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.