Cytological abnormalities and pollen abortion in interspecific hybrids of Nicotiana

Interspecific cross breeding introduces superior agronomic traits into cultivated species; however, problematic pollen sterility occurs in the hybrids. Our previous study obtained interspecific hybrids from the cross between a cytoplasmic male sterility line of Nicotiana tabacum and Nicotiana alata, and some of the hybrids were pollen sterile. Here, we conducted an in-depth cellular study to understand the cytological mechanism of pollen abortion in these hybrids (F1-D sterile) compared with pollen development in the fertile hybrids (F1-S sterile) from the same cross. The ultrastructure observation showed that the membrane of microspore in F1-D sterile hybrid was deficient at all represented developmental stages. Chromosome behavior during meiosis was studied by carbol fuchsin staining, which indicated that cytomixis, chromosome leakage and asymmetric callose wall deposition occurred with high frequency in the microsporocyte of F1-D sterile. The results of the ultrastructure and 6-diamidino-2-phenylindole (DAPI) analyses also showed that the cytoplasm and nucleus were unstable and extruded from F1-D sterile microspore during the developmental process, leading to mature pollen grains that were vacuous and collapsed in the aperture region. In addition, delayed tapetum degradation was also detected in the anther of F1-D sterile, and might be associated with irregular sporopollenin deposition in the aperture region of F1-D sterile pollen. Genetic unbalance and cytomembrane deficiency might both be responsible for the instability of the chromosome, nuclear and cytoplasm, and resulted in pollen abortion in F1-D sterile hybrids, and irregular tapetum degradation might also be related with pollen sterility.


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Hybrid sterility is a widespread postzygotic reproductive barrier between species or subspecies. This 11 effect provides the initial force of genetic differentiation and speciation, and it represents a major barrier 12 to the effective use of inter-subspecific genetic resources in many crops, including tobacco [1-3]. Pollen 13 sterility is one of the most important reasons for interspecific hybrid sterility, along with embryo sac 14 sterility [4,5], and severely prevents the utilization of genetic diversity in hybrids; therefore, it is of great 15 significance to understand the genetic or cytological mechanism of pollen abortion in hybrids.

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The anther locule is where the pollen grain develops, and the developing anther consists of 17 microsporocytes in the center, surrounded by the tapetum and the anther wall [6,7]. The anther wall of 18 Nicotiana belongs to the basic type and is composed of the endothecium, two middle layers and the 19 tapetum [8,9]. The tapetum, which is in direct contact with the microspores, serves as a nutritive and 20 secretory tissue to supply the nutrients and other components required for pollen wall development, and 21 it regulates the development of microspores during pollen mother cell (PMC) meiosis and the subsequent 22 microspore maturation [9][10][11]. Pollen development in most of the species includes the following key 3 1 stages: meiosis, tetrad and primexine development, microspore release, sporopollenin deposition, mitosis, 2 accumulation of starch or lipids, addition of pollenkitt or tryphine to the exine, and dehydration of the 3 cytoplasm [12][13][14]. Before the tetrad stage, the microsporocyte is encased in the callose wall, which is 4 synthesized by the meiocyte and deposited between the plasmalemma and the original cellulosic wall 5 during prophase I of meiosis [7,15]. This callose layer is vital for preventing cell cohesion and fusion 6 and useful as a template for the formation of species-specific exine-sculpting patterns; then, upon its 7 dissolution, the free microspores were released [7, 16-20]. which overcame the pre-fertilization cross-incompatibility encountered when using the corresponding 6 male fertile line as the maternal parent, and F1 offspring with different morphology and fertility were 7 obtained. Offspring named F1-S was morphologically similar to the maternal parent, with well-8 developed stamens and fertile pollen grains. However, hybrids F1-D was morphologically different from 9 the female parent, with the stamens (anthers and filaments) developed, but the pollen grains were 10 completely sterile [33]. We deduced that a fertility restorer from the paternal parent (N. alata) was 11 responsible for male fertility restoration in F1-S fertile, and this hypothesis is undergoing further 12 validation. However, another interesting question is why the F1-D sterile hybrids showed aborted pollen.

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To reveal the cause of pollen abortion in F1-D sterile, we studied the cytological mechanism of pollen 14 abortion through analysis of pollen viability and fertility, microsporogenesis, callose wall deposition, 15 cell ultrastructure, cytomembrane integrity, cytoplasm and nucleus stability, and tapetum development 16 and degradation, which were compared with that in the F1-S fertile hybrids.  Pollen grains from hybrids morphologically different from the maternal parent (F1-D-2 sterile) 3 were utilized to explore the cytological mechanism of male sterility, and pollen from F1-S fertile (F1-4 S-2) was also studied as a control. The genome size of these two hybrids was previously reported [33]. The microspore or pollen cell structure was studied by transmission electron microscopy (TEM).  The anther wall was studied by paraffin sectioning. Anthers at different developmental stages were 10 collected and fixed in FAA (5 mL of formaldehyde and 5 mL of glacial acetic acid in 90 mL of 70% 11 (V/V) ethyl alcohol) at 4 °C overnight，and then dehydrated for 2 h in an ethyl alcohol gradient of 80%, 12 85%, 90%, and 95%, followed by dehydration in 100% ethyl alcohol twice, each time for 1 h. The anther 13 was then hyalinized in 1/2 xylene and 1/2 ethyl alcohol overnight, saturated gradually with paraffin at 37 14°C until saturation, and then immersed in pure paraffin overnight. The saturated materials were embedded, 15 sectioned at 5 μm, dewaxed and then dyed with safranin-Fast Green or toluidine blue.

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In convenience of research and description, the flower buds of the hybrids were classified into nine 19 developmental stages according to the development of the male gametophyte, as shown in Fig. 1. The 20 microsporocyte at stages 1 to stage 2 were undergoing the process of meiosis, and microspore release 8 1 happened before stage 3 ( Fig. 1), followed by the cell wall deposition and microspore mitosis at stage 4.

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The microspores developed further at stages 5 to 7 and matured into bicellular pollen grains at stages 8 3 and 9 (Fig. 1). assay showed that pollen of F1-S fertile at stage 9 germinated well on the in vitro medium (Fig. 2. L), 20 and the germination ratio was higher than 85%. However, the germination ratio was much lower for 21 microspore at stages 7 and 8 ( Fig. 2. M, N), and pollen at stage 6 could not germinate at all (Fig. 2. O). 9 1 In F1-D sterile, pollen at all developmental stages was unable to germinate on in vitro medium.  (Table   17 1). Furthermore, the chromosomes in the meiocytes of F1-D sterile were prone to leak at diakinesis of 18 meiosis I (Table 1), implying that the chromosomes escaped out of the cytoplasm and the callose wall 19 ( Fig. 3. C, D).  (Table 1). Chromosome leakage even resulted in the 10 1 occurrence of cytoplasts-cells that were completely devoid of nuclear material (Fig. 3. C, D). At the 2 tetrad stage, abnormal and vestigial microspores were observed in the anther locule of F1-D sterile, and 3 four microspores within the same callose wall were different in size and shape (Fig. 3. E), unlike those 4 in F1-S fertile. Obviously, these results indicated that cytomixis and chromosome leakage occurred with 5 higher frequency in the meiocytes of F1-D sterile.  (Table 1). In F1-D sterile, asymmetrical callose wall deposition 15 occurred at a high percentage at all developmental stages before microspore release (Fig. 3. A-H), while 16 the callose wall was deposited relatively symmetrically after diakinesis in F1-S fertile (Fig. 3. I, J, K, L).

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Asymmetrical callose deposition was also observed via TEM in the PMCs of F1-D sterile (Fig. 5. C).

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These results indicated that asymmetrical callose wall deposition occurred with higher frequency in F1-

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D sterile. Nuclear material translocation and leakage occurred in the areas of thinned or perforated callose 11 1 wall deposition in the meiocytes (Fig. 3. A, B, C). pollen was totally covered by the exine (Fig. 5. O, P). In fertile pollen, only intine was deposited in the 20 aperture region at the above three developmental stage (Fig. 5. F, J, N).

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Nuclear and cytoplasmic instability in pollen of F1-D 12 1 As cytomembrane deficiency was observed in the microspore/pollen of F1-D sterile, the cell inclusion 2 stability was further studied. TEM observation revealed that the cytoplasm of PMCs in F1-S fertile were 3 covered tightly by the callose cell wall (Fig. 5. A, B), while there was gap between the cytoplasm and 4 callose wall of PMCs in F1-D sterile (Fig. 5. C, D). PMCs of F1-D sterile had abundant cell inclusions 5 ( Fig. 5. C), which were extruded gradually at later developmental stages (Fig. 5. G, K), and the cytoplasm 6 was totally absent from mature pollen grains (Fig. 5. O).

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DAPI staining was used to further examine the microspore/pollen nuclei stability at stage 2 to stage 9 12 ( Fig. 1), which indicated that micronuclei were present in the tetrad phase and the following 13 monokaryotic phase in F1-D sterile (Fig. 6. A, B). normal connectivum and four anther locules of equal size (Fig. 7. A). In F1-D sterile, about 66% of the 2 anthers were normal, as described above (Fig. 7. B), while others had enlarged connectivum and reduced 3 anther locules (Fig. 7. C, D), which might morphologically affect microsporogenesis and pollen 4 development. The anther wall was composed of epidermis, fibrous endothecium, one middle layer and 5 the tapetum, which formed normally in both the sterile and fertile hybrids (Fig. 7. E, F) at the beginning 6 of PMC differentiation. For F1-S fertile hybrids, the tapetum started to degenerate at the early tetrad 7 stage (Fig. 7. G) and mostly disappeared at the release of the microspores (Fig. 7. H). However, the 8 tapetum in F1-D sterile degenerated later than that in F1-S fertile. The tapetum was intact at the tetrad 9 stage (Fig. 7. I), mostly remained after the release of the free microspores ( Fig. 7. J, K), and ultimately 10 degenerated at stage 5 ( Fig. 7. L). Therefore, the tapetum degradation was delayed in the anther of F1-D In this study, microspore and pollen instability in the F1-D sterile was also found. DAPI staining and 6 TEM analysis showed that the cytoplasm and nucleus were extruded from the pollen grain during the 7 processes of microspore maturation. This phenomenon could also be attributed to the plasma membrane 8 deficiency and genetic unbalance of the sterile pollen. In addition, the results also indicated that 9 sporopollenin was irregularly deposited in the aperture region of F1-D sterile pollen, which might be 10 induced by delayed tapetum degeneration after microspore release.

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The timely degradation of the tapetum is necessary for pollen development and fertility, and the timing 12 of tapetum degeneration varies among species. For example, degeneration commenced at the tetrad stage 13 and was completed at the bicellular pollen stage in Brachypodium and rice [11,50]. However, in wheat, 14 barley and Actinidia deliciosa, the breakdown of tapetum cells began during the vacuolated microspore 15 stage [11,34,51]. In this study, tapetum degradation in F1-D sterile was delayed compared with that in 16 F1-S fertile, which might partially cause the observed pollen incapacity.

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The genetic basis of hybrid sterility has been studied in some plants, including Solanum, rice (Oryza),