Fertile Arabidopsis cyp704b1 mutant, defective in sporopollenin biosynthesis, has a normal pollen coat and lipidic organelles in the tapetum

Abstract

The exine acts as a protectant of the pollen from environmental stresses, and the pollen coat plays an important role in the attachment and recognition of the pollen to the stigma. The pollen coat is made of lipidic organelles in the tapetum. The pollen coat is necessary for fertility, as pollen coat-less mutants, such as those deficient in sterol biosynthesis, show severe male sterility. In contrast, the exine is made of sporopollenin precursors that are biosynthesized in the tapetum. Some mutants involved in sporopollenin biosynthesis lose the exine but show the fertile phenotype. One of these mutants, cyp704b1, was reported to lose not only the exine but also the pollen coat. To identify the cause of the fertile phenotype of the cyp704b1 mutant, the detailed structures of the tapetum tissue and pollen surface in the mutant were analyzed. As a result, the cyp704b1 mutant completely lost the normal exine but had high-electron-density granules localized where the exine should be present. Furthermore, normal lipidic organelles in the tapetum and pollen coat embedded between high-electron-density granules on the pollen surface were observed, unlike in a previous report, and the pollen coat was attached to the stigma. Therefore, the pollen coat is necessary for fertility, and the structure that functions like the exine, such as high-electron-density granules, on the pollen surface may play important roles in retaining the pollen coat in the cyp704b1 mutant.

Keywords: exine; pollen coat; sporopollenin; tapetum.

Figure 1. Putative biosynthesis pathway of sporopollenin precursors. ACOS5, acyl-CoA synthetase 5; PKSA/B, polyketide synthase A/B; TKPR1/2, tetraketide α-pyrone reductase 1/2. Dashed arrows indicate multiple reactions.

Figure 2. TEM images of the developmental anther in WT (A–D) and cyp704b1 mutant (E–H). Male gametophyte development in the tetrad stage (A, E), early bicellular pollen stage (B, F), early tricellular pollen stage (C, G), and mature pollen stage (D, H). el, elaioplast; p, pollen; pc, pollen coat; pcl, pollen coat-like; t, tapetum; ta, tapetosome; tet, tetrad. Arrowheads, abnormal high-electron-density granules localized along the tapetal cell wall; arrows, abnormal high-electron-density granules localized on the pollen surface. Scale bar, 5 µm.

Figure 3. TEM images of lipidic organelles and pollen wall in WT (A–E) and cyp704b1 mutant (F–J). Tapetum organelles developed in the early bicellular pollen stage (A, F) and early tricellular pollen stage (C, H). Pollen wall developed in the early bicellular pollen stage (B, G), early tricellular pollen stage (D, I), and mature pollen stage (E, J). el, elaioplast; ex, exine; p, pollen; pc, pollen coat; pcl, pollen coat-like; ta, tapetosome. Arrows, abnormal high-electron-density granules localized on the pollen surface. Scale bar, 500 nm.

Figure 4. SEM images of mature pollen grain of WT (A–C) and cyp704b1 mutant (D–I). Unfixed pollen (A, D, E) and fixed pollen (B, C, F–I). Arrows, abnormal granules localized on the pollen surface; arrowheads, pollen coat. High-magnification images (C, E, H, I) correspond to each square of the images (B, D, F, G), respectively. Scale bar, 5 µm (A, B, D, F, G), 3 µm (C, H), and 1 µm (E, I).

Figure 5. Light microscopy and TEM images of pollen attachment to the papillary cells in WT (A–C) and cyp704b1 mutant (D–F). Light microscopy images (A, D) and TEM images (B, C, E, F). (C, F) High magnification of B and E, respectively. p, pollen; pa, papillary cell; pc, pollen coat; pcl, pollen coat-like. Arrowheads, adhesive surface between the pollen and the papilla. Scale bar, 10 µm (A, D), 5 µm (B, E), and 1 µm (C, F).

Figure 6. SEM images of pollen attachment to the papillary cells in WT (A–C) and cyp704b1 plant (D–F). (B, E) High magnification of A and D, respectively. p, pollen; pa, papillary cell. Arrowheads, adhesive surface between the pollen and the papilla; arrows, pollen tube. Scale bar, 10 µm (A, D), 2 µm (B, E), and 5 µm (C, F).