 2014). After incubation for 30 min, pollen was collected by centrifugation and suspended in 0.05 M potassium phosphate buffer (pH 6.8) containing 2% glutaraldehyde and incubated for 4 h at room temperature and then overnight at 4 ℃ (prefixation). After washing six times with 0.05 M potassium phosphate buffer (pH 6.8), samples were incubated in 0.05 M potassium phosphate buffer (pH 6.8) containing 2% OsO 4 for 2 h at ambient temperature (postfixation). Then, samples were washed once with 0.05 M potassium phosphate buffer (pH 6.8) and then subjected to sequential dehydration for 10 min with 10, 30, 50, 70, 85, 95 and 100% acetone. For this purpose, 100% acetone was prepared by storing over anhydrous sodium sulfite. After two additional washes with 100% acetone, samples were subjected to sequential incubations with 50, 75 and 100% Spurr’s resin solution and incubated overnight with 100% resin solution. After replacing the 100% resin solution with fresh resin solution, samples were incubated for 1 h, then placed in 1.5-ml microtubes and incubated at 70 ℃ for 8 h for resin solidification. Ultrathin sections were made and after staining with uranyl acetate and lead citrate subjected to transmission electron microscopy (Hitachi H-7500, at 80 kV) in the Comprehensive Analysis Center for Science at Saitama University. Cloning of the pollen-specific promoter ACA9pro A gene fragment with restriction enzyme tags for subcloning the pollen-specific promoter ACA9pro (Schiøtt et al. 2004 ) was amplified from Arabidopsis genomic DNA by PCR (Table S1 ) using a KOD-Plus-Neo polymerase (TOYOBO). PCR products were subjected to 1% agarose gel electrophoresis, and a band for ACA9pro was purified using a gel extraction kit (Wizard SV Gel and PCR Clean-up System, Promega; or Gel/PCR Extraction Kit, FastGene). Then, purified ACA 9pro was digested with XbaI (TaKaRa) and HindIII (TaKaRa), and digested samples were subjected to 1% agarose gel electrophoresis and bands were then purified as described above. The resultant XbaI–HindIII fragment of ACA9pro was subcloned into the XbaI–HindIII sites of pBluescriptⅡ SK(+) using a 2×Mighty Mix ligation kit (TaKaRa). The resultant plasmid was designated pBluescriptⅡ SK(+) ACA9pro . Construction of CCT1 expression constructs under the control of ACApro9 Using the pBluescriptⅡ SK(+) ACA9pro as a template, the XbaI–HindIII ACA9pro fragment was amplified by PCR and then subcloned into the XbaI–HindIII sites of pPZP221 35Spro:CCT1cDNA-nosT (100 µg/ml spectinomycin) to create pPZP221 ACA9pro:CCT1cDNA-nosT . However, the resultant plasmid unexpectedly contained inverted ACA9pro ( invACA9pro ). Thus, it was designated pPZP221 invACApro9:CCT1cDNA-nosT (Fig. S1 ). Transformation of Agrobacterium tumefaciens A. tumefaciens GC3101 carrying antibiotic resistance for chloramphenicol, gentamycin, rifampicin and streptomycin was transformed by electroporation using 20 µl competent cells, which were prepared according to a method of Sean Weise ( https://bmb.natsci.msu.edu/sites/_bmb/assets/File/Sharkey_lab/Agrobacterium%20Transformation%20and%20Competent%20Cell%20Preparation.pdf ). Transformants were identified by colony PCR (Table S1 ). Isolation of cct1-3 cct2-5/CCT2 plant lines expressing invACA9pro:CCT1cDNA-nosT Primary shoots (~ 10 cm long) were cut off from cct1-3 cct2-5/CCT2 plants to promote secondary shoot regeneration. When the secondary shoots were ~ 15 cm long, all siliques and opened flowers were removed, and the remaining floral tips and rosette base were inoculated with A. tumefaciens culture according to the floral dip method. T1 seeds were selected on 1/2 MS agar plates containing 50 µg ml –1 gentamycin (FUJIFILM Wako Chemicals, Osaka, Japan) and 10 µg ml –1 meropenem trihydrate (FUJIFILM Wako Chemicals, Osaka, Japan), and cct1-3 cct2-5/CCT2 plant lines expressing invACA9pro:CCT1cDNA-nosT were identified by genotyping for cct1-3 and cct2-5 . To demonstrate co-transmission of cct2-5 with the transgene invACA9pro:CCT1cDNA-nosT via male gametophytes, one of the lines, designated M21, expressing cct1-3 cct2-5/CCT2 invACA9pro:CCT1cDNA-nosT (+/–) was immediately used as a pollen source for pollination over the stigma of emasculated cct1-3 flowers. Results Assessment of segregation in the mutant alleles cct1-3, cct2-3 and cct2-5 in the F2 progeny cct1-3 and cct2-5 plants were crossed in an attempt to isolate cct1-3 cct2-5 double mutants in the F2 progeny. However, no cct1-3 cct2-5 double mutant was obtained. Accordingly, offspring of a cct1-3 cct2-5/CCT2 F2 plant was further analyzed (Table 1 ). Again, no cct1-3 cct2-5 double mutant was obtained in the F3 progeny, suggesting that cct1-3 cct2-5 was eliminated during gametogenesis, fertilization, or early embryogenesis/seed development. Table 1 also shows that a segregation ratio was 1:0.77 for the cross between CCT2/CCT2 and cct2-5/CCT2 in the cct1-3 background, a ratio that differed significantly from the expected Mendelian ratio of 1:2 ( P = 1.48 × 10 –4 < 0.05). This result suggested that the seed yield of cct2-5/CCT2 was decreased by 62% in the cct1-3 background. Table 1 Segregation analysis of offspring of a cct1-3 cct2-5/CCT2 plant Number of offspring χ 2 test for 1:2 a Genotype cct1-3 CCT2 cct1-3 cct2-5/CCT2 cct1-3 cct2-5 χ 2 P Theoretical ratio 1 2 1 Observed number 79 61 0 Observed ratio 1 b 0.77 b 0 14.40 1.48 × 10 –4 c a The χ 2 test was conducted to test the null hypothesis that cct1-3 and cct1-3 cct2-5/CCT2 plants segregate from each other in the predicted 1:2 Mendelian ratio. b The segregation ratio between CCT2 and cct2-5/CCT2 plants in the cct1-3 background was 1:0.77, which differed significantly from the predicted 1:2 Mendelian ratio, suggesting that the seed yield of cct2-5/CCT2 was decreased by 62% in the cct1-3 background. c The P value is small enough ( P < 0.05) to reject the null hypothesis. Similar results were obtained from crossing experiments between cct1-3 and cct2-3 plants (Table S2). No cct1-3 cct2-3 double mutant was obtained from a cct1-3 cct2-3/CCT2 plant in the F3 progeny, and the segregation ratio of CCT2/CCT2 and cct2-3/CCT2 in the cct1-3 background was 1:0.77, which differed significantly from the expected 1:2 Mendelian ratio ( P = 1.02 × 10 –4 < 0.05). Because the T-DNA-tagged mutant cct2-3 carried a drug-resistance gene against ammonium glufosinate (BASTA), the unexpected segregation ratio of 1:0.77 between CCT/CCT2 and cct2-3/CCT2 plants in the cct1-3 background was also confirmed by a survival test on 1/2 MS agar plates containing 40 nmol ml –1 BASTA; among 94 seeds harvested from a cct1-3 cct2-3/CCT2 plant, 53 were dead ( cct1-3 CCT2 ) and 41 survived ( cct1-3 cct2-3/CCT2 ) (Fig. S2), again with a segregation ratio of 1:0.77. We also conducted a segregation test for offspring of a cct2-3 cct1-3/CCT1 plant (Table 2 ). In accordance with results presented in Table S2, no cct1-3 cct2-3 double mutant was obtained from the cct2-3 cct1-3/CCT1 plant. However, the segregation ratio between CCT1/CCT1 and cct1-3/CCT1 in the cct2-3 background was 1:1.2, which did not differ significantly from the expected 1:2 Mendelian ratio ( P = 0.055). These results suggested that cct2-3 cct1-3/CCT1 plants had greater sporophytic potency of seed nursery than cct1-3 cct2-3/CCT2 plants and that cct1-3/CCT1 retained more CCT activity than cct2-3/CCT2 . Table 2 Segregation analysis of offspring of a cct2-3 cct1-3/CCT1 plant Number of offspring χ 2 test for 1:2 a Female CCT1 cct2-3 cct2-3 cct1-3/CCT1 cct1-3 cct2-3 χ 2 P Theoretical ratio 1 2 1 Observed number 29 35 0 Observed ratio 1 1.2 0 5.82 0.055 b a The χ 2 test was conducted to test the null hypothesis that cct1-3 and cct1-3 cct2-5/CCT2 plants segregate from each other in the predicted 1:2 Mendelian ratio. b The P value is large enough ( P > 0.05) to mistakenly reject the null hypothesis. Reciprocal crossing of cct2-5/CCT2 and cct2-3/CCT2 plants in the cct1-3 background To examine if cct1-3 cct2-5 is transmissible to offspring via male or female gametophytes, reciprocal crossing of cct2-5/CCT2 plants was conducted in the cct1-3 background (Table 3 ). When the stigma of emasculated cct1-3 flowers was pollinated with an anther of cct1-3 cct2-5/CCT2 flowers, only cct1-3 but no cct1-3 cct2-5/CCT2 seeds were recovered, indicating that cct2-5 was not transmissible via the male gametophyte in the cct1-3 background. In contrast, when the stigma of emasculated cct1-3 cct2-5/CCT2 flowers was pollinated with an anther of cct1-3 flowers, cct1-3 cct2-5/CCT2 seeds were recovered as well as cct1-3 seeds, and the segregation ratio between CCT2/CCT2 and cct2-5/CCT2 plants in the cct1-3 background (1:0.46) differed significantly from the predicted 1:1 Mendelian ratio ( P = 2.39 × 10 –4 < 0.05), suggesting that cct2-5 is partly transmissible via the female gametophyte in the cct1-3 background. Furthermore, a survival rate of 45.5% (100 × 30/66) was calculated for cct1-3 cct2-5 ovules, demonstrating that cct1-3 cct2-5 ovules were both permissive and fertile. Table 3 Summary of reciprocal crossing of cct2-5/CCT2 plants in the cct1-3 background Reciprocal cross Number of F1 plants χ 2 test for 1:1 a Female Male cct1-3 CCT2/CCT2 cct1-3 cct2-5 / CCT2 χ 2 P cct1-3 CCT2/CCT2 cct1-3 cct2-5 / CCT2 31 0 31.00 2.58 × 10 –8 b cct1-3 cct2-5 / CCT2 cct1-3 CCT2/CCT2 66 30 13.50 2.39 × 10 –4 b In this table, cct2-5 is boldfaced to emphasize transmission via gametophytes. a χ 2 test was conducted to test the null hypothesis that CCT2 / CCT2 and cct2-5 / CCT2 plants segregate from each other in the predicted 1:1 Mendelian ratio. b The P values are small enough ( P < 0.05) to reject the null hypothesis. Similar results were obtained from reciprocal crossing experiments with cct2-3/CCT2 plants in the cct1-3 background (Table S3), in that no cct2-3 allele was transmitted via the male gametophyte in the cct1-3 background whereas it was partly transmitted via the female gametophyte. Alexander’s test for pollen viability We next examined whether cct1-3 cct2-3 and cct1-3 cct2-5 pollen could be established during pollen maturation. To assess the phenotype, we introduced qrt1-1 in the background, which constitutively produces four inseparable pollen grains called a pollen tetrad or a pollen quartet. We first examined pollen viability by Alexander's test. Alexander’s reagent (or acidic fuchsin) stains dead pollen grains green and live grains purple-red (Atlagić et al. 2012 ). Pollen grains in the anthers of qrt1-1 cct2-5 cct1-3/CCT1 (Fig. 2 d) and qrt1-1 cct1-3 cct2-5/CCT2 (Fig. 2 e) plants were all stained purple-red (viable) as were those in the anthers of qrt1-1 (Fig. 2 a), qrt1-1 cct2-5 (Fig. 2 b), and qrt1-1 cct1-3 (Fig. 2 c) plants, and each pollen grain that was a part of a single pollen quartet was equally stained purple-red (Figs. 2 a– 2 e, red arrows). These data suggested that cct1-3 cct2-5 pollen grains would be dead after maturation. Examination of pollen grain shrinkage Observation of pollen quartets under a scanning electron microscope revealed that shrunk pollen grains were included in some pollen quartets from qrt1-1 cct1-3 cct2-5/CCT2 and qrt1-1 cct2-5 cct1-3/CCT1 plants, but few were included in the quartets from qrt1-1 , qrt1-1 cct1-3 , qrt1-1 cct2-5 plants (Fig. S3a). Pollen grain shrinkage was carefully examined during in vitro pollen germination experiments, using qrt1-1 , qrt1-1 cct1-3 , qrt1-1 cct2-5 , qrt1-1 cct1-3 cct2-5/CCT2 and qrt1-1 cct2-5 cct1-3/CCT1 plants (Fig. S3b). On the day of anthesis, harvested anthers were rubbed over the surface of agar plates containing pollen germination medium. After incubation at 23 ℃ for 15 min, the number of quartets having different numbers of shrunken pollen grains was counted under a fluorescence microscope. In qrt1-1 , qrt1-1 cct2-5 and qrt1-1 cct1-3 plants, 88–97% of pollen quartets carried no shrunken pollen grains, and the remaining proportions of the pollen quartets contained only one or two shrunken pollen grains (Fig. S3c). Thus, the proportions of shrunken pollen grains in qrt1-1 , qrt1-1 cct2-5 and qrt1-1 cct1-3 plants were calculated to be 2.6, 4.7, and 0.8%, respectively. By contrast, 83% of quartets from qrt1-1 cct2-5 cct1-3/CCT1 plants and 71% from qrt1-1 cct1-3 cct2-5/CCT2 plants contained no shrunken pollen grains (Fig. S3c). In these plants, no pollen quartets contained more than two shrunken pollen grains, suggesting that qrt1-1 cct2-5 CCT1 and qrt1-1 cct1-3 CCT2 pollen grains were almost intact in these plants. Thus, the proportions of shrunken pollen grains in qrt1-1 cct2-5 cct1-3/CCT1 and qrt1-1 cct1-3 cct2-5/CCT2 plants were calculated to be 6.5 and 13.3%, respectively, and the shrinkage rates of qrt1-1 cct1-3 cct2-5 pollen grains from qrt1-1 cct2-5 cct1-3/CCT1 and qrt1-1 cct1-3 cct2-5/CCT2 plants were calculated to be no more than 13.0 and 26.6%, respectively. Notably, the survival rate of qrt1-1 cct1-3 cct2-5 pollen grains was higher in the qrt1-1 cct2-5 cct1-3/CCT1 plants than in the qrt1-1 cct1-3 cct2-5/CCT2 plants, possibly reflecting higher CCT activity in the parental tissues of the former plants than the latter. Evaluation of pollen maturation by DAPI staining In Arabidopsis, after pollen meiosis, the individual microspore initiates vacuole formation and then divides asymmetrically to produce a vegetative cell and a generative cell (pollen mitosis I). The generative cell then divides into two identical sperm cells (pollen mitosis II) and the vacuoles vanish, resulting in tricellular mature pollen grains (Bolaños-Villegas et al. 2010 ). To examine if the two rounds of pollen mitosis proceeded normally to generate mature pollen, the nuclei were observed after DAPI staining. On the day of flowering, flowers were taken from qrt1-1 , qrt1-1 cct1-3 , qrt1-1 cct2-5 , qrt1-1 cct1-3 cct2-5/CCT2 , and qrt1-1 cct2-5 cct1-3/CCT1 plants and stained with DAPI (Fig. 3 ). In all plants examined, each pollen microspore on a pollen quartet contained three bright spots representing one vegetative nucleus (a less bright spot) and two generative nuclei (GN; two bright spots) (Fig. 3 ), suggesting that the two rounds of pollen mitosis had been completed. Thus, we concluded that cct1-3 cct2-5 microspores on a pollen quartet had matured completely. Examination of pollen germination in vitro Pollen germination was examined on the same pollen germination medium as described above (Fig. 4 a). After incubation at 23 ℃ for 4 h, the number of pollen quartets having different numbers of germinated pollen grains was counted under a fluorescence microscope. Among qrt1-1 , qrt1-1 cct1-3 and qrt1-1 cct2-5 plants, almost 40% of pollen quartets had germinated in all four pollen grains (Fig. 4 b). Thus, the single mutation cct1-3 or cct2-5 did not affect germination rates of pollen quartets from qrt1-1 cct1-3 and qrt1-1 cct2-5 plants. In pollen quartets from qrt1-1 cct2-5 cct1-3/CCT1 and qrt1-1 cct1-3 cct2-5/CCT2 plants, however, no pollen quartets contained more than two germinated microspores, suggesting that, in the pollen quartets from qrt1-1 cct1-3/CCT1 cct2-5 and qrt1-1 cct1-3 cct2-5/CCT2 plants, none of cct1-3 cct2-5 microspores had germinated in vitro. Ultrafine structures of pollen cells from wild-type , cct2-5 and cct1-3 plants The germination rates of pollen grains from wild-type, cct2-5 and cct1-3 plants in the qrt1-1 background were 77.4, 72.3, and 66.0%, respectively. Observation of ultrathin sections of wild-type, cct2-5 , and cct1-3 pollen cells at relatively low magnification showed that the preservation of normal appearance of the cytoplasm coincided with successful pollen germination (Fig. S9). Thus, to figure out which ultrafine structures could be important for the progression of normal pollen germination, we compared the ultrathin sections of pollen cells at higher magnifications. On the day of anthesis, ultrathin sections for TEM were prepared from pollen grains from wild-type, cct2-5 , and cct1-3 plants after 30 min of incubation in pollen germination medium. Results derived from images taken for all the sections of wild-type, cct2-5 , and cct1-3 pollen cells are summarized in Figs. S4, S5, and S6, respectively, and the details of ultrafine structures were described in the supplementary text for the figures. We herein show the images taken for the sections of representative wild-type, cct2-5 , and cct1-3 pollen cells in Fig. 5 a, 5 b, and 5 c, respectively, and enlarged images of the respective pollen cells in Fig. 6 a, 6 b, and 6 c. The following features were collectively confirmed by viewing images taken for the sections of the wild-type pollen cells: (1) the wild-type pollen cells contained one vegetative nucleus and two GN as well as cellular septa enclosing the two GN [Figs. 5 a (S4p), S4a (S4b), S4g], consistent with the results of DAPI staining (Fig. 3 a); (2) lipid bodies (LBs) were seen as dark, electron-dense round structures whose surface looked rather transparent compared with the inner bodies and, hence, may have had a membranous boundary (Figs. 6 a, S4 c, S4 h, S4 m, and S4 q); (3) the cytoplasm contained numerous moderately electron-dense small granular structures (SGSs), which sometimes had a small, semi-transparent domain in the inner area (Fig. 6 a, S4 d, S4 i, S4 n, and S4 r); (4) the cytoplasm had mitochondria and plastids (Figs. 5 a, S4 a, S4 g, S4 l, and S4 p), and plastids often existed in contact with rough endoplasmic reticulum (rER; marked with rE in Figs. 5 a, S4 e, and S4 s); (5) SGSs were sometimes entrapped by rER [Fig. 7 a (S4l), S4g, S4j, and S4k]; (6) there were many, very small vacuolar structures of < 1 µm in diameter (vSVs), some of which contained structures similar to SGSs or LBs (Fig. 6 a, black arrows; Figs. 7 a, S4 f, S4 j, and S4 k); (7) vSVs appeared to fuse with each other to generate a larger vacuolar body of 1 ~ 3 µm in diameter [Figs. 7 a (S4l), S4g, and S4p], which we designated herein the small vacuolar body (SVB); (8) SVBs also ingested LBs for subsequent hydrolysis (series of photos shown in Fig. S4o), while fusing to each other created a larger vacuolar body (Fig. S4u); and (9) rER ran between vSVs or vacuoles to form a network (Figs. 6 a and 7 a, stars; Figs. S4f, S4k, and S4t). These results were consistent with a canonical view that pollen germination requires the development of vacuole to digest LBs and SGSs and control turgor pressure for pollen-tube budding and elongation. The ultrafine structures of wild-type pollen cells, as described above, were also collectively assessed in images of cct2-5 pollen cells from a cct2-5 plant (Fig. S5). However, the following points should be noted. First, although LBs were also electron-dense and surrounded by a semi-transparent boundary (Figs. 6 b, S5 c, S5 h, S5 m, S5 q, and S5 u), they looked smaller in cct2-5 pollen cells than in the wild-type cells. Furthermore, the number of LBs in some sections was much less in cct2-5 pollen cells than that in the wild-type cells. On the other hand, the image of SGSs looked moderately electron-dense or "solid", as seen in the wild-type cells. Because the germination rate of cct2-5 pollen cells (72.3%) was slightly lower than that of the wild-type pollen cells (77.4%), the decreased number and size of LBs in cct2-5 pollen cells compared with the wild-type pollen cells coincided with a slight decrease in pollen germination rate. The ultrafine structures of cct2-5 pollen cells, as described above, were also collectively assessed in images of cct1-3 pollen cells from a cct1-3 plant, except some SGSs retained a moderately electron-dense or "solid" appearance [Figs. 5 c (6c, S6a)] and sometimes had a small, semi-transparent domain [(Figs. 6 c, S6 d, S6 i, and S6 n], and a few SGSs in some cells looked larger and "swollen" (Fig. 6 c, SGS*; Fig. S6r). Because the germination rate of cct1-3 pollen grains from a cct1-3 plant (66%) was further decreased from that of cct2-5 pollen grains from a cct2-5 plant (72.3%), the occurrence of swollen SGSs in cct1-3 pollen cells coincided with the decrease in germination rate of cct1-3 pollen grains compared with that of cct2-5 pollen grains. In summary, after 30 min of pollen incubation in vitro, smaller and fewer LBs were found in cct2-5 and cct1-3 pollen cells than in wild-type pollen cells, and these changes probably caused a ~ 5% decrease in pollen germination rate. The presence of moderately electron-dense or "solid" SGSs in the cytoplasm appeared to be a hallmark of pollen cells competent for germination; moreover, with increasing severity of the cct mutation, i.e., in the order of wild type < cct2-5 < cct1-3 , SGSs deformed to yield SGS*. The occurrence of SGS* coincided with an additional ~ 5% decrease of pollen germination rate. Evaluating abnormalities among cct1-3 cct2-5 pollen grains cct1-3 cct2-5 pollen cells, especially those from cct1-3 cct2-5/CCT2 plants, were anticipated to have the most extreme phenotype considering both gametophytic and sporophytic genetic defects. After 30 min of incubation in pollen germination medium, the proportion of abnormal pollen cells from a cct1-3 cct2-5/CCT2 plant was 37.9% and 43.8%, respectively, as estimated by toluidine blue staining (Fig. S10; Fig. S12, middle) and TEM analysis (Fig. S11; Fig. S12, right). Because these values were less than 50%, i.e., reflecting the expected Mendelian segregation ratio of 1:1 for cct1-3 cct2-5 pollen cells from a cct1-3 cct2-5/CCT2 plant (Fig. S12, left), we concluded that all pollen cells displaying unusual ultrafine structures represented cct1-3 cct2-5 pollen cells. On the other hand, the germination rates of pollen grains from cct2-5 cct1-3/CCT 1 and cct1-3 cct2-5/CCT2 plants in the qrt1-1 background were 32.0 and 30.8%, respectively, and these values were less than half the germination rates of pollen grains from a cct2-5 (72.3%) plant and a cct1-3 (66.0%) plant in the qrt1-1 background, respectively, demonstrating that more than half the pollen grains from cct2-5 cct1-3/CCT 1 or cct1-3 cct2-5/CCT2 plants in the qrt1-1 background did not germinate in vitro. Thus, among the total pollen grains from cct2-5 cct1-3/CCT 1 or cct1-3 cct2-5/CCT2 plants in the qrt1-1 background, although cct2-5 or cct1-3 interfered slightly with pollen germination, cct1-3 cct2-5 completely blocked pollen germination. Accordingly, we speculated that the unusual ultrafine structures found specifically in cct1-3 cct2-5 pollen grains contributed to the defect in pollen germination. Ultrafine structures of cct1-3 cct2-5 pollen cells from cct2-5 cct1-3/CCT 1 and cct1-3 cct2-5/CCT2 plants On the day of anthesis, ultrathin sections of pollen grains were prepared for TEM via a 30-min incubation in pollen germination medium. All the results obtained from images of pollen grain cells from cct2-5 cct1-3/CCT 1 and cct1-3 cct2-5/CCT2 plants are summarized in Figs. S7 and S8, respectively. Ultrathin sections of pollen grains from cct2-5 cct1-3/CCT1 plants (Fig. S7) showed distinct features specific to cct1-3 cct2-5 pollen cells. First, cct1-3 cct2-5 pollen cells from a cct2-5 cct1-3/CCT1 plant contained extremely enlarged LBs together with small or tiny LBs [Figs. 5 d (S7a, 6d), S7c, S7f, and S7h]. Regardless of their size, these LBs were electron-dense, and the greatest density was near the surface. However, they appeared to be further surrounded by a semi-transparent boundary, although the boundary was somewhat discontinuous. Second, the cct1-3 cct2-5 pollen cells contained numerous yet diffuse SGSs (Fig. 6 d, SGS*; Figs. S7e and S7i) and a few "solid" SGSs (Figs. 6 d and S7 i). Third, the remnants of "swollen" SGSs could be seen within a membranous body having a diameter less than ~ 2 µm [Fig. 5 d (6d), black arrows; Fig. S7d], which we referred to as a prevacuolar body (Fig. 6 d, PVB). On the other hand, as seen in wild-type, cct2-5 , and cct1-3 pollen cells, two GN [Figs. 5 d (S7a, S7b) and S7g] and developing cellular septa [Figs. 5 d (S7a, S7b) and S7g; open arrowheads] were evident, consistent with the DAPI staining results (Fig. 3 d); moreover, rER was evident throughout the cytoplasm (Fig. 5 d, rE; Fig. S7b, closed arrowheads) and between cellular components (Fig. 6 d, star; Fig. S7g, closed arrowheads). Some cct1-3 cct2-5 pollen cells from a cct2-5 cct1-3/CCT1 plant displayed the most extreme phenotype (Fig. 8 a). These cells contained two types of PVBs having a diameter less than ~ 2 µm: one type of PVBs contained many undigested SGS*s [Fig. 8 a (S7s), boxes 2, 4, and 5], and another type of PVBs contained various cytoplasmic components [Fig. 8 a (S7s), box 6]. Each type of PVB were gathered on opposite sides of the cytoplasm, as if they were in the process of fusing to generate larger bodies (box 2, SGS*). On the other hand, rER appeared to enclose SGSs, SGS*s, LBs, and other kinds of cellular components together to yield PVBs [Fig. 8 a (S7s), box 6] or the rER entrapped several PVBs (Fig. 7 c; Fig. S8m, arrow). However, fully developed SVBs were scarce [Fig. S7r (S7s)]. The occurrence of different types of PVBs suggested that the whole processes of SVB development from SGSs and/or PVBs might be delayed in cct1-3 cct2-5 pollen cells from a cct1-3 cct2-5/CCT2 plant. The limited development of SVBs might have resulted from a possible delay in the supply or vesicular delivery of degradative enzymes into vSVs and PVBs. Apart from the two types of PVBs described above, i.e., one containing many SGS*s and the other containing various cytoplasmic components other than SGS*s, SGSs, SGS*s, enlarged LBs, and other cellular components were all together entrapped within a different type of membrane body, which we referred to as autophagosome-like bodies [Fig. 8 a (S7s), boxes 3, 5, and 7]. The content of the autophagosome-like bodies remained undigested, possibly because there were no large vacuoles. Thus, the autophagic processes that normally operate in germinating wild-type pollen cells appeared to be suspended in cct1-3 cct2-5 pollen cells from a cct2-5 cct1-3/CCT1 plant. The distinct features of cct1-3 cct2-5 pollen cells from a cct2-5 cct1-3/CCT1 plant, as described above, were also collectively recognized in ultrathin sections of cct1-3 cct2-5 pollen grains from a cct1-3 cct2-5/CCT2 plant. Briefly, extremely enlarged LBs co-existed with small ones [Figs. 5 e (6e, S8a), S8b, S8f, and S8g]; almost all SGSs were swollen, and the two types of PVBs entrapping undigested forms of SGS*s [Figs. 7 c (S8a), S8c, and S8k] and other cellular components (Figs. 7 c, S8 c, S8 d, S8 e, S8 l, S8 n, S8 o, and S8 l) appeared to be halted in their development into SVBs. Notably, a small proportion of cct1-3 cct2-5 pollen cells from a cct1-3 cct2-5/CCT2 plant displayed the most extremely deformed cytoplasmic structures [Figs. 8 b (S8p), S8u, and S8aa]; as shown in Fig. 8 b (S8p), although some LBs were incorporated into vSVs or developing SVBs (Fig. 8 b, box 1; the same as Fig. S8r), extremely enlarged LBs were entrapped by autophagosome-like bodies (Fig. 8 b, boxes 2–4; and Fig. S8q). However, it was uniquely seen in a cct1-3 cct2-5 pollen cell from a cct1-3 cct2-5/CCT2 plant that the autophagosome-like bodies entrapping various cellular components appeared to be incompletely closed and had unusual membrane openings, typically with curling outward at both ends (Fig. 8 b, boxes 5 and 6, asterisks; the same photos in Fig. S8s). Such features were never seen in the wild-type, cct2-5 and cct1-3 pollen cells or even in a cct1-3 cct2-5 pollen cell from a cct2-5 cct1-3/CCT1 plant. In summary, cct1-3 cct2-5 pollen cells from cct2-5 cct1-3/CCT1 and cct1-3 cct2-5/CCT2 plants had two types of PVBs containing undigested SGS*s or various kinds of cytoplasmic components. The former PVBs might have been derived from an extensively swollen form of SGS* (Fig. 8 a, box 2). Nonetheless, these results suggested that the normal process of vacuole development was substantially delayed or suspended in cct1-3 cct2-5 pollen cells. Moreover, the extremely enlarged LBs and other cytoplasmic components were entrapped within autophagosome-like bodies, and in the most extreme case (a cct1-3 cct2-5 pollen cell from a cct1-3 cct2-5/CCT2 plant) the enclosure by autophagosome membranes was incomplete. Because autophagic activity has been reported to be essential for pollen germination (Fujiki et al. 2007 ; Qin et al. 2007 ), the occurrence of autophagosome-like bodies enclosing undigested cellular components or even unclosed autophagosome-like bodies in some cct1-3 cct2-5 pollen cells suggested that the ultimate cellular process required for pollen germination was eventually suspended, possibly owing to PC shortage. Because PC is a bilayer-forming lipid, an extreme degree of PC depletion could cause an excess of PE, which is a non-bilayer lipid, which might cause outward curvature of membranes at the termini of developing autophagosomes. Finally, it should be noted that swollen SGSs were generated in cct1-3 cct2-5 pollen cells partly under the influence of sporophytic defects, i.e., a stronger sporophytic influence was recognized in cct1-3 cct2-5 pollen cells from a cct1-3 cct2-5/CCT2 plant than those from a cct2-5 cct1-3/CCT1 plant. Complementation of the mutant phenotype by expression of a cDNA encoding CCT1 The results of our reciprocal crosses (Tables 3 and S3) showed that cct2-5 and cct2-3 cannot be transmitted via male gametophytes in the cct1-3 background, and such a defect was most likely caused by the lack of CCT1 expression in cct1-3 pollen. We therefore tried to complement this mutant phenotype by expressing CCT1 under the control of a pollen-specific promoter. However, our attempts to create a Ti-plasmid that could drive expression of a CCT1 cDNA under the control of the pollen-specific promoter ACA9pro (pPZP221_ ACA9pro:CCT1cDNA-nosT ) were unsuccessful. Inadvertently, however, we obtained an unusual construct that contained ACA9pro in the inverted orientation ( invACA9pro-CCT1cDNA-nosT ) as shown in Fig. S1 . Interestingly, however, a transgenic cct1-3 cct2-5/CCT2 plant that contained an invACA9pro-CCT1cDNA-nosT construct (+/–) (designated line M21) was created, and pollination of emasculated cct1-3 flowers with pollen from the M21 plant resulted in seeds, some of which carried cct2-5 together with the invACA9pro-CCT1cDNA-nosT construct. Genotyping of seedlings revealed that cct2-5 was not found when the seedlings had no T-DNA; moreover, among the seedlings that had T-DNA, half of them carried cct2-5 (Table 4 ). Although the promoter construct was inverted and its pollen-specific expression was not verified, these results suggested that the expression of CCT1 cDNA was [sufficient to rescue the defect of male gametophytes in cct2-5 transmission to offspring in the cct1-3 background. Thus, we concluded that co-transmission of invACA9pro:CCT1cDNA-nosT with cct2-5 was necessary and sufficient for the male gametophytic transmission of cct2-5 to offspring in the cct1-3 background. Table 4 Pollination of emasculated cct1-3 flowers with pollen from cct1-3 cct2-5/CCT2 invACA9pro:CCT1cDNA-nosT (+/–) plants (T1, M21) a Crossing Number of F1 plants χ 2 test for 1:1 b Female cct1-3 Male M21 T-DNA(+/–) w/o cct2-5 T-DNA(+/–) with cct2-5 Total χ 2 P Observed frequency 7 8 15 0.067 0.796 c Expected frequency 7.5 7.5 15 a Among cct1-3 seedlings examined, 15 carried the T-DNA whereas 8 carried cct2-5 . No cct2-5 was found in cct1-3 seedlings without the T-DNA. b χ 2 test was conducted to test the null hypothesis that cct1-3 individuals carrying T-DNA have CCT2 / CCT2 and cct2-5 / CCT2 in the predicted 1:1 Mendelian ratio (degree of freedom = 1). c The P value was too large to mistakenly reject the null hypothesis. Thus, we concluded that the co-transmission of invACA9pro:CCT1cDNA is necessary and sufficient for transmission of cct2-5 from the pollen of cct1-3 cct2-5 / CCT2 invACA9pro:CCT1cDNA-nosT (+/–) plants (T1, M21) to the ovule of cct1-3 plants. Discussion Because PC plays important roles in membrane biogenesis and temperature acclimation of sporophytes, we anticipated that genetic defects in PC biosynthesis would lead to a biological inferiority in mutant plants compared with wild-type plants. In Arabidopsis, we showed that seeds or seedlings carrying cct1-3 cct2-3 or cct1-3 cct2-5 were inviable, so the transmission of a genetic trait negating the CDP-choline pathway to offspring was avoided. Because PC biosynthesis via the CDP-choline pathway is critical for successful fertilization, we first examined whether cct1-3 cct2-3 and cct1-3 cct2-5 could be transmitted to offspring via male or female gametophytes. Our reciprocal crossing of cct2-3/CCT2 and cct2-5/CCT2 plants in the cct1-3 background (Tables 3 and S3) showed that cct2-3 and cct2-5 could not be transmitted via male gametophytes in the cct1-3 background, whereas they can be transmitted via female gametophytes. We then examined whether cct1-3 cct2-3 and cct1-3 cct2-5 could be maintained or eliminated during gametogenesis. Scanning electron microscopy (Fig. S3a), Alexander’s viability test (Fig. 2 ), and DAPI staining (Fig. 3 ) revealed that cct1-3 cct2-5 did not affect microspore maturation. However, pollen germination experiments in vitro showed that cct1-3 cct2-5 eventually prohibits pollen germination (Fig. 4 b). By contrast, we deduced that cct1-3 cct2-5 partly allows maturation of megaspores and fertilization of female gametophytes (Tables 1 and S2). In the following sections, we discuss how cct1-3 cct2-5 inhibits pollen germination and if there is any biological relevance of differential transmission of mutant genes between male and female gametophytes. In Arabidopsis, storage-type vacuoles form de novo in pollen cells just before anthesis, whereas after germination vacuoles begin to fuse and form an enlarged vacuole that contributes to the generation of turgor pressure to drive pollen-tube elongation (Yamamoto et al. 2003 ). For pollen-tube budding and elongation, pollen cells require energy and osmotic solutes as well as polar membrane lipids. LBs could serve as a source of the diacylglycerol backbone for polar lipids as well as a source of the osmotic solute sucrose, to which glyoxisomal catabolism of TAGs might contribute. Development of vacuoles may be important for catabolism of storage materials as well as accumulation of osmotic solutes. Energy also may be required for intracellular trafficking of pollen-tube materials to the budding point. Our TEM analysis using pollen grains after a 30-min incubation in pollen germination medium showed that LBs in wild-type pollen cells had semi-transparent boundaries (Figs. 6 a, S4 c, S4 h, S4 m, and S4 q), which is consistent with the fact that LB is covered with a phospholipid monolayer embedding oil-body proteins. Thus, semi-transparent boundaries of LBs may contribute to LB fusion with SVBs (Fig. S4o). LBs are then catabolized within SVBs (Figs. 7 a, S4 k, and S4 o). Another source of energy and osmolytes could be derived from SGSs [(Figs. 6 a, S4 d, S4 i, S4 n, and S4 r), which may undergo autodegradation in germinating pollen cells as suggested by the presence of a small transparent body in the center, leading to the development of vSVs. However, SGSs were also found to be entrapped within membranous bodies to form PVBs (Figs. 6 a, S4 f, and S4 k), in which SGSs were digested together with other cellular contents to form SVBs. The developing SVBs likely fuse with one another to form a larger vacuole (Fig. 7 a). Thus, the development of SVBs is one of the important steps during pollen germination. Entrapment of LBs and other cellular components by rER is another pathway to vacuole formation. Compared with wild-type pollen cells (Figs. 5 a, S4 l, and S4 p), we showed that SVBs become less prominent in cct2-5 pollen cells from a cct2-5 plant (Figs. 5 b, S5 g, and S5 p) or cct1-3 pollen cells from a cct1-3 plant (Figs. 5 c, S6 l, and S6 p). For cct1-3 pollen cells (Figs. 5 c, S6 l, and S6 o), vSVs gathered closely to one another. These results suggested that fusion of vSVs into SVBs was delayed in cct2-5 and cct1-3 pollen cells compared with wild-type cells. However, we showed that such a delay only partially slowed the pollen germination rates of cct2-5 and cct1-3 pollen compared with wild-type pollen. In cct2-5 and cct1-3 pollen cells (Figs. 6 c, S6 a, S6 g, S6 l, and S6 p), LBs were both smaller and less numerous than in wild-type pollen cells. This may be attributable to downregulation of LB biogenesis because PC serves as a precursor to TAG, the main component of LBs. However, because the semi-transparent boundaries of LBs were preserved in cct2-5 and cct1-3 pollen cells, we concluded that LBs in these cells could fuse with PVBs or SVBs for subsequent digestion of LB contents. Indeed, our pollen germination experiments showed that the decreased number and size of LBs in cct2-5 and cct1-3 pollen cells correlated with a decrease in pollen germination rate. We showed that cct1-3 cct2-5 pollen cells from a cct2-5 cct1-3/CCT1 plant contain unusually enlarged LBs together with very small LBs (Figs. 6 d, S7 a, S7 f, and S13 b). The disproportionate sizes of LBs in the cct1-3 cct2-5 pollen cells may be similar to what has been reported for seed tissues of oleosin mutants (Shimada et al., 2008 ). Together with phospholipids, oleosins are required as an LB-surface material for sequestrating hydrophobic TAGs within the hydrophilic cytoplasm, and genetic downregulation of oleosin biogenesis causes fusion of LBs so that the mutant cells can save the surface materials. LBs have a phospholipid monolayer, mainly consisting of PC. Thus, the presence of enlarged LBs as well as the very small LBs in cct1-3 cct2-5 pollen cells from a cct2-5 cct1-3/CCT1 plant may have been a consequence of severe PC shortage. The surface of such enlarged LBs looked highly electron-dense or darker compared with LBs in wild-type, cct2-5 , and cct1-3 pollen cells, which may have been caused by a shortage of oil-body surface phospholipids owing to the limited supply of PC. Some LBs retained a semi-transparent, yet fragmentary, boundary (Fig. S7h, closed arrowhead; Fig. 8 a, box 1). The unusually enlarged LBs may have been too large to be incorporated into PVBs (Fig. S7b), and hence an alternative route for LB degradation would be required, as discussed below. By contrast, the smaller LBs may have been incorporated into PVBs (Figs. S7b, S7d). The cct1-3 cct2-5 pollen cells from a cct2-5 cct1-3/CCT1 plant contained many PVBs (Figs. S7d, S7l, S7q, and S13g) but few SVBs or vSVs. The cct1-3 cct2-5 pollen cells from both cct2-5 cct1-3/CCT1 and cct1-3 cct2-5/CCT2 plants contained large, autophagosome-like bodies that entrapped undigested cellular contents such as the unusually enlarged LBs, swollen SGSs, and other cytoplasmic components (Fig. 8 ; Figs. S8aa and S8ac). This may reflect the attempt of such cells to facilitate the degradation of cellular components by autophagy. It has been reported that autophagy is essential for pollen germination, but under physiological conditions the turnover of such autophagosome-like bodies would occur too quickly for analysis by TEM. We also noticed that cct1-3 cct2-5 pollen cells from cct1-3 cct2-5/CCT2 plants contained incompletely closed autophagosome-like bodies (Fig. 8 , boxes 5 and 6; Figs. S8s), suggesting that autophagy is required for pollen germination yet is interrupted owing to the shortage of PC. Thus, cct1-3 cct2-5 has formidable negative consequences for autophagy and, hence, the co-transmission of cct1-3 and cct2-5 via male gametophytes is eventually prohibited. Alternatively, the occurrence of the large, autophagosome-like bodies that entrapped the undigested cellular contents could be a result of a programmed suicide process for eliminating unfavorable pollen grains; in this regard, Yamamoto et al. ( 2003 ) reported that pollen grains not participating in fertilization are eliminated by intracellular lytic bodies. Our reciprocal crossing of cct2-5/CCT2 and cct2-3/CCT2 plants in the cct1-3 background (Tables 3 and S3) revealed that simultaneous transmission of cct2-5 or cct2-3 alleles with cct1-3 via the female gametophyte is largely permissible—the survival rate of cct1-3 cct2-5 ovules was 45.5 and 71.4% in cct1-3 cct2-5/CCT2 and cct1-3 cct2-3/CCT2 plants, respectively. Thus, there are strategical differences between male and female gametophytes regarding the transmission of mutant genes: transmission of the defects in PC biosynthesis via male gametophytes is strictly prohibited because a wide dispersal of unfavorable traits by pollen is not beneficial to the population. By contrast, a portion of ovules carrying cct1-3 and cct2-5 (or cct2-3 ) is maintained so that transmission of the background genome can be ensured upon fertilization with normal pollen. Shockey et al. ( 2016 ) reported that gpat9-2 affects phosphatidic acid biosynthesis in seeds and is also not transmissible via male gametophytes and that gpat9-2 pollen grains are unable to germinate in vitro in the qrt1 background. They concluded that because disruption of phosphatidic acid biosynthesis inhibits TAG biosynthesis, the inhibition of pollen germination is a consequence of TAG shortage (Shockey et al. 2016 ). Our results showed that cct1-3 cct2-5 pollen cells in the qrt1 background had a pollen-germination phenotype similar to that of gpat9-2 , although cct1-3 cct2-5 pollen cells still contained TAGs and that TAG catabolism was inhibited. Furthermore, the autophagy processes that are required for pollen germination are probably inhibited or halted in cct1-3 cct2-5 pollen cells. Little is known about the differential roles of CCT1 and CCT2 in Arabidopsis development. We previously reported that residual CCT activity in rosette-leaf homogenates of cct1-1 and cct2-1 mutants accounted for 29.3 and 78.5%, respectively, of the total CCT activity in the homogenates of the wild-type (WS) ecotype (Inatsugi et al. 2009 ). The Arabidopsis eFP browser predicts relatively higher expression of CCT2 (At4G15130) in pollen than in other tissues ( https://bar.utoronto.ca/efp2/Arabidopsis/Arabidopsis_eFPBrowser2.html ). Our results showed that disruption of both CCT1 and CCT2 abolished pollen germination. We also showed that cct1-3 cct2-5/CCT2 and cct1-3 cct2-3/CCT2 plants exhibited partly restricted development of cct1-3 cct2-5/CCT2 and cct1-3 cct2-3/CCT2 seeds, respectively (Tables 1 and S2), whereas cct2-3 cct1-3/CCT1 plants did not restrict the development of cct2-3 cct1-3/CCT1 seeds (Table 2 ). During seed development, cct1-3 cct2-5/CCT2 and cct1-3 cct2-3/CCT2 seeds contain one copy of CCT2 in the embryo and two copies of CCT2 in the endosperm. However, these copies of CCT2 were found to be insufficient for seed development. In contrast, in cct1-3 plants, in which seed development proceeds normally, developing cct1-3 seeds contain two copies of CCT2 in the embryo and three copies of CCT2 in the endosperm, and these copies of CCT2 were found to be sufficient for seed development. Similarly, in cct2-5 cct1-3/CCT1 plants, developing cct2-5 cct1-3/CCT1 seeds contain one copy of CCT1 in the embryo and two copies of CCT1 in the endosperm, and these copies of CCT1 were found to be sufficient for seed development. Thus, it seems likely that CCT1 and CCT2 differentially contribute to the establishment of Arabidopsis seeds after fertilization. Overall, our results demonstrate that genetic defects that disrupt the CDP-choline pathway towards PC biosynthesis are eliminated before fertilization, but only during pollen germination (Fig. S13). However, future studies should investigate whether complete disruption of the CDP-choline pathway might allow the establishment of Arabidopsis seedlings. For this purpose, it will be essential to engineer a Ti-plasmid construct to specifically drive the expression of CCT1 cDNA in a pollen-specific manner. Declarations Funding information The Japanese Society for the Promotion of Science [Grant-in-Aid for Scientific Research (C) (Nos. 21570034, 24570040 and 16K07392) to I.N.. References Alexander MP (1969) Differential staining of aborted and nonaborted pollen. Stain Technol 44:117–122 Atlagić J, Terzić S, Marjanović-Jeromela A (2012) Staining and fluorescent microscopy methods for pollen viability determination in sunflower and other plant species. Ind Crops Prod 35:88–91 Beaudry FEG, Rifkin JL, Barrett SCH, Wright SI (2020) Evolutionary genomics of plant gametophytic selection. 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Plant Cell Physiol 62:1396–1408 Supplementary Files Supplementarymaterials.docx Cite Share Download PDF Status: Published Journal Publication published 01 Feb, 2026 Read the published version in Journal of Plant Research → Version 1 posted Editorial decision: Minor revision 03 Nov, 2025 Reviewers agreed at journal 15 Oct, 2025 Reviewers invited by journal 15 Oct, 2025 Editor assigned by journal 11 Oct, 2025 First submitted to journal 09 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7822087","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":529843686,"identity":"ac6e5fdf-2cd1-4e1f-8346-1ade1179dbd7","order_by":0,"name":"Momoka Wada","email":"","orcid":"","institution":"Saitama University: Saitama Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Momoka","middleName":"","lastName":"Wada","suffix":""},{"id":529843687,"identity":"22ca7548-89a9-417f-b5a0-b3fd5e230b59","order_by":1,"name":"Chiaki Kuga","email":"","orcid":"","institution":"Saitama University: Saitama 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13:41:06","extension":"png","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":689765,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig6.png","url":"https://assets-eu.researchsquare.com/files/rs-7822087/v1/1cd9b902293913382256b9ae.png"},{"id":94664444,"identity":"49f77ff8-7142-4920-bb8d-9db11af7b83c","added_by":"auto","created_at":"2025-10-29 12:22:03","extension":"png","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":775039,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig7.png","url":"https://assets-eu.researchsquare.com/files/rs-7822087/v1/99bd36432ced983f4c2b67e5.png"},{"id":94672702,"identity":"766cb278-89a1-410f-b01d-940d70f862b4","added_by":"auto","created_at":"2025-10-29 13:40:51","extension":"png","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3496626,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig8.png","url":"https://assets-eu.researchsquare.com/files/rs-7822087/v1/064142ca2b08fa5a125b7849.png"},{"id":94664443,"identity":"cace9684-f353-4022-804a-198603d80da4","added_by":"auto","created_at":"2025-10-29 12:22:03","extension":"xml","order_by":30,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":170403,"visible":true,"origin":"","legend":"","description":"","filename":"JPRED25010000structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7822087/v1/e6d7f8f18aa5e40338cfb6fe.xml"},{"id":94664439,"identity":"40482f95-754a-4fbc-9133-2b9d597ab699","added_by":"auto","created_at":"2025-10-29 12:22:03","extension":"html","order_by":31,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":194751,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7822087/v1/5f12ace77bee6388023e8122.html"},{"id":94672900,"identity":"63088205-d2f1-4f5e-a277-b2364f2af8ab","added_by":"auto","created_at":"2025-10-29 13:41:04","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":167451,"visible":true,"origin":"","legend":"\u003cp\u003eStructures of the genes \u003cem\u003ecct1-3\u003c/em\u003e, \u003cem\u003ecct2-3\u003c/em\u003eand \u003cem\u003ecct2-5\u003c/em\u003e. \u003cem\u003ecct1-3\u003c/em\u003e contains a T-DNA insertion within exon 4, \u003cem\u003ecct2-3\u003c/em\u003econtains an insertion in intron 4, and \u003cem\u003ecct2-5\u003c/em\u003e contains an insertion in intron 7.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7822087/v1/25e8c9387a6a8bf70dd5fa87.jpg"},{"id":94673189,"identity":"b2aa8136-5008-4409-8a63-fbbb1a71e4a5","added_by":"auto","created_at":"2025-10-29 13:41:16","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":655215,"visible":true,"origin":"","legend":"\u003cp\u003eAlexander's test for pollen viability. Pollen tetrads in anthers from \u003cem\u003eqrt1-1\u003c/em\u003e (\u003cstrong\u003ea\u003c/strong\u003e), \u003cem\u003eqrt1-1 cct2-5\u003c/em\u003e (\u003cstrong\u003eb\u003c/strong\u003e), \u003cem\u003eqrt1-1 cct1-3 \u003c/em\u003e(\u003cstrong\u003ec\u003c/strong\u003e), \u003cem\u003eqrt1-1 cct2-5 cct1-3/CCT1 \u003c/em\u003e(\u003cstrong\u003ed\u003c/strong\u003e) and \u003cem\u003eqrt1-1 cct1-3 cct2-5/CCT2\u003c/em\u003e (\u003cstrong\u003ee\u003c/strong\u003e) plants were stained with Alexander's reagent. Arrows indicate pollen tetrads with four viable microspores. Bars = 1.0 mm.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7822087/v1/5d514432492f4218343370d9.jpg"},{"id":94672685,"identity":"20cff0a0-2871-43ec-a557-71e9bf4f99ad","added_by":"auto","created_at":"2025-10-29 13:40:50","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":377106,"visible":true,"origin":"","legend":"\u003cp\u003eDAPI staining of pollen grains. Pollen tetrads from\u003cem\u003e qrt1-1\u003c/em\u003e(\u003cstrong\u003ea\u003c/strong\u003e), \u003cem\u003eqrt1-1 cct2-5 \u003c/em\u003e(\u003cstrong\u003eb\u003c/strong\u003e), \u003cem\u003eqrt1-1 cct1-3 \u003c/em\u003e(\u003cstrong\u003ec\u003c/strong\u003e),\u003cem\u003e qrt1-1 cct2-5 cct1-3/CCT1 \u003c/em\u003e(\u003cstrong\u003ed\u003c/strong\u003e) and\u003cem\u003eqrt1-1 cct1-3 cct2-5/CCT2\u003c/em\u003e (\u003cstrong\u003ee\u003c/strong\u003e) plants were stained.\u003cem\u003e \u003c/em\u003eArrows indicate microspores with two bright fluorescing spots as well as one faint spot, implying completion of the two rounds of pollen mitosis. Bars = 25 µm.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7822087/v1/8dbbc5f5e87622b7b44f8415.jpg"},{"id":94664426,"identity":"25e2c2fa-012e-44a9-bafb-70bcaeffde53","added_by":"auto","created_at":"2025-10-29 12:22:03","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":515699,"visible":true,"origin":"","legend":"\u003cp\u003eObservation of pollen germination in vitro.\u003cstrong\u003e \u003c/strong\u003eAnthers from \u003cem\u003eqrt1-1\u003c/em\u003e, \u003cem\u003eqrt1-1 cct2-5\u003c/em\u003e, \u003cem\u003eqrt1-1 cct1-3\u003c/em\u003e, \u003cem\u003eqrt1-1 cct2-5 cct1-3/CCT1\u003c/em\u003e and \u003cem\u003eqrt1-1 cct1-3 cct2-5/CCT2\u003c/em\u003eplants were rubbed over the surface of agar plates containing pollen germination medium. After a 4-h incubation at 23 ℃, pollen quartets were observed under a fluorescence microscope.\u003cstrong\u003e a \u003c/strong\u003eArrowheads denote germinated pollen grains.\u003cstrong\u003e \u003c/strong\u003eBar = 10 µm. \u003cstrong\u003eb\u003c/strong\u003e The proportion of pollen quartets carrying different numbers of germinated pollen grains. In pollen quartets from \u003cem\u003eqrt1-1 cct1-3 cct2-5/CCT2\u003c/em\u003e and \u003cem\u003eqrt1-1 cct2-5 cct1-3/CCT1 \u003c/em\u003eplants, no pollen quartets carried more than two germinated pollen grains.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7822087/v1/ca3453aa1a6f33b7e0ca49ec.jpg"},{"id":94664415,"identity":"ac66c881-c8da-4e97-9ee6-312a4f4db97a","added_by":"auto","created_at":"2025-10-29 12:22:02","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2142364,"visible":true,"origin":"","legend":"\u003cp\u003eTEM analysis of ultrathin sections of pollen grain cells from Arabidopsis wild-type, \u003cem\u003ecct2-5\u003c/em\u003e, \u003cem\u003ecct1-3\u003c/em\u003e, \u003cem\u003ecct2-5 cct1-3/CCT1\u003c/em\u003e, and \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e plants. Panels show photos of representative pollen grain cells from wild-type (\u003cstrong\u003ea\u003c/strong\u003e), \u003cem\u003ecct2-5 \u003c/em\u003e(\u003cstrong\u003eb\u003c/strong\u003e), \u003cem\u003ecct1-3 \u003c/em\u003e(\u003cstrong\u003ec\u003c/strong\u003e), \u003cem\u003ecct2-5 cct1-3/CCT1 \u003c/em\u003e(\u003cstrong\u003ed\u003c/strong\u003e), \u003cem\u003ecct1-3 cct2-5/CCT2 \u003c/em\u003e(\u003cstrong\u003ee\u003c/strong\u003e) plants. Panels \u003cstrong\u003ea\u003c/strong\u003e and \u003cstrong\u003eb \u003c/strong\u003eare the same as in\u003cstrong\u003e \u003c/strong\u003eFigs. S4p and S5t, respectively, and panels \u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ed,\u003c/strong\u003eand\u003cstrong\u003e e \u003c/strong\u003eare modified from Fig. S6a, Fig. S7a, and Fig. S8a, respectively. Open arrowheads indicate cell septa for GN. GN, generative nuclei; LB, lipid body; M, mitochondrion; P, plastid; rE, rough endoplasmic reticulum; SGS, small granular structure; SGS*, swollen SGS; vSV, very small vacuolar structure. Bars = 1 µm.\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7822087/v1/eb551067d7408d1fd82f3fc1.jpg"},{"id":94664420,"identity":"a9263e35-6bb4-4894-9bc0-9ce59634c82a","added_by":"auto","created_at":"2025-10-29 12:22:02","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1875617,"visible":true,"origin":"","legend":"\u003cp\u003eTEM analysis of typical ultrafine structures in\u003cstrong\u003e \u003c/strong\u003ethe cytoplasm of\u003cstrong\u003e \u003c/strong\u003epollen grain cells from Arabidopsis wild-type, \u003cem\u003ecct2-5\u003c/em\u003e, \u003cem\u003ecct1-3\u003c/em\u003e, \u003cem\u003ecct2-5 cct1-3/CCT1\u003c/em\u003e, \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e plants. Panels \u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ed,\u003c/strong\u003e and\u003cstrong\u003e e \u003c/strong\u003eshow enlarged photos of Figs. 5a, 5b, 5c, 5d, and 5e, respectively. LB, lipid body; M, mitochondrion; P, plastid; PVB, prevacuolar body; rE, rough endoplasmic reticulum; SGS, small granular structure; SGS*, swollen SGS; SVB, small vacuolar body; and vSV, very small vacuolar structure. Stars represent the site where the rough endoplasmic reticulum entraps other cellular components. Bars = 1 µm.\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7822087/v1/381cd23a7a8526b06bd989ff.jpg"},{"id":94664418,"identity":"56f39d2e-c0b3-4069-b6fd-6a17091bf2be","added_by":"auto","created_at":"2025-10-29 12:22:02","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2171118,"visible":true,"origin":"","legend":"\u003cp\u003eDevelopmental processes of SVBs from vSVs and PVBs are delayed in putative \u003cem\u003ecct1-3 cct2-5\u003c/em\u003epollen cells compared with those in wild-type and \u003cem\u003ecct2-5\u003c/em\u003e pollen cells. Panels \u003cstrong\u003ea\u003c/strong\u003e and \u003cstrong\u003eb\u003c/strong\u003e show enlarged photos of wild-type (Fig. S4l) and \u003cem\u003ecct2-5\u003c/em\u003e (Fig. S5l) pollen cells, and panel \u003cstrong\u003ec \u003c/strong\u003eshows an enlarged photo of a putative \u003cem\u003ecct1-3\u003c/em\u003e \u003cem\u003ecct2-5 \u003c/em\u003epollen cell (Fig. S8a) from \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e plants. LB, lipid body; P, plastid; PVB, prevacuolar body; rE, rough endoplasmic reticulum; SGS, small granular structure; SGS*, swollen SGS; SVB, small vacuolar body; and vSV, very small vacuolar structure. The star in \u003cstrong\u003eb\u003c/strong\u003e represents the site where the rough endoplasmic reticulum entraps various cellular components including vSVs and SVBs, which are ingesting SGSs and LBs. Bars = 1 µm.\u003c/p\u003e","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7822087/v1/f8049231ae9c8669093c7b98.jpg"},{"id":94673523,"identity":"d998b0dc-0a24-4097-9a49-1ac767f219d9","added_by":"auto","created_at":"2025-10-29 13:41:27","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2359420,"visible":true,"origin":"","legend":"\u003cp\u003eUnusual ultrafine structures observed by TEM in \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen cells. Panel a shows images taken from 7 regions of a \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen cell from a \u003cem\u003ecct2-5 cct1-3/CCT1\u003c/em\u003e plant, shown in Fig. S7r (S7s). Box 1: some enlarged LBs were enclosed within a membrane boundary with discontinuous electron-dense staining. Boxes 2, 4, and 5: many SGSs were entrapped within PVBs of ~2 µm in diameter, within which entrapped SGSs looked diffused and undigested. Two types of PVBs could be seen: one containing the cytosol (box 2) might be a very swollen or fused form of SGS*, and the other containing different types of cytoplasmic components (box 6) was a typical PVB. Apart from these PVBs, the SGSs, enlarged LBs, and other cellular components were all entrapped within autophagosome-like bodies (boxes 3, 5, and 7). Panel \u003cstrong\u003eb\u003c/strong\u003e shows images\u003cstrong\u003e \u003c/strong\u003efrom a \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen cell from a \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e plant, shown in Fig. S8p. Box 1: vSV or SVB appears to be ingesting a small LB. Boxes 2–4: LBs are in the process of being entrapped by membranous, autophagosome-like bodies, which were already ingesting other cellular components. Boxes 5 and 6: the endoplasmic reticulum or the autophagosome-like bodies are in the process of entrapping LBs and other cellular components, while the termini of the developing membranes displayed an unusual outward curvature as shown by asterisks. Bars = 1 µm.\u003c/p\u003e","description":"","filename":"Fig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7822087/v1/195fbcc22909000302f7e379.jpg"},{"id":101690434,"identity":"9c4048e2-2b9a-4800-a057-f13eb13366ff","added_by":"auto","created_at":"2026-02-02 16:02:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11893009,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7822087/v1/72326a10-546f-4739-8e00-14b63776f4d7.pdf"},{"id":94664446,"identity":"e3e2d2ee-b57a-4a39-9be2-56f6b83bdbae","added_by":"auto","created_at":"2025-10-29 12:22:03","extension":"docx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":22933363,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-7822087/v1/86b45fe4af73c8a3aad15365.docx"}],"financialInterests":"","formattedTitle":"Genetic defects in the CDP-choline pathway for phosphatidylcholine biosynthesis cannot be transmitted to offspring via male gametophytes owing to interruption of autophagy-like processes required for pollen germination in Arabidopsis thaliana","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRecent studies on gametogenesis have focused on how gametophytic selection has contributed to diverse evolutionary processes (Beaudry et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In mammals, gametogenesis refers to a process that produces a haploid cell (n) from a diploid cell (2n) through meiosis. In plants, however, meiosis is not sufficient for the completion of gametogenesis: for maturation, both microspores and megaspores generally require mitosis and differentiation, which depend on the successful progression of certain fundamental processes such as biosynthesis of cellular components, polarized vacuolation, catabolism of storage materials, vesicular trafficking and membrane biogenesis (McCormick \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Skinner and Sundaresan \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Furthermore, autophagy is required for pollen germination (Fujiki et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Qin et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Some of these processes may be essential for sporophyte development. In this context, any mutation that is unfavorable for sporophytes must be eliminated during gametogenesis and/or before fertilization. Mutations in essential genes may have similar consequences such as the lethality of male and female gametophytes, which ensures that unfavorable mutant genes will not be transmitted to offspring. However, certain mutations are transmitted at different rates via male or female gametophytes (Bola\u0026ntilde;os‑Villegas et al. 2015; El-Kasmi 2011; Liu et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). For example, functional loss of the canonical α-SNAP in Arabidopsis results in gametophytic lethality by arresting first mitosis during gametogenesis, but reciprocal crosses revealed that transmission of the mutation via female gametophytes is leaky whereas that via male gametophytes is strictly prohibited (Liu et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Lipids are essential components of biomembranes, and some of genes responsible for lipid biosynthesis are among the essential genes (Meinke et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). However, it is not well understood how mutations in lipid genes are eliminated before fertilization or if there is any difference in the rate of transmission of mutant lipid-associated genes via male or female gametophytes.\u003c/p\u003e\u003cp\u003eIn plant cells, phosphatidylcholine (PC) is a major membrane phospholipid that serves as not only a biosynthetic precursor to galactolipids (Kobayashi el al. 2013) that are abundant in photosynthetic membranes but also a substrate for fatty-acid desaturases that regulate membrane fluidity (Ohlrogge and Browse \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Thus, any failure of PC biosynthesis is anticipated to negatively impact sporophyte growth and development. In this context, any mutation that affects PC biosynthesis must be eliminated from the genome before fertilization or during the early stages of embryogenesis. However, no critical studies have been reported to evaluate the importance of PC biosynthesis in plants or to examine if and how mutations in PC biosynthesis could be eliminated before fertilization.\u003c/p\u003e\u003cp\u003eIn \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, PC biosynthesis occurs via the CDP-choline pathway, which is regulated by the genes \u003cem\u003eCCT1\u003c/em\u003e (AT2G32260) and \u003cem\u003eCCT2\u003c/em\u003e (AT4G15130), each of which encodes CTP:phosphorylcholine cytidylyltransferase (CCT, E.C. 2.7.7.15) (Inatsugi et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). CCT synthesizes CDP-choline, a precursor to the polar-head group of PC, from CTP and phosphorylcholine; amino alcohol phosphotransferases (AAPT; E.C. 2.7.8.2) then transfer the phosphorylcholine residue of CDP-choline to the \u003cem\u003esn\u003c/em\u003e-3 position of \u003cem\u003esn\u003c/em\u003e-1,2-diacylglycerol to produce PC. Apart from the CDP-choline pathway, other eukaryotes like yeast can synthesize PC via triple methylation of phosphatidylethanolamine (PE) (Vance and Ridgeway \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Carman and Zeimets \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). However, the Arabidopsis genome does not have a gene responsible for the first methylation step (Ohlrogge and Browse \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Keogh et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and, hence, the PE methylation pathway towards PC does not complement the deficiency of the CDP-choline pathway towards PC in Arabidopsis.\u003c/p\u003e\u003cp\u003eSeveral studies have revealed the roles of phospholipids in reproductive development of Arabidopsis. For \u003cem\u003eS\u003c/em\u003e-adenosyl-\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-methionine:phosphoethanolamine \u003cem\u003eN\u003c/em\u003e-methyltransferase (EC 2.1.1.103), which is required for phosphorylcholine biosynthesis, the temperature-sensitive mutant \u003cem\u003et365\u003c/em\u003e exhibits male sterility (Mou et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). For CTP:phosphorylethanolamine cytidylyltransferase (PECT1; EC 2.7.7.14), which is required for PE biosynthesis, the null mutant \u003cem\u003epect1-6\u003c/em\u003e is unable to proceed to embryo development beyond the early globular stage (Mizoi et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), whereas the \u003cem\u003epect1-4\u003c/em\u003e mutant that retains 26% of the PECT1 activity causes a delay in anther development. Thus, downregulation of the CDP-ethanolamine pathway towards PE biosynthesis causes a delay in anther development, but disruption of the CDP-ethanolamine pathway does not completely abolish the development of male and female gametophytes. AAPT1 and AAPT2 are required for the final step in PC and PE biosynthesis. Disruption of both AAPT1 and AAPT2 abolishes the establishment of \u003cem\u003eaapt1 aapt2\u003c/em\u003e seeds, and 50% of pollen grains from \u003cem\u003eaapt1 aapt2\u003c/em\u003e/\u003cem\u003eAAPT2\u003c/em\u003e or \u003cem\u003eaapt1/AAPT1 aapt2\u003c/em\u003e plants having the \u003cem\u003eqrt1\u003c/em\u003e background die, suggesting that disruption of both PC and PE biosynthesis induces the lethality of male gametophytes (Liu et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Phosphatidic acid is the common precursor to glycerolipids, and glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.2.15) catalyzes the first step of phosphatidic acid biosynthesis. GPAT9 is responsible for the production of phosphatidic acid that is necessary for cytoplasmic glycerolipids, and disruption of \u003cem\u003eGPAT9\u003c/em\u003e abolishes the establishment of \u003cem\u003egpat9\u003c/em\u003e seeds (Shockey et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Reciprocal crosses of \u003cem\u003egpat9-2/GPAT9\u003c/em\u003e plants demonstrated that \u003cem\u003egpat9-2\u003c/em\u003e is not transmissible via male gametophytes, and \u003cem\u003egpat9-2\u003c/em\u003e pollen grains are unable to germinate in vitro in the \u003cem\u003eqrt1\u003c/em\u003e background. In the \u003cem\u003eqrt1\u003c/em\u003e background, \u003cem\u003egpat9-2\u003c/em\u003e pollen grains are smaller than \u003cem\u003eGPAT9\u003c/em\u003e pollen grains (Shockey et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Because disruption of phosphatidic acid biosynthesis inhibits triacylglycerol (TAG) biosynthesis, the inhibition of pollen germination is thought to be caused by TAG shortage (Shockey et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Choline kinase is required for the biosynthesis of phosphorylcholine, the substrate of CCT, and the choline/ethanolamine kinase CEK4 is involved in embryo development (Lin et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Finally, phosphatidylserine is involved in vesicular trafficking and required for normal progression of cell plate formation (Yamaoka et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Disruption of phosphatidylserine biosynthesis affects embryo development and causes pollen lethality, but even the null mutant \u003cem\u003epss1\u003c/em\u003e can escape pollen lethality and establish \u003cem\u003epss1\u003c/em\u003e seedlings (Yamaoka et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eUsing T-DNA-tagged mutants of \u003cem\u003eA. thaliana\u003c/em\u003e, designated \u003cem\u003ecct1-3\u003c/em\u003e, \u003cem\u003ecct2-3\u003c/em\u003e and \u003cem\u003ecct2-5\u003c/em\u003e, we investigated whether the double mutation \u003cem\u003ecct1-3 cct2-3\u003c/em\u003e or \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e can be carried over to the F3 progeny. Our results show that neither \u003cem\u003ecct1-3 cct2-3\u003c/em\u003e nor \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e seedlings are present in F3 progeny. We then investigated the mechanism by which the \u003cem\u003ecct1-3 cct2-3\u003c/em\u003e or \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e mutation can be eliminated from the F3 seed population by conducting reciprocal crossing, Alexander\u0026rsquo;s pollen viability test, in vitro pollen germination test, and observation of ultrathin sections of germinating pollen grains by transmission electron microscopy (TEM). We conclude that \u003cem\u003ecct1-3 cct2-3\u003c/em\u003e and \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e are strictly eliminated during pollen germination owing to PC shortage; this elimination inhibits the progression of autophagy required for pollen germination. We also showed that \u003cem\u003ecct1-3 cct2-3\u003c/em\u003e and \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e are partly transmissible via female gametophytes. Our results suggest that plants utilize distinct transmission strategies between male and female gametophytes, at least in regards to mutant genes in PC biosynthesis: transmission of the mutant genes via male gametophytes is strictly prohibited by inhibition of pollen germination so that a wide dispersal of deleterious mutation among the progeny is prevented, whereas transmission of the same mutant genes via female gametophytes is partly permissive so that the background genome of the escaped mutant ovules can be rescued by fertilization with normal pollen.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePlant materials\u003c/h2\u003e\u003cp\u003e\u003cem\u003eArabidopsis thaliana\u003c/em\u003e (L.) Heynh. ecotype Columbia was obtained from Lehle seeds (Round Rock, TX, U.S.A., \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.arabidopsis.com/\u003c/span\u003e\u003cspan address=\"http://www.arabidopsis.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). \u003cem\u003ecct1-3\u003c/em\u003e (GK-349C03-016244) was obtained from GABI-Kat (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eG\u003c/span\u003eenom\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ea\u003c/span\u003enalyse im \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ebi\u003c/span\u003eologischen System Pflanze - \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eK\u003c/span\u003e\u0026ouml;lner \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eA\u003c/span\u003erabidopsis \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eT\u003c/span\u003e-DNA lines) via the Arabidopsis Biological Resource Center (ABRC). \u003cem\u003ecct2-3\u003c/em\u003e (SK34804, CS1012739) was obtained from ABRC, whereas \u003cem\u003ecct2-5\u003c/em\u003e (SALK_200207, originally distributed by ABRC) was a gift from Peter Moffett (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePlant growth\u003c/h3\u003e\n\u003cp\u003eArabidopsis seeds were sown on soil (Supermix A, Sakata, Kanagawa, Japan) packed in a stainless steel pan (185 mm W \u0026times; 140 mm L \u0026times; 30 mm D) or a plastic pot (60 mmφ \u0026times; 60 mm H). After a 2-day incubation at 4 ℃ under 100% relative humidity, pans (or pots) were incubated in a growth room regulated at 23 ℃ under a 16-h light/8-h dark photo regime at a photon flux density of 110 \u0026micro;mol m\u003csup\u003e\u0026ndash;2\u003c/sup\u003es\u003csup\u003e\u0026ndash;1\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eArabidopsis transformation and selection for the transformants\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eA. thaliana\u003c/em\u003e was transformed by the floral dip method (Clough and Bent \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) using \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV3101. Seeds were sterilized by immersion in 70% ethanol for 1 min then in a sterilizing mixture containing 5% (v/v) sodium hypochlorite and 0.02% (w/v) Triton X-100 for 5 min twice. After washing with sterilized water, seeds were aseptically sown on 1/2 MS plates containing a half-strength Murashige-Skoog salts (Company, City, Japan), 1\u0026times; Gamborg B5 vitamins (Gamborg et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1968\u003c/span\u003e), 0.5% MES (pH 5.7), and 0.7% agar for plant growth (Company, City, Japan).\u003c/p\u003e\n\u003ch3\u003eGenotyping by PCR\u003c/h3\u003e\n\u003cp\u003eOne cotyledon or an equivalent size of leaf was homogenized in 400 \u0026micro;l DNA extraction buffer containing 0.2 M Tris-HCl (pH 9.0), 0.4 M LiCl, 25 mM EDTAཥ2Na and 1% SDS in a round-bottomed 1.5-ml microtube (As one, Osaka, Japan) using a homogenizer pestle (CT1.5 3-325-0268, Kenis, Osaka, Japan). After centrifugation at 16,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 1 min, a 300-\u0026micro;l portion of the supernatant was recovered for DNA precipitation with an equivalent volume of 2-propanol. Each DNA pellet was recovered by centrifugation twice at 16,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 5 min, carefully eliminating the residual supernatant. After a 15-min evacuation under vacuum, DNA was dissolved in 100 \u0026micro;l of buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTAཥ2Na) to make a template DNA solution.\u003c/p\u003e\u003cp\u003ePCR for genotyping was conducted using a Thermal cycler (2720, Applied Biosystems, Tokyo, Japan) under reaction conditions summarized in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Each reaction mixture contained 5 \u0026micro;l 2\u0026times;Quick Taq\u0026reg; HS DyeMix (TOYOBO, Tokyo, Japan), 1 \u0026micro;l template DNA solution, and 1 \u0026micro;l each of 10 pmol \u0026micro;l\u003csup\u003e\u0026ndash;1\u003c/sup\u003e primer solutions (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eConstruction of\u003c/b\u003e \u003cb\u003ecct\u003c/b\u003e \u003cb\u003emutants in the\u003c/b\u003e \u003cb\u003eqrt1-1\u003c/b\u003e \u003cb\u003ebackground\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eqrt1-1\u003c/em\u003e plants form inseparable pollen tetrads and hence are useful for meiotic segregation analysis of mutant phenotypes. To construct \u003cem\u003eqrt1-1 cct1-3/CCT1 cct2-3/CCT2\u003c/em\u003e and \u003cem\u003eqrt1-1 cct1-3/CCT1 cct2-5/CCT2\u003c/em\u003e plants, \u003cem\u003eqrt1-1\u003c/em\u003e plants (♂) were first crossed with \u003cem\u003ecct1-3 cct2-3/CCT2\u003c/em\u003e and \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e plants (♀) to obtain \u003cem\u003eqrt1-1/QRT1 cct1-3/CCT1 cct2-3/CCT2\u003c/em\u003e and \u003cem\u003eqrt1-1/QRT1 cct1-3/CCT1 cct2-5/CCT2\u003c/em\u003e plants, respectively, from which seedlings with the \u003cem\u003ecct1-3/CCT1 cct2-3/CCT2\u003c/em\u003e and \u003cem\u003ecct1-3/CCT1 cct2-5/CCT2\u003c/em\u003e genotypes were identified in the respective offspring by PCR. Finally, seedlings with \u003cem\u003eqrt1-1\u003c/em\u003e were identified under a scanning electron microscope (TM-1000, HITACHI, Tokyo, Japan).\u003c/p\u003e\n\u003ch3\u003eReciprocal crosses\u003c/h3\u003e\n\u003cp\u003eReciprocal crosses of \u003cem\u003ecct2-3/CCT\u003c/em\u003e and \u003cem\u003ecct2-5/CCT2\u003c/em\u003e in the \u003cem\u003ecct1-3\u003c/em\u003e background were conducted by crossing \u003cem\u003ecct1-3\u003c/em\u003e vs. \u003cem\u003ecct1-3 cct2-3/CCT2\u003c/em\u003e and \u003cem\u003ecct1-3\u003c/em\u003e vs. \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e, respectively. Segregation ratios of mutant alleles in the F1 offspring were determined by PCR.\u003c/p\u003e\n\u003ch3\u003eAlexander’s staining to assess pollen survival\u003c/h3\u003e\n\u003cp\u003ePollen viability was determined by Alexander staining (Alexander \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1969\u003c/span\u003e, Atlagić et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). On the day of flowering, open flowers were sampled in a 1.5-ml microtube and immediately submerged in Alexander\u0026rsquo;s solution. After 15 min, samples were washed with water, and anther and pollen grains were observed under a fluorescence microscope (LEICA DMR, Leica Microsystems, Tokyo, Japan).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003ePollen germination in vitro\u003c/h2\u003e\u003cp\u003ePollen collected on the day of flowering was spread over a 1.5% agar plate made up with pollen germination medium containing 0.01% boric acid, 5 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 5 mM KCl, 1 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 10% sucrose, 10 \u0026micro;M brassinosteroid (a gift from Dr. Miho Ikeda at Saitama University) and 0.5% ethanol (Vogler et al. 2014) and incubated in a growth cabinet (BiOTRON LPH-240, NK Systems Limited, Tokyo, Japan) regulated at 23℃ in darkness. Pollen germination and pollen-tube elongation were observed under a fluorescence microscope (LEICA DMR, Leica Microsystems, Tokyo, Japan).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eObservation of pollen morphology by scanning electron microscopy\u003c/h3\u003e\n\u003cp\u003eOn the day of anthesis, anthers were harvested, and pollen was spread over a glass slide. The morphology of the pollen surface was observed under a scanning electron microscope.\u003c/p\u003e\n\u003ch3\u003eDAPI staining for evaluation of mitosis in pollen\u003c/h3\u003e\n\u003cp\u003eFour to five opened flowers sampled on the day of flowering were immersed in DAPI staining medium containing 0.4 \u0026micro;g ml\u003csup\u003e\u0026ndash;1\u003c/sup\u003e DAPI, 0.1% (w/v) Triton X-100, 1 mM EDTAཥ2Na and 100 mM sodium phosphate pH 7 (Schnedl et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1977\u003c/span\u003e). Pollen grains were collected by centrifugation and observed under a fluorescence microscope (LEICA DMR, Leica Microsystems, Tokyo, Japan).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eTransmission electron microscopy\u003c/h2\u003e\u003cp\u003eSamples for transmission electron microscopy were made according to Kaneko (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Pollen collected on the day of anthesis was spread over an agar plate made up with pollen germination medium (Vogler et al. 2014). After incubation for 30 min, pollen was collected by centrifugation and suspended in 0.05 M potassium phosphate buffer (pH 6.8) containing 2% glutaraldehyde and incubated for 4 h at room temperature and then overnight at 4 ℃ (prefixation). After washing six times with 0.05 M potassium phosphate buffer (pH 6.8), samples were incubated in 0.05 M potassium phosphate buffer (pH 6.8) containing 2% OsO\u003csub\u003e4\u003c/sub\u003e for 2 h at ambient temperature (postfixation). Then, samples were washed once with 0.05 M potassium phosphate buffer (pH 6.8) and then subjected to sequential dehydration for 10 min with 10, 30, 50, 70, 85, 95 and 100% acetone. For this purpose, 100% acetone was prepared by storing over anhydrous sodium sulfite. After two additional washes with 100% acetone, samples were subjected to sequential incubations with 50, 75 and 100% Spurr\u0026rsquo;s resin solution and incubated overnight with 100% resin solution. After replacing the 100% resin solution with fresh resin solution, samples were incubated for 1 h, then placed in 1.5-ml microtubes and incubated at 70 ℃ for 8 h for resin solidification. Ultrathin sections were made and after staining with uranyl acetate and lead citrate subjected to transmission electron microscopy (Hitachi H-7500, at 80 kV) in the Comprehensive Analysis Center for Science at Saitama University.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCloning of the pollen-specific promoter\u003c/b\u003e \u003cb\u003eACA9pro\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA gene fragment with restriction enzyme tags for subcloning the pollen-specific promoter \u003cem\u003eACA9pro\u003c/em\u003e (Schi\u0026oslash;tt et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) was amplified from Arabidopsis genomic DNA by PCR (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) using a KOD-Plus-Neo polymerase (TOYOBO). PCR products were subjected to 1% agarose gel electrophoresis, and a band for \u003cem\u003eACA9pro\u003c/em\u003e was purified using a gel extraction kit (Wizard SV Gel and PCR Clean-up System, Promega; or Gel/PCR Extraction Kit, FastGene). Then, purified \u003cem\u003eACA\u003c/em\u003e9pro was digested with XbaI (TaKaRa) and HindIII (TaKaRa), and digested samples were subjected to 1% agarose gel electrophoresis and bands were then purified as described above. The resultant XbaI\u0026ndash;HindIII fragment of \u003cem\u003eACA9pro\u003c/em\u003e was subcloned into the XbaI\u0026ndash;HindIII sites of \u003cem\u003epBluescriptⅡ\u003c/em\u003e SK(+) using a 2\u0026times;Mighty Mix ligation kit (TaKaRa). The resultant plasmid was designated \u003cem\u003epBluescriptⅡ\u003c/em\u003e SK(+) \u003cem\u003eACA9pro\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConstruction of\u003c/b\u003e \u003cb\u003eCCT1\u003c/b\u003e \u003cb\u003eexpression constructs under the control of\u003c/b\u003e \u003cb\u003eACApro9\u003c/b\u003e\u003c/p\u003e\u003cp\u003eUsing the \u003cem\u003epBluescriptⅡ\u003c/em\u003e SK(+) \u003cem\u003eACA9pro\u003c/em\u003e as a template, the XbaI\u0026ndash;HindIII \u003cem\u003eACA9pro\u003c/em\u003e fragment was amplified by PCR and then subcloned into the XbaI\u0026ndash;HindIII sites of pPZP221 \u003cem\u003e35Spro:CCT1cDNA-nosT\u003c/em\u003e (100 \u0026micro;g/ml spectinomycin) to create pPZP221 \u003cem\u003eACA9pro:CCT1cDNA-nosT\u003c/em\u003e. However, the resultant plasmid unexpectedly contained inverted \u003cem\u003eACA9pro\u003c/em\u003e (\u003cem\u003einvACA9pro\u003c/em\u003e). Thus, it was designated pPZP221 \u003cem\u003einvACApro9:CCT1cDNA-nosT\u003c/em\u003e (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eTransformation of\u003c/b\u003e \u003cb\u003eAgrobacterium tumefaciens\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eA. tumefaciens\u003c/em\u003e GC3101 carrying antibiotic resistance for chloramphenicol, gentamycin, rifampicin and streptomycin was transformed by electroporation using 20 \u0026micro;l competent cells, which were prepared according to a method of Sean Weise (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bmb.natsci.msu.edu/sites/_bmb/assets/File/Sharkey_lab/Agrobacterium%20Transformation%20and%20Competent%20Cell%20Preparation.pdf\u003c/span\u003e\u003cspan address=\"https://bmb.natsci.msu.edu/sites/_bmb/assets/File/Sharkey_lab/Agrobacterium%20Transformation%20and%20Competent%20Cell%20Preparation.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Transformants were identified by colony PCR (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eIsolation of\u003c/b\u003e \u003cb\u003ecct1-3 cct2-5/CCT2\u003c/b\u003e \u003cb\u003eplant lines expressing\u003c/b\u003e \u003cb\u003einvACA9pro:CCT1cDNA-nosT\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePrimary shoots (~\u0026thinsp;10 cm long) were cut off from \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e plants to promote secondary shoot regeneration. When the secondary shoots were ~\u0026thinsp;15 cm long, all siliques and opened flowers were removed, and the remaining floral tips and rosette base were inoculated with \u003cem\u003eA. tumefaciens\u003c/em\u003e culture according to the floral dip method. T1 seeds were selected on 1/2 MS agar plates containing 50 \u0026micro;g ml\u003csup\u003e\u0026ndash;1\u003c/sup\u003e gentamycin (FUJIFILM Wako Chemicals, Osaka, Japan) and 10 \u0026micro;g ml\u003csup\u003e\u0026ndash;1\u003c/sup\u003e meropenem trihydrate (FUJIFILM Wako Chemicals, Osaka, Japan), and \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e plant lines expressing \u003cem\u003einvACA9pro:CCT1cDNA-nosT\u003c/em\u003e were identified by genotyping for \u003cem\u003ecct1-3\u003c/em\u003e and \u003cem\u003ecct2-5\u003c/em\u003e. To demonstrate co-transmission of \u003cem\u003ecct2-5\u003c/em\u003e with the transgene \u003cem\u003einvACA9pro:CCT1cDNA-nosT\u003c/em\u003e via male gametophytes, one of the lines, designated M21, expressing \u003cem\u003ecct1-3 cct2-5/CCT2 invACA9pro:CCT1cDNA-nosT (+/\u0026ndash;)\u003c/em\u003e was immediately used as a pollen source for pollination over the stigma of emasculated \u003cem\u003ecct1-3\u003c/em\u003e flowers.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eAssessment of segregation in the mutant alleles\u003c/b\u003e \u003cb\u003ecct1-3, cct2-3\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ecct2-5\u003c/b\u003e \u003cb\u003ein the F2 progeny\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003ecct1-3\u003c/em\u003e and \u003cem\u003ecct2-5\u003c/em\u003e plants were crossed in an attempt to isolate \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e double mutants in the F2 progeny. However, no \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e double mutant was obtained. Accordingly, offspring of a \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e F2 plant was further analyzed (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Again, no \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e double mutant was obtained in the F3 progeny, suggesting that \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e was eliminated during gametogenesis, fertilization, or early embryogenesis/seed development. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e also shows that a segregation ratio was 1:0.77 for the cross between \u003cem\u003eCCT2/CCT2\u003c/em\u003e and \u003cem\u003ecct2-5/CCT2\u003c/em\u003e in the \u003cem\u003ecct1-3\u003c/em\u003e background, a ratio that differed significantly from the expected Mendelian ratio of 1:2 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.48 \u0026times; 10\u003csup\u003e\u0026ndash;4\u003c/sup\u003e \u0026lt; 0.05). This result suggested that the seed yield of \u003cem\u003ecct2-5/CCT2\u003c/em\u003e was decreased by 62% in the \u003cem\u003ecct1-3\u003c/em\u003e background.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSegregation analysis of offspring of a cct1-3 cct2-5/CCT2 plant\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eNumber of offspring\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eχ\u003csup\u003e2\u003c/sup\u003e test for 1:2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGenotype\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ecct1-3 CCT2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003ecct1-3 cct2-5\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eχ\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTheoretical ratio\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eObserved number\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eObserved ratio\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.77 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e14.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.48 \u0026times; 10\u003csup\u003e\u0026ndash;4 c\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e The χ\u003csup\u003e2\u003c/sup\u003e test was conducted to test the null hypothesis that \u003cem\u003ecct1-3\u003c/em\u003e and \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e plants segregate from each other in the predicted 1:2 Mendelian ratio.\u003c/p\u003e\u003cp\u003e\u003csup\u003eb\u003c/sup\u003e The segregation ratio between \u003cem\u003eCCT2\u003c/em\u003e and \u003cem\u003ecct2-5/CCT2\u003c/em\u003e plants in the \u003cem\u003ecct1-3\u003c/em\u003e background was 1:0.77, which differed significantly from the predicted 1:2 Mendelian ratio, suggesting that the seed yield of \u003cem\u003ecct2-5/CCT2\u003c/em\u003e was decreased by 62% in the \u003cem\u003ecct1-3\u003c/em\u003e background.\u003c/p\u003e\u003cp\u003e\u003csup\u003ec\u003c/sup\u003e The \u003cem\u003eP\u003c/em\u003e value is small enough (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) to reject the null hypothesis.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eSimilar results were obtained from crossing experiments between \u003cem\u003ecct1-3\u003c/em\u003e and \u003cem\u003ecct2-3\u003c/em\u003e plants (Table S2). No \u003cem\u003ecct1-3 cct2-3\u003c/em\u003e double mutant was obtained from a \u003cem\u003ecct1-3 cct2-3/CCT2\u003c/em\u003e plant in the F3 progeny, and the segregation ratio of \u003cem\u003eCCT2/CCT2\u003c/em\u003e and \u003cem\u003ecct2-3/CCT2\u003c/em\u003e in the \u003cem\u003ecct1-3\u003c/em\u003e background was 1:0.77, which differed significantly from the expected 1:2 Mendelian ratio (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.02 \u0026times; 10\u003csup\u003e\u0026ndash;4\u003c/sup\u003e \u0026lt; 0.05). Because the T-DNA-tagged mutant \u003cem\u003ecct2-3\u003c/em\u003e carried a drug-resistance gene against ammonium glufosinate (BASTA), the unexpected segregation ratio of 1:0.77 between \u003cem\u003eCCT/CCT2\u003c/em\u003e and \u003cem\u003ecct2-3/CCT2\u003c/em\u003e plants in the \u003cem\u003ecct1-3\u003c/em\u003e background was also confirmed by a survival test on 1/2 MS agar plates containing 40 nmol ml\u003csup\u003e\u0026ndash;1\u003c/sup\u003e BASTA; among 94 seeds harvested from a \u003cem\u003ecct1-3 cct2-3/CCT2\u003c/em\u003e plant, 53 were dead (\u003cem\u003ecct1-3 CCT2\u003c/em\u003e) and 41 survived (\u003cem\u003ecct1-3 cct2-3/CCT2\u003c/em\u003e) (Fig. S2), again with a segregation ratio of 1:0.77.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe also conducted a segregation test for offspring of a \u003cem\u003ecct2-3 cct1-3/CCT1\u003c/em\u003e plant (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In accordance with results presented in Table S2, no \u003cem\u003ecct1-3 cct2-3\u003c/em\u003e double mutant was obtained from the \u003cem\u003ecct2-3 cct1-3/CCT1\u003c/em\u003e plant. However, the segregation ratio between \u003cem\u003eCCT1/CCT1\u003c/em\u003e and \u003cem\u003ecct1-3/CCT1\u003c/em\u003e in the \u003cem\u003ecct2-3\u003c/em\u003e background was 1:1.2, which did not differ significantly from the expected 1:2 Mendelian ratio (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.055). These results suggested that \u003cem\u003ecct2-3 cct1-3/CCT1\u003c/em\u003e plants had greater sporophytic potency of seed nursery than \u003cem\u003ecct1-3 cct2-3/CCT2\u003c/em\u003e plants and that \u003cem\u003ecct1-3/CCT1\u003c/em\u003e retained more CCT activity than \u003cem\u003ecct2-3/CCT2\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSegregation analysis of offspring of a cct2-3 cct1-3/CCT1 plant\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eNumber of offspring\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eχ\u003csup\u003e2\u003c/sup\u003e test for 1:2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eCCT1 cct2-3\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ecct2-3 cct1-3/CCT1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003ecct1-3 cct2-3\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eχ\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTheoretical ratio\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eObserved number\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eObserved ratio\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.055 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e The χ\u003csup\u003e2\u003c/sup\u003e test was conducted to test the null hypothesis that \u003cem\u003ecct1-3\u003c/em\u003e and \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e plants segregate from each other in the predicted 1:2 Mendelian ratio.\u003c/p\u003e\u003cp\u003e\u003csup\u003eb\u003c/sup\u003e The \u003cem\u003eP\u003c/em\u003e value is large enough (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) to mistakenly reject the null hypothesis.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eReciprocal crossing of\u003c/b\u003e \u003cb\u003ecct2-5/CCT2\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ecct2-3/CCT2\u003c/b\u003e \u003cb\u003eplants in the\u003c/b\u003e \u003cb\u003ecct1-3\u003c/b\u003e \u003cb\u003ebackground\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo examine if \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e is transmissible to offspring via male or female gametophytes, reciprocal crossing of \u003cem\u003ecct2-5/CCT2\u003c/em\u003e plants was conducted in the \u003cem\u003ecct1-3\u003c/em\u003e background (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). When the stigma of emasculated \u003cem\u003ecct1-3\u003c/em\u003e flowers was pollinated with an anther of \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e flowers, only \u003cem\u003ecct1-3\u003c/em\u003e but no \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e seeds were recovered, indicating that \u003cem\u003ecct2-5\u003c/em\u003e was not transmissible via the male gametophyte in the \u003cem\u003ecct1-3\u003c/em\u003e background. In contrast, when the stigma of emasculated \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e flowers was pollinated with an anther of \u003cem\u003ecct1-3\u003c/em\u003e flowers, \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e seeds were recovered as well as \u003cem\u003ecct1-3\u003c/em\u003e seeds, and the segregation ratio between \u003cem\u003eCCT2/CCT2\u003c/em\u003e and \u003cem\u003ecct2-5/CCT2\u003c/em\u003e plants in the \u003cem\u003ecct1-3\u003c/em\u003e background (1:0.46) differed significantly from the predicted 1:1 Mendelian ratio (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.39 \u0026times; 10\u003csup\u003e\u0026ndash;4\u003c/sup\u003e \u0026lt; 0.05), suggesting that \u003cem\u003ecct2-5\u003c/em\u003e is partly transmissible via the female gametophyte in the \u003cem\u003ecct1-3\u003c/em\u003e background. Furthermore, a survival rate of 45.5% (100 \u0026times; 30/66) was calculated for \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e ovules, demonstrating that \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e ovules were both permissive and fertile.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSummary of reciprocal crossing of cct2-5/CCT2 plants in the cct1-3 background\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eReciprocal cross\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eNumber of F1 plants\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eχ\u003csup\u003e2\u003c/sup\u003e test for 1:1 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ecct1-3\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eCCT2/CCT2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003ecct1-3\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ecct2-5\u003c/b\u003e/\u003cem\u003eCCT2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eχ\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ecct1-3\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eCCT2/CCT2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ecct1-3\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ecct2-5\u003c/b\u003e/\u003cem\u003eCCT2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e31.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.58 \u0026times; 10\u003csup\u003e\u0026ndash;8 b\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ecct1-3\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ecct2-5\u003c/b\u003e/\u003cem\u003eCCT2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ecct1-3\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eCCT2/CCT2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e13.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.39 \u0026times; 10\u003csup\u003e\u0026ndash;4 b\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e\u003cp\u003eIn this table, \u003cem\u003ecct2-5\u003c/em\u003e is boldfaced to emphasize transmission via gametophytes.\u003c/p\u003e\u003cp\u003e\u003csup\u003ea\u003c/sup\u003eχ\u003csup\u003e2\u003c/sup\u003e test was conducted to test the null hypothesis that \u003cem\u003eCCT2\u003c/em\u003e/\u003cem\u003eCCT2\u003c/em\u003e and \u003cem\u003ecct2-5\u003c/em\u003e/\u003cem\u003eCCT2\u003c/em\u003e plants segregate from each other in the predicted 1:1 Mendelian ratio.\u003c/p\u003e\u003cp\u003e\u003csup\u003eb\u003c/sup\u003eThe \u003cem\u003eP\u003c/em\u003e values are small enough (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) to reject the null hypothesis.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eSimilar results were obtained from reciprocal crossing experiments with \u003cem\u003ecct2-3/CCT2\u003c/em\u003e plants in the \u003cem\u003ecct1-3\u003c/em\u003e background (Table S3), in that no \u003cem\u003ecct2-3\u003c/em\u003e allele was transmitted via the male gametophyte in the \u003cem\u003ecct1-3\u003c/em\u003e background whereas it was partly transmitted via the female gametophyte.\u003c/p\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eAlexander\u0026rsquo;s test for pollen viability\u003c/h2\u003e\u003cp\u003eWe next examined whether \u003cem\u003ecct1-3 cct2-3\u003c/em\u003e and \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen could be established during pollen maturation. To assess the phenotype, we introduced \u003cem\u003eqrt1-1\u003c/em\u003e in the background, which constitutively produces four inseparable pollen grains called a pollen tetrad or a pollen quartet.\u003c/p\u003e\u003cp\u003eWe first examined pollen viability by Alexander's test. Alexander\u0026rsquo;s reagent (or acidic fuchsin) stains dead pollen grains green and live grains purple-red (Atlagić et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Pollen grains in the anthers of \u003cem\u003eqrt1-1 cct2-5 cct1-3/CCT1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003ed) and \u003cem\u003eqrt1-1 cct1-3 cct2-5/CCT2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003ee) plants were all stained purple-red (viable) as were those in the anthers of \u003cem\u003eqrt1-1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), \u003cem\u003eqrt1-1 cct2-5\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), and \u003cem\u003eqrt1-1 cct1-3\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003ec) plants, and each pollen grain that was a part of a single pollen quartet was equally stained purple-red (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, red arrows). These data suggested that \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen grains would be dead after maturation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eExamination of pollen grain shrinkage\u003c/h2\u003e\u003cp\u003eObservation of pollen quartets under a scanning electron microscope revealed that shrunk pollen grains were included in some pollen quartets from \u003cem\u003eqrt1-1 cct1-3 cct2-5/CCT2\u003c/em\u003e and \u003cem\u003eqrt1-1 cct2-5 cct1-3/CCT1\u003c/em\u003e plants, but few were included in the quartets from \u003cem\u003eqrt1-1\u003c/em\u003e, \u003cem\u003eqrt1-1 cct1-3\u003c/em\u003e, \u003cem\u003eqrt1-1 cct2-5\u003c/em\u003e plants (Fig. S3a). Pollen grain shrinkage was carefully examined during in vitro pollen germination experiments, using \u003cem\u003eqrt1-1\u003c/em\u003e, \u003cem\u003eqrt1-1 cct1-3\u003c/em\u003e, \u003cem\u003eqrt1-1 cct2-5\u003c/em\u003e, \u003cem\u003eqrt1-1 cct1-3 cct2-5/CCT2\u003c/em\u003e and \u003cem\u003eqrt1-1 cct2-5 cct1-3/CCT1\u003c/em\u003e plants (Fig. S3b). On the day of anthesis, harvested anthers were rubbed over the surface of agar plates containing pollen germination medium. After incubation at 23 ℃ for 15 min, the number of quartets having different numbers of shrunken pollen grains was counted under a fluorescence microscope. In \u003cem\u003eqrt1-1\u003c/em\u003e, \u003cem\u003eqrt1-1 cct2-5\u003c/em\u003e and \u003cem\u003eqrt1-1 cct1-3\u003c/em\u003e plants, 88\u0026ndash;97% of pollen quartets carried no shrunken pollen grains, and the remaining proportions of the pollen quartets contained only one or two shrunken pollen grains (Fig. S3c). Thus, the proportions of shrunken pollen grains in \u003cem\u003eqrt1-1\u003c/em\u003e, \u003cem\u003eqrt1-1 cct2-5\u003c/em\u003e and \u003cem\u003eqrt1-1 cct1-3\u003c/em\u003e plants were calculated to be 2.6, 4.7, and 0.8%, respectively. By contrast, 83% of quartets from \u003cem\u003eqrt1-1 cct2-5 cct1-3/CCT1\u003c/em\u003e plants and 71% from \u003cem\u003eqrt1-1 cct1-3 cct2-5/CCT2\u003c/em\u003e plants contained no shrunken pollen grains (Fig. S3c). In these plants, no pollen quartets contained more than two shrunken pollen grains, suggesting that \u003cem\u003eqrt1-1 cct2-5 CCT1\u003c/em\u003e and \u003cem\u003eqrt1-1 cct1-3 CCT2\u003c/em\u003e pollen grains were almost intact in these plants. Thus, the proportions of shrunken pollen grains in \u003cem\u003eqrt1-1 cct2-5 cct1-3/CCT1\u003c/em\u003e and \u003cem\u003eqrt1-1 cct1-3 cct2-5/CCT2\u003c/em\u003e plants were calculated to be 6.5 and 13.3%, respectively, and the shrinkage rates of \u003cem\u003eqrt1-1 cct1-3 cct2-5\u003c/em\u003e pollen grains from \u003cem\u003eqrt1-1 cct2-5 cct1-3/CCT1\u003c/em\u003e and \u003cem\u003eqrt1-1 cct1-3 cct2-5/CCT2\u003c/em\u003e plants were calculated to be no more than 13.0 and 26.6%, respectively. Notably, the survival rate of \u003cem\u003eqrt1-1 cct1-3 cct2-5\u003c/em\u003e pollen grains was higher in the \u003cem\u003eqrt1-1 cct2-5 cct1-3/CCT1\u003c/em\u003e plants than in the \u003cem\u003eqrt1-1 cct1-3 cct2-5/CCT2\u003c/em\u003e plants, possibly reflecting higher CCT activity in the parental tissues of the former plants than the latter.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eEvaluation of pollen maturation by DAPI staining\u003c/h2\u003e\u003cp\u003eIn Arabidopsis, after pollen meiosis, the individual microspore initiates vacuole formation and then divides asymmetrically to produce a vegetative cell and a generative cell (pollen mitosis I). The generative cell then divides into two identical sperm cells (pollen mitosis II) and the vacuoles vanish, resulting in tricellular mature pollen grains (Bola\u0026ntilde;os-Villegas et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo examine if the two rounds of pollen mitosis proceeded normally to generate mature pollen, the nuclei were observed after DAPI staining. On the day of flowering, flowers were taken from \u003cem\u003eqrt1-1\u003c/em\u003e, \u003cem\u003eqrt1-1 cct1-3\u003c/em\u003e, \u003cem\u003eqrt1-1 cct2-5\u003c/em\u003e, \u003cem\u003eqrt1-1 cct1-3 cct2-5/CCT2\u003c/em\u003e, and \u003cem\u003eqrt1-1 cct2-5 cct1-3/CCT1\u003c/em\u003e plants and stained with DAPI (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In all plants examined, each pollen microspore on a pollen quartet contained three bright spots representing one vegetative nucleus (a less bright spot) and two generative nuclei (GN; two bright spots) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003e), suggesting that the two rounds of pollen mitosis had been completed. Thus, we concluded that \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e microspores on a pollen quartet had matured completely.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eExamination of pollen germination in vitro\u003c/h2\u003e\u003cp\u003ePollen germination was examined on the same pollen germination medium as described above (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). After incubation at 23 ℃ for 4 h, the number of pollen quartets having different numbers of germinated pollen grains was counted under a fluorescence microscope. Among \u003cem\u003eqrt1-1\u003c/em\u003e, \u003cem\u003eqrt1-1 cct1-3\u003c/em\u003e and \u003cem\u003eqrt1-1 cct2-5\u003c/em\u003e plants, almost 40% of pollen quartets had germinated in all four pollen grains (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Thus, the single mutation \u003cem\u003ecct1-3\u003c/em\u003e or \u003cem\u003ecct2-5\u003c/em\u003e did not affect germination rates of pollen quartets from \u003cem\u003eqrt1-1 cct1-3\u003c/em\u003e and \u003cem\u003eqrt1-1 cct2-5\u003c/em\u003e plants. In pollen quartets from \u003cem\u003eqrt1-1 cct2-5 cct1-3/CCT1\u003c/em\u003e and \u003cem\u003eqrt1-1 cct1-3 cct2-5/CCT2\u003c/em\u003e plants, however, no pollen quartets contained more than two germinated microspores, suggesting that, in the pollen quartets from \u003cem\u003eqrt1-1 cct1-3/CCT1 cct2-5\u003c/em\u003e and \u003cem\u003eqrt1-1 cct1-3 cct2-5/CCT2\u003c/em\u003e plants, none of \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e microspores had germinated in vitro.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eUltrafine structures of pollen cells from wild-type\u003c/b\u003e, \u003cb\u003ecct2-5\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ecct1-3\u003c/b\u003e \u003cb\u003eplants\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe germination rates of pollen grains from wild-type, \u003cem\u003ecct2-5\u003c/em\u003e and \u003cem\u003ecct1-3\u003c/em\u003e plants in the \u003cem\u003eqrt1-1\u003c/em\u003e background were 77.4, 72.3, and 66.0%, respectively. Observation of ultrathin sections of wild-type, \u003cem\u003ecct2-5\u003c/em\u003e, and \u003cem\u003ecct1-3\u003c/em\u003e pollen cells at relatively low magnification showed that the preservation of normal appearance of the cytoplasm coincided with successful pollen germination (Fig. S9). Thus, to figure out which ultrafine structures could be important for the progression of normal pollen germination, we compared the ultrathin sections of pollen cells at higher magnifications.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOn the day of anthesis, ultrathin sections for TEM were prepared from pollen grains from wild-type, \u003cem\u003ecct2-5\u003c/em\u003e, and \u003cem\u003ecct1-3\u003c/em\u003e plants after 30 min of incubation in pollen germination medium. Results derived from images taken for all the sections of wild-type, \u003cem\u003ecct2-5\u003c/em\u003e, and \u003cem\u003ecct1-3\u003c/em\u003e pollen cells are summarized in Figs. S4, S5, and S6, respectively, and the details of ultrafine structures were described in the supplementary text for the figures. We herein show the images taken for the sections of representative wild-type, \u003cem\u003ecct2-5\u003c/em\u003e, and \u003cem\u003ecct1-3\u003c/em\u003e pollen cells in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, respectively, and enlarged images of the respective pollen cells in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, and \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003ec.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe following features were collectively confirmed by viewing images taken for the sections of the wild-type pollen cells: (1) the wild-type pollen cells contained one vegetative nucleus and two GN as well as cellular septa enclosing the two GN [Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003ea (S4p), S4a (S4b), S4g], consistent with the results of DAPI staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003ea); (2) lipid bodies (LBs) were seen as dark, electron-dense round structures whose surface looked rather transparent compared with the inner bodies and, hence, may have had a membranous boundary (Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS4\u003c/span\u003ec, \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS4\u003c/span\u003eh, \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS4\u003c/span\u003em, and \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS4\u003c/span\u003eq); (3) the cytoplasm contained numerous moderately electron-dense small granular structures (SGSs), which sometimes had a small, semi-transparent domain in the inner area (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS4\u003c/span\u003ed, \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS4\u003c/span\u003ei, \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS4\u003c/span\u003en, and \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS4\u003c/span\u003er); (4) the cytoplasm had mitochondria and plastids (Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS4\u003c/span\u003ea, \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS4\u003c/span\u003eg, \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS4\u003c/span\u003el, and \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS4\u003c/span\u003ep), and plastids often existed in contact with rough endoplasmic reticulum (rER; marked with rE in Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS4\u003c/span\u003ee, and \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS4\u003c/span\u003es); (5) SGSs were sometimes entrapped by rER [Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e7\u003c/span\u003ea (S4l), S4g, S4j, and S4k]; (6) there were many, very small vacuolar structures of \u0026lt;\u0026thinsp;1 \u0026micro;m in diameter (vSVs), some of which contained structures similar to SGSs or LBs (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, black arrows; Figs.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS4\u003c/span\u003ef, \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS4\u003c/span\u003ej, and \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS4\u003c/span\u003ek); (7) vSVs appeared to fuse with each other to generate a larger vacuolar body of 1\u0026thinsp;~\u0026thinsp;3 \u0026micro;m in diameter [Figs.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e7\u003c/span\u003ea (S4l), S4g, and S4p], which we designated herein the small vacuolar body (SVB); (8) SVBs also ingested LBs for subsequent hydrolysis (series of photos shown in Fig. S4o), while fusing to each other created a larger vacuolar body (Fig. S4u); and (9) rER ran between vSVs or vacuoles to form a network (Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003ea and \u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, stars; Figs. S4f, S4k, and S4t). These results were consistent with a canonical view that pollen germination requires the development of vacuole to digest LBs and SGSs and control turgor pressure for pollen-tube budding and elongation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe ultrafine structures of wild-type pollen cells, as described above, were also collectively assessed in images of \u003cem\u003ecct2-5\u003c/em\u003e pollen cells from a \u003cem\u003ecct2-5\u003c/em\u003e plant (Fig. S5). However, the following points should be noted. First, although LBs were also electron-dense and surrounded by a semi-transparent boundary (Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, \u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003eS5\u003c/span\u003ec, \u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003eS5\u003c/span\u003eh, \u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003eS5\u003c/span\u003em, \u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003eS5\u003c/span\u003eq, and \u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003eS5\u003c/span\u003eu), they looked smaller in \u003cem\u003ecct2-5\u003c/em\u003e pollen cells than in the wild-type cells. Furthermore, the number of LBs in some sections was much less in \u003cem\u003ecct2-5\u003c/em\u003e pollen cells than that in the wild-type cells. On the other hand, the image of SGSs looked moderately electron-dense or \"solid\", as seen in the wild-type cells. Because the germination rate of \u003cem\u003ecct2-5\u003c/em\u003e pollen cells (72.3%) was slightly lower than that of the wild-type pollen cells (77.4%), the decreased number and size of LBs in \u003cem\u003ecct2-5\u003c/em\u003e pollen cells compared with the wild-type pollen cells coincided with a slight decrease in pollen germination rate.\u003c/p\u003e\u003cp\u003eThe ultrafine structures of \u003cem\u003ecct2-5\u003c/em\u003e pollen cells, as described above, were also collectively assessed in images of \u003cem\u003ecct1-3\u003c/em\u003e pollen cells from a \u003cem\u003ecct1-3\u003c/em\u003e plant, except some SGSs retained a moderately electron-dense or \"solid\" appearance [Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003ec (6c, S6a)] and sometimes had a small, semi-transparent domain [(Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, \u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003eS6\u003c/span\u003ed, \u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003eS6\u003c/span\u003ei, and \u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003eS6\u003c/span\u003en], and a few SGSs in some cells looked larger and \"swollen\" (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, SGS*; Fig. S6r). Because the germination rate of \u003cem\u003ecct1-3\u003c/em\u003e pollen grains from a \u003cem\u003ecct1-3\u003c/em\u003e plant (66%) was further decreased from that of \u003cem\u003ecct2-5\u003c/em\u003e pollen grains from a \u003cem\u003ecct2-5\u003c/em\u003e plant (72.3%), the occurrence of swollen SGSs in \u003cem\u003ecct1-3\u003c/em\u003e pollen cells coincided with the decrease in germination rate of \u003cem\u003ecct1-3\u003c/em\u003e pollen grains compared with that of \u003cem\u003ecct2-5\u003c/em\u003e pollen grains.\u003c/p\u003e\u003cp\u003eIn summary, after 30 min of pollen incubation in vitro, smaller and fewer LBs were found in \u003cem\u003ecct2-5\u003c/em\u003e and \u003cem\u003ecct1-3\u003c/em\u003e pollen cells than in wild-type pollen cells, and these changes probably caused a\u0026thinsp;~\u0026thinsp;5% decrease in pollen germination rate. The presence of moderately electron-dense or \"solid\" SGSs in the cytoplasm appeared to be a hallmark of pollen cells competent for germination; moreover, with increasing severity of the \u003cem\u003ecct\u003c/em\u003e mutation, i.e., in the order of wild type\u0026thinsp;\u0026lt;\u0026thinsp;\u003cem\u003ecct2-5\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;\u003cem\u003ecct1-3\u003c/em\u003e, SGSs deformed to yield SGS*. The occurrence of SGS* coincided with an additional\u0026thinsp;~\u0026thinsp;5% decrease of pollen germination rate.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEvaluating abnormalities among\u003c/b\u003e \u003cb\u003ecct1-3 cct2-5\u003c/b\u003e \u003cb\u003epollen grains\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen cells, especially those from \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e plants, were anticipated to have the most extreme phenotype considering both gametophytic and sporophytic genetic defects. After 30 min of incubation in pollen germination medium, the proportion of abnormal pollen cells from a \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e plant was 37.9% and 43.8%, respectively, as estimated by toluidine blue staining (Fig. S10; Fig. S12, middle) and TEM analysis (Fig. S11; Fig. S12, right). Because these values were less than 50%, i.e., reflecting the expected Mendelian segregation ratio of 1:1 for \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen cells from a \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e plant (Fig. S12, left), we concluded that all pollen cells displaying unusual ultrafine structures represented \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen cells. On the other hand, the germination rates of pollen grains from \u003cem\u003ecct2-5 cct1-3/CCT\u003c/em\u003e1 and \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e plants in the \u003cem\u003eqrt1-1\u003c/em\u003e background were 32.0 and 30.8%, respectively, and these values were less than half the germination rates of pollen grains from a \u003cem\u003ecct2-5\u003c/em\u003e (72.3%) plant and a \u003cem\u003ecct1-3\u003c/em\u003e (66.0%) plant in the \u003cem\u003eqrt1-1\u003c/em\u003e background, respectively, demonstrating that more than half the pollen grains from \u003cem\u003ecct2-5 cct1-3/CCT\u003c/em\u003e1 or \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e plants in the \u003cem\u003eqrt1-1\u003c/em\u003e background did not germinate in vitro. Thus, among the total pollen grains from \u003cem\u003ecct2-5 cct1-3/CCT\u003c/em\u003e1 or \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e plants in the \u003cem\u003eqrt1-1\u003c/em\u003e background, although \u003cem\u003ecct2-5\u003c/em\u003e or \u003cem\u003ecct1-3\u003c/em\u003e interfered slightly with pollen germination, \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e completely blocked pollen germination. Accordingly, we speculated that the unusual ultrafine structures found specifically in \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen grains contributed to the defect in pollen germination.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eUltrafine structures of\u003c/b\u003e \u003cb\u003ecct1-3 cct2-5\u003c/b\u003e \u003cb\u003epollen cells from\u003c/b\u003e \u003cb\u003ecct2-5 cct1-3/CCT\u003c/b\u003e\u003cb\u003e1 and\u003c/b\u003e \u003cb\u003ecct1-3 cct2-5/CCT2\u003c/b\u003e \u003cb\u003eplants\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOn the day of anthesis, ultrathin sections of pollen grains were prepared for TEM via a 30-min incubation in pollen germination medium. All the results obtained from images of pollen grain cells from \u003cem\u003ecct2-5 cct1-3/CCT\u003c/em\u003e1 and \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e plants are summarized in Figs. S7 and S8, respectively.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eUltrathin sections of pollen grains from \u003cem\u003ecct2-5 cct1-3/CCT1\u003c/em\u003e plants (Fig. S7) showed distinct features specific to \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen cells. First, \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen cells from a \u003cem\u003ecct2-5 cct1-3/CCT1\u003c/em\u003e plant contained extremely enlarged LBs together with small or tiny LBs [Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003ed (S7a, 6d), S7c, S7f, and S7h]. Regardless of their size, these LBs were electron-dense, and the greatest density was near the surface. However, they appeared to be further surrounded by a semi-transparent boundary, although the boundary was somewhat discontinuous. Second, the \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen cells contained numerous yet diffuse SGSs (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003ed, SGS*; Figs. S7e and S7i) and a few \"solid\" SGSs (Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003ed and \u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003eS7\u003c/span\u003ei). Third, the remnants of \"swollen\" SGSs could be seen within a membranous body having a diameter less than ~\u0026thinsp;2 \u0026micro;m [Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003ed (6d), black arrows; Fig. S7d], which we referred to as a prevacuolar body (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003ed, PVB). On the other hand, as seen in wild-type, \u003cem\u003ecct2-5\u003c/em\u003e, and \u003cem\u003ecct1-3\u003c/em\u003e pollen cells, two GN [Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003ed (S7a, S7b) and S7g] and developing cellular septa [Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003ed (S7a, S7b) and S7g; open arrowheads] were evident, consistent with the DAPI staining results (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003ed); moreover, rER was evident throughout the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003ed, rE; Fig. S7b, closed arrowheads) and between cellular components (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003ed, star; Fig. S7g, closed arrowheads).\u003c/p\u003e\u003cp\u003eSome \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen cells from a \u003cem\u003ecct2-5 cct1-3/CCT1\u003c/em\u003e plant displayed the most extreme phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). These cells contained two types of PVBs having a diameter less than ~\u0026thinsp;2 \u0026micro;m: one type of PVBs contained many undigested SGS*s [Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e8\u003c/span\u003ea (S7s), boxes 2, 4, and 5], and another type of PVBs contained various cytoplasmic components [Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e8\u003c/span\u003ea (S7s), box 6]. Each type of PVB were gathered on opposite sides of the cytoplasm, as if they were in the process of fusing to generate larger bodies (box 2, SGS*). On the other hand, rER appeared to enclose SGSs, SGS*s, LBs, and other kinds of cellular components together to yield PVBs [Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e8\u003c/span\u003ea (S7s), box 6] or the rER entrapped several PVBs (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e7\u003c/span\u003ec; Fig. S8m, arrow). However, fully developed SVBs were scarce [Fig. S7r (S7s)]. The occurrence of different types of PVBs suggested that the whole processes of SVB development from SGSs and/or PVBs might be delayed in \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen cells from a \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e plant. The limited development of SVBs might have resulted from a possible delay in the supply or vesicular delivery of degradative enzymes into vSVs and PVBs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eApart from the two types of PVBs described above, i.e., one containing many SGS*s and the other containing various cytoplasmic components other than SGS*s, SGSs, SGS*s, enlarged LBs, and other cellular components were all together entrapped within a different type of membrane body, which we referred to as autophagosome-like bodies [Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e8\u003c/span\u003ea (S7s), boxes 3, 5, and 7]. The content of the autophagosome-like bodies remained undigested, possibly because there were no large vacuoles. Thus, the autophagic processes that normally operate in germinating wild-type pollen cells appeared to be suspended in \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen cells from a \u003cem\u003ecct2-5 cct1-3/CCT1\u003c/em\u003e plant.\u003c/p\u003e\u003cp\u003eThe distinct features of \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen cells from a \u003cem\u003ecct2-5 cct1-3/CCT1\u003c/em\u003e plant, as described above, were also collectively recognized in ultrathin sections of \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen grains from a \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e plant. Briefly, extremely enlarged LBs co-existed with small ones [Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003ee (6e, S8a), S8b, S8f, and S8g]; almost all SGSs were swollen, and the two types of PVBs entrapping undigested forms of SGS*s [Figs.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e7\u003c/span\u003ec (S8a), S8c, and S8k] and other cellular components (Figs.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e7\u003c/span\u003ec, \u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003eS8\u003c/span\u003ec, \u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003eS8\u003c/span\u003ed, \u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003eS8\u003c/span\u003ee, \u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003eS8\u003c/span\u003el, \u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003eS8\u003c/span\u003en, \u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003eS8\u003c/span\u003eo, and \u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003eS8\u003c/span\u003el) appeared to be halted in their development into SVBs. Notably, a small proportion of \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen cells from a \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e plant displayed the most extremely deformed cytoplasmic structures [Figs.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e8\u003c/span\u003eb (S8p), S8u, and S8aa]; as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e8\u003c/span\u003eb (S8p), although some LBs were incorporated into vSVs or developing SVBs (Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e8\u003c/span\u003eb, box 1; the same as Fig. S8r), extremely enlarged LBs were entrapped by autophagosome-like bodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e8\u003c/span\u003eb, boxes 2\u0026ndash;4; and Fig. S8q). However, it was uniquely seen in a \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen cell from a \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e plant that the autophagosome-like bodies entrapping various cellular components appeared to be incompletely closed and had unusual membrane openings, typically with curling outward at both ends (Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e8\u003c/span\u003eb, boxes 5 and 6, asterisks; the same photos in Fig. S8s). Such features were never seen in the wild-type, \u003cem\u003ecct2-5\u003c/em\u003e and \u003cem\u003ecct1-3\u003c/em\u003e pollen cells or even in a \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen cell from a \u003cem\u003ecct2-5 cct1-3/CCT1\u003c/em\u003e plant.\u003c/p\u003e\u003cp\u003eIn summary, \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen cells from \u003cem\u003ecct2-5 cct1-3/CCT1\u003c/em\u003e and \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e plants had two types of PVBs containing undigested SGS*s or various kinds of cytoplasmic components. The former PVBs might have been derived from an extensively swollen form of SGS* (Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e8\u003c/span\u003ea, box 2). Nonetheless, these results suggested that the normal process of vacuole development was substantially delayed or suspended in \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen cells. Moreover, the extremely enlarged LBs and other cytoplasmic components were entrapped within autophagosome-like bodies, and in the most extreme case (a \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen cell from a \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e plant) the enclosure by autophagosome membranes was incomplete. Because autophagic activity has been reported to be essential for pollen germination (Fujiki et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Qin et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), the occurrence of autophagosome-like bodies enclosing undigested cellular components or even unclosed autophagosome-like bodies in some \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen cells suggested that the ultimate cellular process required for pollen germination was eventually suspended, possibly owing to PC shortage. Because PC is a bilayer-forming lipid, an extreme degree of PC depletion could cause an excess of PE, which is a non-bilayer lipid, which might cause outward curvature of membranes at the termini of developing autophagosomes. Finally, it should be noted that swollen SGSs were generated in \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen cells partly under the influence of sporophytic defects, i.e., a stronger sporophytic influence was recognized in \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen cells from a \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e plant than those from a \u003cem\u003ecct2-5 cct1-3/CCT1\u003c/em\u003e plant.\u003c/p\u003e\u003cp\u003e\u003cb\u003eComplementation of the mutant phenotype by expression of a cDNA encoding\u003c/b\u003e \u003cb\u003eCCT1\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe results of our reciprocal crosses (Tables\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and S3) showed that \u003cem\u003ecct2-5\u003c/em\u003e and \u003cem\u003ecct2-3\u003c/em\u003e cannot be transmitted via male gametophytes in the \u003cem\u003ecct1-3\u003c/em\u003e background, and such a defect was most likely caused by the lack of \u003cem\u003eCCT1\u003c/em\u003e expression in \u003cem\u003ecct1-3\u003c/em\u003e pollen. We therefore tried to complement this mutant phenotype by expressing \u003cem\u003eCCT1\u003c/em\u003e under the control of a pollen-specific promoter. However, our attempts to create a Ti-plasmid that could drive expression of a \u003cem\u003eCCT1\u003c/em\u003e cDNA under the control of the pollen-specific promoter \u003cem\u003eACA9pro\u003c/em\u003e (pPZP221_\u003cem\u003eACA9pro:CCT1cDNA-nosT\u003c/em\u003e) were unsuccessful. Inadvertently, however, we obtained an unusual construct that contained \u003cem\u003eACA9pro\u003c/em\u003e in the inverted orientation (\u003cem\u003einvACA9pro-CCT1cDNA-nosT\u003c/em\u003e) as shown in Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Interestingly, however, a transgenic \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e plant that contained an \u003cem\u003einvACA9pro-CCT1cDNA-nosT\u003c/em\u003e construct (+/\u0026ndash;) (designated line M21) was created, and pollination of emasculated \u003cem\u003ecct1-3\u003c/em\u003e flowers with pollen from the M21 plant resulted in seeds, some of which carried \u003cem\u003ecct2-5\u003c/em\u003e together with the \u003cem\u003einvACA9pro-CCT1cDNA-nosT\u003c/em\u003e construct. Genotyping of seedlings revealed that \u003cem\u003ecct2-5\u003c/em\u003e was not found when the seedlings had no T-DNA; moreover, among the seedlings that had T-DNA, half of them carried \u003cem\u003ecct2-5\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Although the promoter construct was inverted and its pollen-specific expression was not verified, these results suggested that the expression of \u003cem\u003eCCT1\u003c/em\u003e cDNA was [sufficient to rescue the defect of male gametophytes in \u003cem\u003ecct2-5\u003c/em\u003e transmission to offspring in the \u003cem\u003ecct1-3\u003c/em\u003e background. Thus, we concluded that co-transmission of \u003cem\u003einvACA9pro:CCT1cDNA-nosT\u003c/em\u003e with \u003cem\u003ecct2-5\u003c/em\u003e was necessary and sufficient for the male gametophytic transmission of \u003cem\u003ecct2-5\u003c/em\u003e to offspring in the \u003cem\u003ecct1-3\u003c/em\u003e background.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePollination of emasculated cct1-3 flowers with pollen from cct1-3 cct2-5/CCT2 invACA9pro:CCT1cDNA-nosT (+/\u0026ndash;) plants (T1, M21) a\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eCrossing\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eNumber of F1 plants\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003eχ\u003csup\u003e2\u003c/sup\u003e test for 1:1 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003cp\u003e\u003cem\u003ecct1-3\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003cp\u003eM21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eT-DNA(+/\u0026ndash;)\u003c/em\u003e \u003cb\u003ew/o cct2-5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eT-DNA(+/\u0026ndash;)\u003c/em\u003e \u003cb\u003ewith cct2-5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eχ\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eObserved frequency\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.067\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.796 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eExpected frequency\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e Among \u003cem\u003ecct1-3\u003c/em\u003e seedlings examined, 15 carried the T-DNA whereas 8 carried \u003cem\u003ecct2-5\u003c/em\u003e. No \u003cem\u003ecct2-5\u003c/em\u003e was found in \u003cem\u003ecct1-3\u003c/em\u003e seedlings without the T-DNA.\u003c/p\u003e\u003cp\u003e\u003csup\u003eb\u003c/sup\u003e χ\u003csup\u003e2\u003c/sup\u003e test was conducted to test the null hypothesis that \u003cem\u003ecct1-3\u003c/em\u003e individuals carrying T-DNA have \u003cem\u003eCCT2\u003c/em\u003e/\u003cem\u003eCCT2\u003c/em\u003e and \u003cem\u003ecct2-5\u003c/em\u003e/\u003cem\u003eCCT2\u003c/em\u003e in the predicted 1:1 Mendelian ratio (degree of freedom\u0026thinsp;=\u0026thinsp;1).\u003c/p\u003e\u003cp\u003e\u003csup\u003ec\u003c/sup\u003e The \u003cem\u003eP\u003c/em\u003e value was too large to mistakenly reject the null hypothesis.\u003c/p\u003e\u003cp\u003eThus, we concluded that the co-transmission of \u003cem\u003einvACA9pro:CCT1cDNA\u003c/em\u003e is necessary and sufficient for transmission of \u003cem\u003ecct2-5\u003c/em\u003e from the pollen of \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e/\u003cem\u003eCCT2 invACA9pro:CCT1cDNA-nosT\u003c/em\u003e (+/\u0026ndash;) plants (T1, M21) to the ovule of \u003cem\u003ecct1-3\u003c/em\u003e plants.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eBecause PC plays important roles in membrane biogenesis and temperature acclimation of sporophytes, we anticipated that genetic defects in PC biosynthesis would lead to a biological inferiority in mutant plants compared with wild-type plants. In Arabidopsis, we showed that seeds or seedlings carrying \u003cem\u003ecct1-3 cct2-3\u003c/em\u003e or \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e were inviable, so the transmission of a genetic trait negating the CDP-choline pathway to offspring was avoided. Because PC biosynthesis via the CDP-choline pathway is critical for successful fertilization, we first examined whether \u003cem\u003ecct1-3 cct2-3\u003c/em\u003e and \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e could be transmitted to offspring via male or female gametophytes. Our reciprocal crossing of \u003cem\u003ecct2-3/CCT2\u003c/em\u003e and \u003cem\u003ecct2-5/CCT2\u003c/em\u003e plants in the \u003cem\u003ecct1-3\u003c/em\u003e background (Tables\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and S3) showed that \u003cem\u003ecct2-3\u003c/em\u003e and \u003cem\u003ecct2-5\u003c/em\u003e could not be transmitted via male gametophytes in the \u003cem\u003ecct1-3\u003c/em\u003e background, whereas they can be transmitted via female gametophytes. We then examined whether \u003cem\u003ecct1-3 cct2-3\u003c/em\u003e and \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e could be maintained or eliminated during gametogenesis. Scanning electron microscopy (Fig. S3a), Alexander\u0026rsquo;s viability test (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e), and DAPI staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003e) revealed that \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e did not affect microspore maturation. However, pollen germination experiments in vitro showed that \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e eventually prohibits pollen germination (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). By contrast, we deduced that \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e partly allows maturation of megaspores and fertilization of female gametophytes (Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and S2). In the following sections, we discuss how \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e inhibits pollen germination and if there is any biological relevance of differential transmission of mutant genes between male and female gametophytes.\u003c/p\u003e\u003cp\u003eIn Arabidopsis, storage-type vacuoles form de novo in pollen cells just before anthesis, whereas after germination vacuoles begin to fuse and form an enlarged vacuole that contributes to the generation of turgor pressure to drive pollen-tube elongation (Yamamoto et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). For pollen-tube budding and elongation, pollen cells require energy and osmotic solutes as well as polar membrane lipids. LBs could serve as a source of the diacylglycerol backbone for polar lipids as well as a source of the osmotic solute sucrose, to which glyoxisomal catabolism of TAGs might contribute. Development of vacuoles may be important for catabolism of storage materials as well as accumulation of osmotic solutes. Energy also may be required for intracellular trafficking of pollen-tube materials to the budding point. Our TEM analysis using pollen grains after a 30-min incubation in pollen germination medium showed that LBs in wild-type pollen cells had semi-transparent boundaries (Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS4\u003c/span\u003ec, \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS4\u003c/span\u003eh, \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS4\u003c/span\u003em, and \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS4\u003c/span\u003eq), which is consistent with the fact that LB is covered with a phospholipid monolayer embedding oil-body proteins. Thus, semi-transparent boundaries of LBs may contribute to LB fusion with SVBs (Fig. S4o). LBs are then catabolized within SVBs (Figs.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS4\u003c/span\u003ek, and \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS4\u003c/span\u003eo). Another source of energy and osmolytes could be derived from SGSs [(Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS4\u003c/span\u003ed, \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS4\u003c/span\u003ei, \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS4\u003c/span\u003en, and \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS4\u003c/span\u003er), which may undergo autodegradation in germinating pollen cells as suggested by the presence of a small transparent body in the center, leading to the development of vSVs. However, SGSs were also found to be entrapped within membranous bodies to form PVBs (Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS4\u003c/span\u003ef, and \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS4\u003c/span\u003ek), in which SGSs were digested together with other cellular contents to form SVBs. The developing SVBs likely fuse with one another to form a larger vacuole (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Thus, the development of SVBs is one of the important steps during pollen germination. Entrapment of LBs and other cellular components by rER is another pathway to vacuole formation.\u003c/p\u003e\u003cp\u003eCompared with wild-type pollen cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS4\u003c/span\u003el, and \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003eS4\u003c/span\u003ep), we showed that SVBs become less prominent in \u003cem\u003ecct2-5\u003c/em\u003e pollen cells from a \u003cem\u003ecct2-5\u003c/em\u003e plant (Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, \u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003eS5\u003c/span\u003eg, and \u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003eS5\u003c/span\u003ep) or \u003cem\u003ecct1-3\u003c/em\u003e pollen cells from a \u003cem\u003ecct1-3\u003c/em\u003e plant (Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, \u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003eS6\u003c/span\u003el, and \u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003eS6\u003c/span\u003ep). For \u003cem\u003ecct1-3\u003c/em\u003e pollen cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, \u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003eS6\u003c/span\u003el, and \u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003eS6\u003c/span\u003eo), vSVs gathered closely to one another. These results suggested that fusion of vSVs into SVBs was delayed in \u003cem\u003ecct2-5\u003c/em\u003e and \u003cem\u003ecct1-3\u003c/em\u003e pollen cells compared with wild-type cells. However, we showed that such a delay only partially slowed the pollen germination rates of \u003cem\u003ecct2-5\u003c/em\u003e and \u003cem\u003ecct1-3\u003c/em\u003e pollen compared with wild-type pollen. In \u003cem\u003ecct2-5\u003c/em\u003e and \u003cem\u003ecct1-3\u003c/em\u003e pollen cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, \u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003eS6\u003c/span\u003ea, \u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003eS6\u003c/span\u003eg, \u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003eS6\u003c/span\u003el, and \u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003eS6\u003c/span\u003ep), LBs were both smaller and less numerous than in wild-type pollen cells. This may be attributable to downregulation of LB biogenesis because PC serves as a precursor to TAG, the main component of LBs. However, because the semi-transparent boundaries of LBs were preserved in \u003cem\u003ecct2-5\u003c/em\u003e and \u003cem\u003ecct1-3\u003c/em\u003e pollen cells, we concluded that LBs in these cells could fuse with PVBs or SVBs for subsequent digestion of LB contents. Indeed, our pollen germination experiments showed that the decreased number and size of LBs in \u003cem\u003ecct2-5\u003c/em\u003e and \u003cem\u003ecct1-3\u003c/em\u003e pollen cells correlated with a decrease in pollen germination rate.\u003c/p\u003e\u003cp\u003eWe showed that \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen cells from a \u003cem\u003ecct2-5 cct1-3/CCT1\u003c/em\u003e plant contain unusually enlarged LBs together with very small LBs (Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003ed, \u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003eS7\u003c/span\u003ea, \u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003eS7\u003c/span\u003ef, and \u003cspan refid=\"Fig21\" class=\"InternalRef\"\u003eS13\u003c/span\u003eb). The disproportionate sizes of LBs in the \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen cells may be similar to what has been reported for seed tissues of oleosin mutants (Shimada et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Together with phospholipids, oleosins are required as an LB-surface material for sequestrating hydrophobic TAGs within the hydrophilic cytoplasm, and genetic downregulation of oleosin biogenesis causes fusion of LBs so that the mutant cells can save the surface materials. LBs have a phospholipid monolayer, mainly consisting of PC. Thus, the presence of enlarged LBs as well as the very small LBs in \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen cells from a \u003cem\u003ecct2-5 cct1-3/CCT1\u003c/em\u003e plant may have been a consequence of severe PC shortage. The surface of such enlarged LBs looked highly electron-dense or darker compared with LBs in wild-type, \u003cem\u003ecct2-5\u003c/em\u003e, and \u003cem\u003ecct1-3\u003c/em\u003e pollen cells, which may have been caused by a shortage of oil-body surface phospholipids owing to the limited supply of PC. Some LBs retained a semi-transparent, yet fragmentary, boundary (Fig. S7h, closed arrowhead; Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e8\u003c/span\u003ea, box 1). The unusually enlarged LBs may have been too large to be incorporated into PVBs (Fig. S7b), and hence an alternative route for LB degradation would be required, as discussed below. By contrast, the smaller LBs may have been incorporated into PVBs (Figs. S7b, S7d). The \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen cells from a \u003cem\u003ecct2-5 cct1-3/CCT1\u003c/em\u003e plant contained many PVBs (Figs. S7d, S7l, S7q, and S13g) but few SVBs or vSVs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen cells from both \u003cem\u003ecct2-5 cct1-3/CCT1\u003c/em\u003e and \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e plants contained large, autophagosome-like bodies that entrapped undigested cellular contents such as the unusually enlarged LBs, swollen SGSs, and other cytoplasmic components (Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e8\u003c/span\u003e; Figs. S8aa and S8ac). This may reflect the attempt of such cells to facilitate the degradation of cellular components by autophagy. It has been reported that autophagy is essential for pollen germination, but under physiological conditions the turnover of such autophagosome-like bodies would occur too quickly for analysis by TEM. We also noticed that \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen cells from \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e plants contained incompletely closed autophagosome-like bodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e8\u003c/span\u003e, boxes 5 and 6; Figs. S8s), suggesting that autophagy is required for pollen germination yet is interrupted owing to the shortage of PC. Thus, \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e has formidable negative consequences for autophagy and, hence, the co-transmission of \u003cem\u003ecct1-3\u003c/em\u003e and \u003cem\u003ecct2-5\u003c/em\u003e via male gametophytes is eventually prohibited. Alternatively, the occurrence of the large, autophagosome-like bodies that entrapped the undigested cellular contents could be a result of a programmed suicide process for eliminating unfavorable pollen grains; in this regard, Yamamoto et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) reported that pollen grains not participating in fertilization are eliminated by intracellular lytic bodies.\u003c/p\u003e\u003cp\u003eOur reciprocal crossing of \u003cem\u003ecct2-5/CCT2\u003c/em\u003e and \u003cem\u003ecct2-3/CCT2\u003c/em\u003e plants in the \u003cem\u003ecct1-3\u003c/em\u003e background (Tables\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and S3) revealed that simultaneous transmission of \u003cem\u003ecct2-5\u003c/em\u003e or \u003cem\u003ecct2-3\u003c/em\u003e alleles with \u003cem\u003ecct1-3\u003c/em\u003e via the female gametophyte is largely permissible\u0026mdash;the survival rate of \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e ovules was 45.5 and 71.4% in \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e and \u003cem\u003ecct1-3 cct2-3/CCT2\u003c/em\u003e plants, respectively. Thus, there are strategical differences between male and female gametophytes regarding the transmission of mutant genes: transmission of the defects in PC biosynthesis via male gametophytes is strictly prohibited because a wide dispersal of unfavorable traits by pollen is not beneficial to the population. By contrast, a portion of ovules carrying \u003cem\u003ecct1-3\u003c/em\u003e and \u003cem\u003ecct2-5\u003c/em\u003e (or \u003cem\u003ecct2-3\u003c/em\u003e) is maintained so that transmission of the background genome can be ensured upon fertilization with normal pollen.\u003c/p\u003e\u003cp\u003eShockey et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) reported that \u003cem\u003egpat9-2\u003c/em\u003e affects phosphatidic acid biosynthesis in seeds and is also not transmissible via male gametophytes and that \u003cem\u003egpat9-2\u003c/em\u003e pollen grains are unable to germinate in vitro in the \u003cem\u003eqrt1\u003c/em\u003e background. They concluded that because disruption of phosphatidic acid biosynthesis inhibits TAG biosynthesis, the inhibition of pollen germination is a consequence of TAG shortage (Shockey et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Our results showed that \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen cells in the \u003cem\u003eqrt1\u003c/em\u003e background had a pollen-germination phenotype similar to that of \u003cem\u003egpat9-2\u003c/em\u003e, although \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen cells still contained TAGs and that TAG catabolism was inhibited. Furthermore, the autophagy processes that are required for pollen germination are probably inhibited or halted in \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen cells.\u003c/p\u003e\u003cp\u003eLittle is known about the differential roles of \u003cem\u003eCCT1\u003c/em\u003e and \u003cem\u003eCCT2\u003c/em\u003e in Arabidopsis development. We previously reported that residual CCT activity in rosette-leaf homogenates of \u003cem\u003ecct1-1\u003c/em\u003e and \u003cem\u003ecct2-1\u003c/em\u003e mutants accounted for 29.3 and 78.5%, respectively, of the total CCT activity in the homogenates of the wild-type (WS) ecotype (Inatsugi et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The Arabidopsis eFP browser predicts relatively higher expression of \u003cem\u003eCCT2\u003c/em\u003e (At4G15130) in pollen than in other tissues (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bar.utoronto.ca/efp2/Arabidopsis/Arabidopsis_eFPBrowser2.html\u003c/span\u003e\u003cspan address=\"https://bar.utoronto.ca/efp2/Arabidopsis/Arabidopsis_eFPBrowser2.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Our results showed that disruption of both \u003cem\u003eCCT1\u003c/em\u003e and \u003cem\u003eCCT2\u003c/em\u003e abolished pollen germination. We also showed that \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e and \u003cem\u003ecct1-3 cct2-3/CCT2\u003c/em\u003e plants exhibited partly restricted development of \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e and \u003cem\u003ecct1-3 cct2-3/CCT2\u003c/em\u003e seeds, respectively (Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and S2), whereas \u003cem\u003ecct2-3 cct1-3/CCT1\u003c/em\u003e plants did not restrict the development of \u003cem\u003ecct2-3 cct1-3/CCT1\u003c/em\u003e seeds (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). During seed development, \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e and \u003cem\u003ecct1-3 cct2-3/CCT2\u003c/em\u003e seeds contain one copy of \u003cem\u003eCCT2\u003c/em\u003e in the embryo and two copies of \u003cem\u003eCCT2\u003c/em\u003e in the endosperm. However, these copies of \u003cem\u003eCCT2\u003c/em\u003e were found to be insufficient for seed development. In contrast, in \u003cem\u003ecct1-3\u003c/em\u003e plants, in which seed development proceeds normally, developing \u003cem\u003ecct1-3\u003c/em\u003e seeds contain two copies of \u003cem\u003eCCT2\u003c/em\u003e in the embryo and three copies of \u003cem\u003eCCT2\u003c/em\u003e in the endosperm, and these copies of \u003cem\u003eCCT2\u003c/em\u003e were found to be sufficient for seed development. Similarly, in \u003cem\u003ecct2-5 cct1-3/CCT1\u003c/em\u003e plants, developing \u003cem\u003ecct2-5 cct1-3/CCT1\u003c/em\u003e seeds contain one copy of \u003cem\u003eCCT1\u003c/em\u003e in the embryo and two copies of \u003cem\u003eCCT1\u003c/em\u003e in the endosperm, and these copies of \u003cem\u003eCCT1\u003c/em\u003e were found to be sufficient for seed development. Thus, it seems likely that \u003cem\u003eCCT1\u003c/em\u003e and \u003cem\u003eCCT2\u003c/em\u003e differentially contribute to the establishment of Arabidopsis seeds after fertilization.\u003c/p\u003e\u003cp\u003eOverall, our results demonstrate that genetic defects that disrupt the CDP-choline pathway towards PC biosynthesis are eliminated before fertilization, but only during pollen germination (Fig. S13). However, future studies should investigate whether complete disruption of the CDP-choline pathway might allow the establishment of Arabidopsis seedlings. For this purpose, it will be essential to engineer a Ti-plasmid construct to specifically drive the expression of \u003cem\u003eCCT1\u003c/em\u003e cDNA in a pollen-specific manner.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding information\u003c/h2\u003e\u003cp\u003eThe Japanese Society for the Promotion of Science [Grant-in-Aid for Scientific Research (C) (Nos. 21570034, 24570040 and 16K07392) to I.N..\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlexander MP (1969) Differential staining of aborted and nonaborted pollen. Stain Technol 44:117\u0026ndash;122\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAtlagić J, Terzić S, Marjanović-Jeromela A (2012) Staining and fluorescent microscopy methods for pollen viability determination in sunflower and other plant species. 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Plant J 55:798\u0026ndash;809\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShockey J, Regmi A, Cotton K, Adhikari N, Browse J, Bates PD (2016) Identification of Arabidopsis GPAT9 (At5g60620) as an essential gene involved in triacylglycerol biosynthesis. Plant Physiol 170:163\u0026ndash;179\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSkinner DJ, Sundaresan V (2018) Recent advances in understanding female gametophyte development. F1000Research 7:804\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVance JE, Ridgeway ND (1988) The methylation of phosphatidylethanolamine. Prog Lipid Res 27:61\u0026ndash;79\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVogler F, Schmalzl C, Englhart M, Bircheneder M, Sprunck S (2014) Brassinosteroids promote Arabidopsis pollen germination and growth. Plant Reprod 27: 153\u0026ndash;167\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYamamoto Y, Nishimura M, Hara-Nishimura I, Noguchi T (2003) Behavior of vacuoles during microspore and pollen development in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. Plant Cell Physiol 44:1192\u0026ndash;1201\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYamaoka Y, Yu Y, Mizoi J, Fujiki Y, Saito K, Nishijima M, Lee Y, Nishida I (2011) \u003cem\u003ePHOSPHATIDYLSERINE SYNTHASE1\u003c/em\u003e is required for microspore development in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. Plant J 67:648\u0026ndash;661\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYamaoka Y, Shin S, Lee Y, Ito M, Lee Y, Nishida I (2021) Phosphatidylserine is required for the normal progression of cell plate formation in \u003cem\u003eArabidopsis\u003c/em\u003e root meristems. Plant Cell Physiol 62:1396\u0026ndash;1408\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-plant-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpre","sideBox":"Learn more about [Journal of Plant Research](http://link.springer.com/journal/10265)","snPcode":"10265","submissionUrl":"https://www.editorialmanager.com/jpre/default2.aspx","title":"Journal of Plant Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Autophagy, Essential genes, Phosphatidylcholine, Pollen germination, Reciprocal cross","lastPublishedDoi":"10.21203/rs.3.rs-7822087/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7822087/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePhosphatidylcholine is a major plant membrane phospholipid that contributes to the biogenesis and desaturation of membrane lipids and storage lipids. Thus, to ensure reproductive capacity, any genetic defect that affects phosphatidylcholine biosynthesis must be eliminated before fertilization. In \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, phosphatidylcholine biosynthesis depends on \u003cem\u003eCCT1\u003c/em\u003e and \u003cem\u003eCCT2\u003c/em\u003e, both encoding CTP:phosphorylcholine cytidylyltransferase. Using \u003cem\u003eA. thaliana\u003c/em\u003e T-DNA-tagged mutants, we demonstrate that neither \u003cem\u003ecct1-3 cct2-3\u003c/em\u003e nor \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e seedlings are viable. Reciprocal crosses of \u003cem\u003ecct2-3/CCT2\u003c/em\u003e or \u003cem\u003ecct2-5/CCT2\u003c/em\u003e plants in the \u003cem\u003ecct1-3\u003c/em\u003e background revealed that neither \u003cem\u003ecct2-3\u003c/em\u003e nor \u003cem\u003ecct2-5\u003c/em\u003e was transmitted via \u003cem\u003ecct1-3\u003c/em\u003e male gametophytes, although each allele was transmitted via \u003cem\u003ecct1-3\u003c/em\u003e female gametophytes. Although all pollen grains on a pollen quartet from \u003cem\u003eqrt1-1 cct1-3 cct2-5/CCT2\u003c/em\u003e plants were viable, none of \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen grains from \u003cem\u003ecct2-5 cct1-3/CCT1\u003c/em\u003e and \u003cem\u003ecct1-3 cct2-5/CCT2\u003c/em\u003e plants were able to germinate in vitro. Transmission electron microscopy analysis of pollen grains subjected to pollen germination revealed that \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e pollen grains developed unusual ultrafine structures, such as lipid bodies of disproportionate size including extremely enlarged ones, swelling of small granular structures, inhibition of vacuole development, and accumulation of incomplete autophagosome-like bodies enclosing various intracellular compartments. Thus, transmission of \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e to offspring via male gametophytes appears to be strictly prohibited by interruption of autophagic processes required for pollen germination, thereby preventing the widespread dispersal of deleterious mutations among the progeny. By contrast, \u003cem\u003ecct1-3 cct2-5\u003c/em\u003e was partly transmissible via female gametophytes, so the background genome could be rescued by fertilization.\u003c/p\u003e","manuscriptTitle":"Genetic defects in the CDP-choline pathway for phosphatidylcholine biosynthesis cannot be transmitted to offspring via male gametophytes owing to interruption of autophagy-like processes required for pollen germination in Arabidopsis thaliana","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-29 12:21:58","doi":"10.21203/rs.3.rs-7822087/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revision","date":"2025-11-03T23:53:29+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-10-15T19:43:01+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-15T06:30:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-11T10:53:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Plant Research","date":"2025-10-09T23:16:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-plant-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpre","sideBox":"Learn more about [Journal of Plant Research](http://link.springer.com/journal/10265)","snPcode":"10265","submissionUrl":"https://www.editorialmanager.com/jpre/default2.aspx","title":"Journal of Plant Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a81922b5-457b-455c-b153-17de95b15332","owner":[],"postedDate":"October 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-02T16:00:29+00:00","versionOfRecord":{"articleIdentity":"rs-7822087","link":"https://doi.org/10.1007/s10265-025-01690-0","journal":{"identity":"journal-of-plant-research","isVorOnly":false,"title":"Journal of Plant Research"},"publishedOn":"2026-02-01 15:58:04","publishedOnDateReadable":"February 1st, 2026"},"versionCreatedAt":"2025-10-29 12:21:58","video":"","vorDoi":"10.1007/s10265-025-01690-0","vorDoiUrl":"https://doi.org/10.1007/s10265-025-01690-0","workflowStages":[]},"version":"v1","identity":"rs-7822087","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7822087","identity":"rs-7822087","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}