3.3 Histo-cytological analysis of anther development under drought stress and recovery conditions
To understand details of the effects of drought stress on the male development, anthers at six developmental stages as earlier described (Chen et al., 2018; Polowick and Sawhney, 1992, 1993a, b, 1995) were examined under light microscope (LM) and transmission electron microscope (TEM). The six stages included: meiotic (MEI), tetrad (TED), early uninucleate microspore (EUM), vacuolated uninucleate microspore (VUM), binucleate (BIN) and mature pollen (MP).
Pre-meiotic anthers from DS plants exhibited no distinct alterations from WW anthers (Not shown). However, defects were obvious at all of the later stages examined after 4 d drought-stress treatment and rewatering (Figure 3A). At MEI stage, the sporogenous and tapetum tissues were constricted and moved apart especially in the outer tapetum region after 4 d of DS anther (arrow 1) so that the sporogenous tissue no longer filled the locular space; the type-3 anther wall layers and the sporogenous tissue were collapsed and not distinctly demarcated. At other stages, after 4 d of DS, abnormalities included: degenerated and enlarged pollen (arrows 2 and 4 respectively); ectopic callose wall dissolution and precocious released of microspores at TED stage; compressed and compactly parked microspores at EUM stage; dwindled tapetum layer at TED and EUM stages and its enhanced disintegration and dissolution at VUM stage; premature induction of anther dehiscence (arrow 3), partial degeneration of stomia cells (arrow 5) and the large number of pollen grains retained in anther locules due to inefficient dehiscence at MP stage. In the type-2 anther, the tapetum was hypertrophic, persistent at VUM stage and exhibited delayed degeneration at BIN stage; together with type-3 anther, the anthers were collapsed at BIN stage so that two instead of a common locule in each anther lobe were observed (arrow 6) and a total lack of dehiscence due to persistent stomia cells at MP stage (arrow 7). Additionally, in the type-3 anthers, premature disintegration and dissolution of the callose wall, tapetum and sporogenous tissues were observed with conspicuous empty locule (arrow 8) from TED to MP stages, although debris of degenerated microspores (arrow 9) was sometimes observed in the locule at some stages).
The effects of DS on pollen development were further revealed by TEM observations (Figure 3B, Figure S1E). Microspores were completely dissolved and absent in the locule from TED to MP stages in the type-3 anther indicating that drought during mid-meiotic stage can prevent microsporogenesis. Developing pollen cells at different stages of development in anther of DS (4 d), type-2 and type-3 were highly plasmolyzed with constricted protoplasm separated from the pollen cell wall (arrow 10) and had abnormally numerous small or large vacuoles (Figure 3B). Other abnormities observed in the developing pollen included expansion of the middle lamella after 4 d of DS at MEI stage (Figure S1E); premature microspore wall formation at TED stage after 4 d DS and at MEI stage in type-3 anther (Figure 3B, Figure S1E); in the type-2 anthers at TED stage, failure of tetrad formation and lack of callose wall around individual tetrad of microspores’ nuclei were observed indicating delayed cleavage of meiotic mother cell’s (MMC) cytoplasm and impaired callose deposition and/or ectopic callose dissolution, although evidence of meiosis was the presence of multiple nucleoli in the cytoplasm of MMC (Figure 3B) suggesting that drought during early meiotic stage can prolong microsporogenesis and delay microspore mother cell cleavage.
In addition, TEM observations revealed disturbances in tapetum development caused by drought stress (Figure 3C). Under well-watered condition, the tapetum degeneration occurred at VUM stage. However, in DS (4 d) anthers, from MEI to EUM stages the tapetum underwent ectopic degeneration with constricted protoplasm at MEI stage (Arrow 11) and completely devoid of protoplasmic contents and cellular structure at TED and EUM stages respectively. After re-watering, in the type-2 anthers, excessive vacuolation of tapetum cells was observed at MEI and TED stages, and the tapetum persisted and maintained cellular structure up to VUM stage and degeneration was delayed until BIN stage. Although persistent and developed under well-watered condition between 10-12 DARW, the tapetum in the type-2 anthers exhibited signs of plasmolysis, with cells contracted and separated from the tapetum cell wall (arrow 11), adjacent tapetum cells (arrow 12) and adjacent middle layer cells (arrow 13). In the type-3 anthers, obvious abnormalities included ectopic degeneration of the tapetum cells beginning at MEI stage and their complete absence from anthers at TED to VUM stages. In short, drought stress spanning the period from early meiotic to binucleate stages affects anther and pollen development through induction of tapetum/pollen degeneration and dissolution, delayed tapetum degeneration and stomia persistence.