pp 1–17 | Cite as

Pollen grain development and male sterility in the perfect flowers of Maytenus obtusifolia Mart. (Celastraceae)

  • Isabella Veríssimo Nader HaddadEmail author
  • Bárbara de Sá-Haiad
  • Lygia Dolores Ribeiro de Santiago-Fernandes
  • Silvia Rodrigues Machado
Original Article


Perfect flowers of Maytenus obtusifolia have partial sterility of pollen grains, resulting in collapsed and developed free microspores. However, the cellular events resulting in partial male sterility have not been determined. In pistillate flowers of this species, male sterility is related to the premature programmed cell death (PCD) in tapetum and sporogenic cells. The process occurs through autophagy via macroautophagy and massive autophagy and is associated with sporophytic cytoplasmic male sterility (CMS). Here, we characterised the development of pollen grains and investigated the cellular events that result in tapetum cells and free microspores PCD in perfect flowers, using light and transmission electron microscopy combined with the TUNEL (Terminal deoxynucleotidyl transferase mediated dUDP end-Labeling) assay and the ZIO (Zinc iodide-osmium tetroxide) method. Pollen grain development in perfect flowers was divided into eight developmental stages based on the characteristics of the pollen grains. Tapetum cells undergo PCD at the free microspore stage, through a macroautophagic process, by formation of autophagosomes and by autophagosomes giving rise to lytic vacuoles at maturity. In the final stage of PCD, massive autophagy occurs by rupture of the tonoplast. The development of viable and inviable microspores diverges at the vacuolated microspore stage, when PCD occurs in some free microspores, causing interruption of pollen development through necrosis. These events result in the observed partial male sterility. Viable microspores undergo mitosis and develop into tricellular pollen grains. Male sterility in hermaphrodite individuals is here interpreted as gametophytic CMS.


Anatomy Autophagy Gametophytic CMS Immunocytochemistry PCD Ultrastructure 



We thank the Electron Microscopy Center (CME) IBB, UNESP and its technical staff, Dr. Elton Scudeler and Ana Silvia Garcia, for assistance with the TUNEL analyses. This study is part of the Ph.D. Thesis of I. V. N. H., carried out in the Programa de Pós-graduação em Ciências Biológicas (Botânica) of the Universidade Federal do Rio de Janeiro (UFRJ), Museu Nacional, Rio de Janeiro, Brazil. The study was supported financially by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior). B. S. H. was supported by research grants from FAPERJ APQ1 (Proc. E-26/111.207/2014) and MCTI/CNPq/Universal (Proc. 447624/2014-8). S. R. M. receives a scholarship from CNPq (Proc. 304396/2015-0).


  1. Benevides CR, Haddad IVN, Barreira NP, Rodarte ATA, Galetto L, Santiago-Fernandes LDR, Lima HA (2013) Maytenus obtusifolia Mart. (Celastraceae): a tropical woody species in a transitional evolutionary stage of the gynodioecy–dioecy pathway. Plant Syst Evol 299(9):1693–1707. CrossRefGoogle Scholar
  2. Bergman P, Edqvist J, Farbos I, Glimelius K (2000) Male-sterile tobacco displays abnormal mitochondrial atp1 transcript accumulation and reduced floral ATP/ADP ratio. Plant Mol Biol 42:531–544. CrossRefGoogle Scholar
  3. Carvalho-Okano RM (1992) Estudos taxonômicos do gênero Maytenus Mol. emend. Mol. (Celastraceae) do Brasil extra-amazônico. Dissertation, Universidade Estadual de CampinasGoogle Scholar
  4. Chase CD (2006) Cytoplasmic male sterility: a window to the world of plant mitochondrial-nuclear interactions. Trends Genet 23(2):81–90. CrossRefGoogle Scholar
  5. Ehlers BK, Maurice S, Bataillon T (2005) Sex inheritance in gynodioecious species: a polygenic view. Proc R Soc B 272:1795–1802. CrossRefGoogle Scholar
  6. Gabe M (1968) Techniques histologiques. Masson & Cie, ParisGoogle Scholar
  7. Gahan PB (1984) Plant histochemistry and cytochemistry: an introduction. Academic Press, Inc., LondonGoogle Scholar
  8. González-Melendi P, Uyttewaal M, Morcillo CN, Mora JRH, Fajardo S, Budar F, Lucas MM (2008) A light and electron microscopy analysis of the events leading to male sterility in Ogu-INRA CMS of rapeseed (Brassica napus). J Exp Bot 59(4):827–838. CrossRefGoogle Scholar
  9. Gravieli Y, Sherman Y, Ben-Sasson SA (1992) Identification of programmed cell death in-situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:493–501. CrossRefGoogle Scholar
  10. Grigorjeva VV, Gabarayeva N (2017) Pollen wall ontogeny in Polemonium caeruleum (Polemoniaceae) and suggested underlying mechanisms of development. Protoplasma 255(1):109–128. CrossRefGoogle Scholar
  11. Haddad IVN, Santiago-Fernandes LDR, Machado SR (2018) Autophagy is associated with male sterility in pistillate flowers of Maytenus obtusifolia (Celastraceae). Aust J Bot 66:108–115. Scholar
  12. Hu J, Huang W, Huang Q, Qin X, Yu C, Wang L, Li S, Zhu R, Zhu Y (2014) Mitochondria and cytoplasmic male sterility in plants. Mitochondrion 19(B):282–288. CrossRefGoogle Scholar
  13. Karnovsky MJ (1965) A formaldehyde-gluraldehyde fixative of high osmolality for use in electron microscopy. J Cell Biol 27:137–138 Retrieved from Google Scholar
  14. Kawanabe T, Ariizumi T, Kawai-Yamada M, Uchimiya H, Toriyama K (2006) Abolition of the tapetum suicide program ruins microsporogenesis. Plant Cell Physiol 47:784–787. CrossRefGoogle Scholar
  15. Kirasak K, Ketsa S, Imsabai W, van Doorn WG (2010) Do mitochondria in Dendrobium petal mesophyll cells form vacuole-like vesicles? Protoplasma 241:51–61. CrossRefGoogle Scholar
  16. Ku S, Yoon H, Suh HS, Chung Y-Y (2003) Male-sterility of thermosensitive genic male-sterile rice is associated with premature programmed cell death of the tapetum. Planta 217:559–565. CrossRefGoogle Scholar
  17. Li N, Zhang D-S, Liu H-S, Yin C-S, Li X-x, Liang W, Yuan Z, Xu B, Chu H-W, Wang J, Wen T-Q, Huang H, Luo D, Ma H, Zhang DB (2006) The rice tapetum degeneration retardation gene is required for tapetum degradation and anther development. Plant Cell 18:2999–3014. CrossRefGoogle Scholar
  18. Liu Y, Bassham D (2012) Autophagy: pathways for self-eating in plant cells. Annu Rev Plant Biol 63:215–237. CrossRefGoogle Scholar
  19. Lockshin RA, Zakeri Z (2004) Apoptosis, autophagy, and more. Int J Biochem Cell B 36:2405–2419. CrossRefGoogle Scholar
  20. Luo XD, Dai LF, Wang SB, Wolukau JN, Jahn M, Chen JF (2006) Male gamete development and early tapetal degeneration in cytoplasmic male-sterile pepper investigated by meiotic, anatomical and ultrastructural analyses. Plant Breed 125:395–399. CrossRefGoogle Scholar
  21. Majewska-Sawka A, Rodriguez-Garcia MI, Nakashima H, Jassen B (1993) Ultrastructural expression of cytoplasmic male sterility in sugar beet (Beta vulgaris L.). Sex Plant Reprod 6:22–32. CrossRefGoogle Scholar
  22. Marty F (1978) Cytochemical studies on GERL, provacuoles, and vacuoles in root meristematic cells of Euphorbia. PNAS 75(2):852–856. CrossRefGoogle Scholar
  23. Minina EA, Bozhkov PV, Hofius D (2014) Autophagy as initiator or executioner of cell death. Trends Plant Sci 19(11):692–697. CrossRefGoogle Scholar
  24. Mogensen HL, Wagner VT (1987) Associations among components of the male germ unit following in vivo pollination in barley. Protoplasma 138:161–172. CrossRefGoogle Scholar
  25. Nakonechnaya OV, Kalachev AV (2018) Pollen ultrastructure in Aristolochia manshuriensis and A. contorta (Aristolochiaceae). Protoplasma 255(5):1309–1316. CrossRefGoogle Scholar
  26. Papini A, Mosti S, Brighigna L (1999) Programmed-cell-death events during tapetum development of angiosperms. Protoplasma 207:213–221. CrossRefGoogle Scholar
  27. Peled-Zehavi H, Galili G (2018) Fluorescence imaging of autophagy mediated ER-to-vacuole trafficking in plants. In: The plant endoplasmic reticulum, Vol. 1691 (Eds C Hawes, V Kriechbaumer), Humana Press, New York, pp 239–249Google Scholar
  28. Reape T, Molony E, McCabe P (2008) Programmed cell death in plants: distinguishing between different modes. J Exp Bot 59:435–444. CrossRefGoogle Scholar
  29. Reinecke M, Walther C (1978) Aspects of turnover and biogenesis of synaptic vesicles at locust neuromuscular junctions as reveled by iodide-osmium tetroxide (ZIO) reacting with intravesicular SH-groups. J Cell Biol 78:839–855CrossRefGoogle Scholar
  30. Reynolds ES (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17:208–212. CrossRefGoogle Scholar
  31. Russell SD, Cass DD (1981) Ultrastructure of the sperms of Plumbago zeylanica. I. Cytology and association with the vegetative nucleus. Protoplasma 107:85–107. CrossRefGoogle Scholar
  32. Sabar M, Gagliardi D, Balk J, Leaver CJ (2003) ORFB is a subunit of F1FO-ATP synthase: insight into the basis of cytoplasmic male sterility in sunflower. EMBO Rep 4:381–386. CrossRefGoogle Scholar
  33. Salvesen GS, Hempel A, Coll NS (2015) Protease signaling in animal and plant-regulated cell death. FEBS J 283:2577–2598. CrossRefGoogle Scholar
  34. Shi S, Ding D, Mei S, Wang J (2010) A comparative light and electron microscopic analysis of microspore and tapetum development in fertile and cytoplasmic male sterile radish. Protoplasma 241:37–49. CrossRefGoogle Scholar
  35. Tadege M, Kuhlemeier C (1997) Aerobic fermentation during tobacco pollen development. Plant Mol Biol 35:343–354. CrossRefGoogle Scholar
  36. Tanaka I (1993) Development of male gametes in flowering plants. J Plant Res 106:55–63. CrossRefGoogle Scholar
  37. van Doorn W, Papini A (2013) Ultrastructure of autophagy in plant cells. Autophagy 9:1922–1936. doi:
  38. van Doorn W, Beers E, Dangl J, Franklin-Tong V, Gallois P, Hara-Nishimura I, Jones A, Kawai-Yamada M, Lam E, Mundy J, Mur L, Petersen M, Smertenko A, Taliansky M, Breusegem V, Wolpert T, Woltering E, Zhivotovsky B, Bozhkov P (2011) Morphological classification of plant cell deaths. Cell Death Differ 18:1241–1246. CrossRefGoogle Scholar
  39. Van Hautegem T, Waters AJ, Goodrich J, Nowack MK (2015) Only in dying, life: programmed cell death during plant development. Trends Plant Sci 20(2):102–113. CrossRefGoogle Scholar
  40. Wu H, Cheung AY (2000) Programmed cell death in plant reproduction. Plant Mol Biol 44:267–281. CrossRefGoogle Scholar
  41. Xu P, Yang Y, Zhang Z, Chen W, Zhang C, Zhang L, Zou S, Ma Z (2008) Expression of the nuclear gene TaFAd is under mitochondrial retrograde regulation in anthers of male sterile wheat plants with timopheevii cytoplasm. J Exp Bot 59(6):1375–1381. CrossRefGoogle Scholar
  42. Yang S, Peng L, Bao H, Tian H (2018) Cytological features of developing anthers in rose balsam. J Am Soc Hortic Sci 143(2):95–100. CrossRefGoogle Scholar
  43. Yu H-S, Hu S-Y, Zhu C (1989) Ultrastructure of sperm cells and the male germ unit in pollen tubes of Nicotiana tabacum. Protoplasma 152:29–36. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Departamento de Botânica, Museu NacionalUniversidade Federal do Rio de JaneiroRio de JaneiroBrazil
  2. 2.Departamento de Botânica, Instituto de BiociênciasUniversidade Estadual PaulistaBotucatuBrazil

Personalised recommendations