Tubulin cytoskeleton during microsporogenesis in the male-sterile genotype of Allium sativum and fertile Allium ampeloprasum L

Abstract

Microsporogenesis in garlic. The male-sterile Allium sativum (garlic) reproduces exclusively in the vegetative mode, and anthropogenic factors seem to be the cause of the loss of sexual reproduction capability. There are many different hypotheses concerning the causes of male sterility in A.sativum; however, the mechanisms underlying this phenomenon have not been comprehensively elucidated.Numerous attempts have been undertaken to understand the causes of male sterility, but the tubulin cytoskeleton in meiotically dividing cells during microsporogenesis has never been investigated in this species. Using sterile A.sativum genotype L13 and its fertile close relative A. ampeloprasum (leek), we have analysed the distribution of the tubulin cytoskeleton during microsporogenesis. We observed that during karyokinesis and cytokinesis, in both meiotic divisions I and II, the microtubular cytoskeleton in garlic L13 formed configurations that resembled tubulin arrangement typical of monocots. However, the tubulin cytoskeleton in garlic was distinctly poorer (composed of a few MT filaments) compared with that found in meiotically dividing cells in A. ampeloprasum. These differences did not affect the course of karyogenesis, chondriokinesis, and cytokinesis, which contributed to completion of microsporogenesis, but there was no further development of the male gametophyte. At the very beginning of the successive stage of development of fertile pollen grains, i.e. gametogenesis, there were disorders involving the absence of a normal cortical cytoskeleton and dramatically progressive degeneration of the cytoplasm in garlic. Therefore,we suggest that, due to disturbances in cortical cytoskeleton formation at the very beginning of gametogenesis, the intracellular transport governed by the cytoskeleton might be perturbed, leading to microspore decay in the male-sterile garlic genotype.

Fig. 1

Consensus tree of maximum parsimony analyses based on the ITS sequences data set of different accession of A. ameloprasum and their closely related A. sativum species. The underline indicates sequences of A. ameloprasum L. and A. sativum L13 obtained in the present study. Allium fistulosum and A. cepa were used as an outgroup in the analysis. Bootstrap values higher than 50 % (1000 replicates) are shown at each branch

Fig. 2

A. sativum L13: A inflorescence a-flower, b-topsets, c-leaf-like bracts. B Mitotically dividing meristematic root cell in early anaphase. MTs of the karyokinetic spindle visualised by indirect immunofluorescence (green colour). Chromosomes stained with DAPI (blue colour). C–H Microsporocytes of A. sativum during meiosis I. C, D Prophase I, C visible short fragments of MTs surrounding the cell nucleus (arrows) and parietal cytoplasm devoid of MTs (star), D tangential section—visible crossing MTs (arrow) around the nucleus, the absence of visible nuclear chromatin, as it is strongly condensed and distributed irregularly within the nucleus. E Metaphase I, visible karyokinetic spindle and metaphase chromosomes. F–H Telophase I, F tangential section—visible phragmoplast (arrows) and one of the two telophase nuclei, G diagonal section—visible one of the two telophase nuclei and radially arranged MTs of the phragmoplast, only a small fragment of the parietal cytoplasm was devoid of MTs (arrows). H An undulating primary septum visible between two nuclei and MTs in the parietal cytoplasm (arrows). Figures B–H are in the same magnification

Fig. 3

Microsporocytes of A. sativum during meiosis II. A–G MTs visualised by indirect immunofluorescence (green colour). Nuclear DNA stained with DAPI (blue colour). H, I TEM. A, B Prophase II, long and thick filaments of MTs surrounding the nucleus, arrowheads—black spots devoid of the cytoplasm and indicating cytoplasm decay. B Visible one of the two dyad cells. C Metaphase II—visible one of the two dyad cells with a karyokinetic spindle and metaphase chromosomes. D Telophase II, diagonal section—visible one of the four telophase nuclei and irregularly arranged filaments of the MTs forming the phragmoplast. E–G Tetrads of microspores, E visible two of the four tetrad cells with short fragments of MTs scattered in the cytoplasm. G Tubulin visible as single points dispersed in the cytoplasm—arrow. E–G Arrowheads—black spots devoid of the cytoplasm and indicating cytoplasm decay. Figures A–G are in the same magnification. H, I Tetrads of microspores visualised under TEM, I visibly lytic vacuoles (arrow) and electron-dense deposits (star). J Tetrads of A. ampeloprasum, visible two of the four microspores with normally developed cell organelles (TEM)

Fig. 4

A. ampeloprasum L. A inflorescence B–I Microsporocytes during meiosis I and II. MTs visualised by indirect immunofluorescence (green colour). Nuclear DNA stained with DAPI (blue colour). B, C Prophase I, visible dense network that was evenly distributed across the cytoplasm (arrows). D Metaphase I, visible more numerous and clearly thinner MT filaments and metaphase chromosomes. E, F Telophase I, E a well-developed phragmoplast with a well-visible primary septum between two telophase nuclei (arrow), F visible MTs in the parietal cytoplasm (arrows). G Metaphase II, the diagonal section shows one of the two cells with the karyokinetic spindle and chromosomes forming a metaphase plate. H, I Tetrads of microspores with developing cortical MTs. J Single microspore with visible, well-developed cortical MTs arranged radially around the nucleus. Figures B–J are in the same magnification. K Pollen grains stained with the Alexander assay; viable grains stained purple, dead pollen grains stained green (arrows). L Germination of pollen grains on the stigma—visible fluorescence of the callose wall of the pollen tube stained with aniline blue