© 2019 The Japan Mendel Society Cytologia 84(3): 221–225 Wax-Deficient Arabidopsis Mutant cer1-1 Shows Abnormal Tapetosomes, Elaioplasts, and Pollen Coat Keiko Kobayashi1, Masashi Suzuki2 and Noriko Nagata1* 1 Department of Chemical and Biological Sciences, Faculty of Science, Japan Women’s University, 2–8–1 Mejirodai, Bunkyo-ku, Tokyo 112–8681, Japan 2 The Chemical Biology Laboratory, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113–8657, Japan Received February 28, 2019; accepted March 14, 2019 Summary Normal maturation of the pollen coat is important for pollination in higher plants. The pollen coat contains abundant lipids and proteins, which are derived from tapetum cell-specific organelles, i.e., the tapeto- some and elaioplast. The wax-deficient mutant eceriferum1 (cer1) shows a conditional male sterile phenotype. It is considered that this phenotype is caused by dysfunction of the pollen coat, although cer1 mutant pollen coats appear normal at a particular stage. In the present study, abnormalities in the tapetum cells and pollen coat of cer1-1 mutants were observed by scanning electron microscope and light microscopic analysis. To investigate the effect of wax composition changes on the structure of the pollen coat, tapetosome, and elaioplast in cer1-1 mutants, we observed the ultrastructure of the cer1-1 male gametophyte at various developmental stages. We ob- served abnormal giant aggregation of the lipids, which might be due to a fusion of the tapetosome and elaioplast in the early bicellular pollen stage tapetum cell. On the contrary, in wild-type plants, the fusion of the tapetosome and elaioplast occurred at the late bicellular pollen stage. In cer1-1, abnormalities in the tapetum cell and the pol- len coat might occur because of the unusual timing of fusion of the tapetosome and the elaioplast. This is the first report showing the role of tapetum wax in the maturation of the tapetosome and elaioplast, which may affect pol- len coat function. Key words Elaioplast, Pollen coat, Tapetosome, Tapetum, Wax, Arabidopsis. The pollen surface of the higher plants is covered C18 fatty acids, which are produced in plastids. These by the exine and pollen coat. The pollen coat contains fatty acids are elongated to C24–C36 at the endoplasmic abundant lipids and proteins and has many functions, reticulum and then converted to a mixture of waxes in- such as initiating hydration of the pollen at the stigma cluding ketones, aldehydes, alkanes, and alcohols (Kunst (Preuss et al. 1993, Hülskamp et al. 1995) and attaching and Samuels 2009). These long-chain lipids (above C29) to vector insects (Piffanelli et al. 1998) in addition to are decreased in the pollen of cer6 mutants (Preuss et al. its role in self-incompatibility (Shiba et al. 2001). Spe- 1993). These mutants show two phenotypes: one has a cific lipidic organelles, i.e., tapetosomes and elaioplasts, pollen coat with fewer, smaller lipid droplets; the other which are located in the innermost part of the anther, are loses the pollen coat at an early stage (Preuss et al. 1993, involved in the formation of the pollen coat (Hernández- Fiebig et al. 2000). These mutants are sterile because Pinzón et al. 1999). In Brassica napus, the tapetosome pollen hydration fails regardless of whether the pollen contains triacylglycerol and wax alkanes, while the coat exists. This suggests that the pollen coat of cer6 elaioplast contains sterol esters (Hernández-Pinzón et al. mutants is dysfunctional, even though it appears normal 1999, Hsieh and Huang 2007). Sterols are biosynthesized (Preuss et al. 1993, Fiebig et al. 2000). via the cytosolic mevalonate pathway. Sterol-deficient Long-chain acyl-CoA synthetase (LACS) was shown Arabidopsis mutants show a sterile phenotype because to provide CoA-activated very long-chain fatty acids to the pollen coat in these mutants is defective. They also the wax biosynthesis pathways. The pollen coat lipids show abnormal development of the tapetosome and the (alkanes, ketones, and alkenes) were reduced signifi- elaioplast, which are the locations of the biosynthesis cantly in lacs1/lacs4 double knockout mutant lines. It is and accumulation of pollen coat components (Ishiguro reported that the mutant was also conditionally sterile, et al. 2010, Jin et al. 2012, Kobayashi et al. 2018). though its pollen surface seemed to be normal (Jessen Mutants deficient in pollen coat wax also show the et al. 2011). sterile phenotype. Waxes are biosynthesized via C16 or It is thought that CER1 is involved in catalyzing al- kane formation (Aarts et al. 1995, Bourdenx et al. 2011, * Corresponding author, e-mail: [email protected] Bernard et al. 2012). The composition of the cuticular DOI: 10.1508/cytologia.84.221 waxes of cer1 mutants stems has previously been char- 222 K. Kobayashi et al. Cytologia 84(3) acterized by a dramatically lower content of products Materials and methods of the alkane-forming pathway (e.g., alkanes, secondary alcohols, and ketones; Bourdenx et al. 2011, Sakuradani Plant growth conditions et al. 2013). The cer1 mutants were reported to have pol- Seeds were sown on 1/2 MS medium (Wako Pure len coats, although their lipid droplets were abnormal in Chemical Industries, Ltd., Japan) supplemented with number and size. The sterile phenotype of these mutants 1.5% (w/v) sucrose and stored at 4°C for more than 2 may be caused by the abnormal function of their pollen days. After vernalization, wild-type (WT; Ler) and coats (Aarts et al. 1995, Hülskamp et al. 1995). cer1-1 plants (Aarts et al. 1995), obtained from the Ara- Analysis of these mutants suggested that wax compo- bidopsis Biological Resource Center, were grown for sition is important for the normal function of the pollen 2 weeks on 1/2 MS agar medium and then transferred coat, and a normal pollen coat is necessary for the fertil- to soil. All growth occurred under a 16 : 8 h light : dark ity of the pollen. It is thought that the waxes are biosyn- cycle at 23°C in a growth chamber until the flowering thesized in tapetum cells, which are the source of the stage. pollen coat, and accumulate in the elaioplast and the ta- petosome. However, detailed observations of the tapeto- Microscopic analyses somes and elaioplasts of wax-deficient mutants have not For scanning electron microscopy (SEM), the anthers been reported previously. Therefore, we considered that of inflorescence samples were fixed with 2% glutaral- the pollen coat and tapetum-specific organelles might dehyde in a 20 mM sodium cacodylate buffer, pH 7.0, show abnormal phenotypes in wax-deficient mutants. In at 4°C for 2 h. They were then washed with the buffer the present study, we characterized, in detail, the ultra- for 4 h at 4°C. Then, they were post-fixed with 2% OsO4 structure of the pollen coat and the developmental stage in the buffer at 4°C for 1 h. The fixed samples were run of tapetum cells in an Arabidopsis cer1-1 mutant to re- through an ethanol series and a t-butyl alcohol series. veal which steps of pollen coat development are affected The samples were lyophilized for 30 min at -10°C using by abnormal wax composition. a freeze-drier, coated with platinum, and observed with an S-800 scanning electron microscope (Jeol, Tokyo, Fig. 1. SEM images of the pollen coat of WT plant (A, C) and cer1-1 mutant (B, D). Scale bars=5 µm (A, B) and 1 µm (C, D). 2019 Arabidopsis cer1 Abnormal Tapetum and Pollen Coat 223 Japan). Transmission electron microscopic (TEM) analy- shed pollen from WT, cerl-m, and cerl-7 plants all ap- sis was performed as described previously (Kobayashi peared to contain tryphine, which generally was found et al. 2018). For light microscope analysis, sections to cover the entire pollen grain. Since the result of SEM (1 µm thick) were mounted on slide glasses, stained with analysis of WT pollen was not shown, the difference in a toluidine blue solution, and observed under a light mi- pollen coat between WT and cer1 plants remains to be croscope. elucidated. In the present study, we identified the abnor- mal expansion of the pollen coat on part of the surface Results and discussion of cer1-1 pollen grains while other parts lacked a pollen coat; both of these phenotypes were rarely seen in WT It has been reported that Arabidopsis cer1-1 mutants pollen grains (Fig. 1). show a male sterile phenotype because of pollen coat To investigate the developmental stage at which WT dysfunction, despite a normal appearance (Aarts et al. and cer1-1 pollen grains diverge, anthers of WT and 1995). In a previous study of wax-deficient mutants, the cer1-1 plants in the middle bicellular pollen stage to the pollen coat surfaces of these mutants were not clearly tricellular pollen stage were observed by microscopy. observed because the fixation methods were not opti- At the middle bicellular pollen stage, the thickness of mized for SEM-based pollen coat observation (Preuss the tapetum of the cer1-1 plant was more heterogeneous et al. 1993, Hülskamp et al. 1995, Jessen et al. 2011). than that of the WT plant, although tapetum was shown Aarts et al. (1995) reported that the exines of mature, in both the WT and cer1-1 (Fig. 2A, B). The tapetum of Fig. 2. Light microscopic images of the tapetum and pollen in WT plant (A, C, E) and cer1-1 mutant (B, D, F). Tapetum and pol- len in the middle bicellular pollen stage (A, B), late bicellular pollen stage (C, D), and tricellular pollen stage (E, F). Scale bars=50 µm.