4. DISCUSSION
Successful development of the anther in flowering plants is very crucial
to ensuring plant fertility and productivity because it produces and
delivers the male gamete to the female gametophyte for efficient
fertilization (Borg et al., 2009).
However, anther development is often perturbed by abiotic stresses such
as drought resulting in male sterility and yield reduction
(Jin et al., 2013;
Nguyen and Sutton, 2009). Nevertheless,
the developmental flaws and the underlying physiological and molecular
mechanisms remain unclear in tomato. In this study, we examined the
effect of drought stress on anther development using
morpho-physiological and molecular analyses in tomato. Reproductive
development is extremely sensitive to abiotic stresses but during flower
ontogeny, some stages are more sensitive
(Sato et al., 2002). In our study,
drought induced bud abortion specifically at tetrad (TED), early
uninucleate (EUM) and vacuolated uninucleate microspore (VUM) stages
(Figure 1 C) whereas more advanced and younger buds in the proximal and
distal regions of a truss respectively, were drought tolerant. The
advanced buds proceeded to anthesis during or immediately after the
stress, before bud abortion set in while the younger buds recovered and
developed to anthesis after rewatering. This indicates that the break in
flowering is the result of bud abortion and the inherent drought
tolerance of the younger buds permitted growth resumption after
transient growth arrest, led to the second peak and extended period of
flower production. Thus, drought altered flowering phenology (Figure 1B)
through induction of irreversible and reversible arrest of anther bud
development, and delayed anthesis. Drought-induced reduction in yield is
attributed to flower and pod abortions
(Fang et al., 2010;
Kokubun et al., 2001). However, we,
demonstrated that in addition to flower abortion, bud abortion is a
crucial factor limiting yield in tomato. Additionally, all preanthesis
DS anthers had reduced length but the reduction in stamen length was
much more severe in type-3 flower that the stigma extended above the
anther cone (Figures 1E, Figure S1D), similar to heat-induced stigma
exsertion (Pan et al., 2019). Suggesting
that the underlying mechanisms involved in stamen shortening leading to
stigma exsertion under both stresses in tomato might be similar.
Male sterility due to poor pollen viability is the most important
limiting factor affecting yield under drought stress
(Jin et al., 2013). In the present study,
drought stress caused marked reduction in pollen viability (Figure 2A)
and subsequently low fruit set in accordance with previous reports
(Saini and Aspinall, 1981). However, the
effect of DS on pollen viability varied with stage of anther
development. It was more severe for anthers between meiotic and
binucleate stages which produced anthers that lacked pollen or had
pollen that were severely malformed (Figures 3A and B). Further, male
sterility due to anther indehiscence and stigma exsertion were observed
in our study (Figure 1E and Figure 3A) in line with previous reports
under low and high temperatures (Kiran et
al., 2019; Pan et al., 2019). Our
results suggest that reduction in fruit yield is due mainly to the
reduction in pollen viability but secondary factors including anther
indehiscence and stigma exsertion also play role. Importantly, the
drought-tolerant and susceptible anthers identified are of significant
importance for identifying major players involved in drought stress
tolerance that can facilitate drought tolerance breeding in tomato.
The production of viable pollen grains depends on the normal development
and function of the tapetum (Kawanabe et
al., 2006). However, abiotic stresses especially extreme temperatures
can cause early or delayed tapetum degeneration leading to pollen
abortion (Oda et al., 2010). The current
study revealed that drought stress induced both precocious and delayed
tapetum degeneration (Figure 3A and C) in agreement with previous
reports (Jin et al., 2013;
Oda et al., 2010 ). The early tapetum
degeneration might have led to the early dissolution of the callose wall
and subsequently, the ectopic release of microspores and abnormal
microspore wall formation at meiotic (type-3) and tetrad stages (Figure
3B, S1), in line with earlier reports under low-temperature stress
(Gothandam et al., 2007;
Mamun et al., 2006). Moreover, it might
constitute the main underlying cause of pollen and flower bud abortions
at tetrad and early uninucleate stages. However, at later stages, pollen
abortion might not be related to tapetum degeneration since it is
already in a degenerate state at these stages even in WW anthers (Figure
3A and C). Therefore, other factors including but not limited to defects
in pollen structure such as plasma membrane and organelles malfunction
might be involved in bud and pollen abortions at later stages,
consistent with the high number of GO terms in the cellular component
significantly enriched in down-regulated DEGs (Table S6).
Drought-induced altered expression of genes involved in
tapetum/microspore development causes abnormal tapetum development and
male sterility (Jin et al., 2013). In our
study, except for SlMYB80 which was markedly induced at PMC-MEI
stage, DS repressed the examined tapetum expressed genes at PMC-MEI and
TED-VUM stages (Figure 5). The synthesis and deposition of callose
provide a temporary wall that separates microsporocytes, meiotic cells
and microspores of the tetrad and subsequently degraded by the enzyme
callase secreted by the tapetum to release the microspore
(Lu et al., 2014;
Scott et al., 2004). SlCDM1 , a C3H
zinger finger TF and SlMYB80 regulate callose metabolism during
microsporogenesis (Lu et al., 2014;
Zhang et al., 2007). The large number of
tapetum and microspore expressed genes down-regulated with conspicuous
defects in tapetum, pollen and callose wall in this study, is consistent
with (Jin et al., 2013). Additionally,
mutants of MYB80 are male sterile with altered tapetum and pollen
development in many crops (Phan et al.,
2012; Xu et al., 2014). Therefore, the
up-regulation of SlMYB80 at PMC-MEI stage is consistent with
normal pollen development (Figure 2A) of type-1 anthers. Suggesting a
positive and overriding role of SlMYB80 in early anther
development under drought stress. Our results suggest that drought
stress between meiosis and early uninucleate microspore stages is
detrimental and can induce irreversible arrest of tapetum and pollen
development in tomato.
In flowering crops starch forms the principal storage food reserve in
mature male gametophyte (Dorion et al.,
1996; Jin et al., 2013). Storage of
starch in mature pollen relies on uninterrupted sucrose supply to the
anther and its unimpeded cleavage principally by cell wall invertases
into monosaccharides which are conveyed into cells via monosaccharide
transporters and subsequently used in starch biosynthesis
(Dorion et al., 1996;
Ruan et al., 2010). Our study clearly
showed that sucrose, glucose, fructose, and starch accumulated
abnormally in developing drought-stressed anthers (Figure 6 and S3),
suggesting that drought affected carbon allocation and processes
implicated in starch biosynthesis. Sucrose and starch metabolic genes
were differentially drought-regulated. The sucrose cleavage genesSlCWINV3-like, and TOMSSF at PMC-MEI stage, andSlSUS6 at BIN-MP stage were noticeably down-regulated, resulting
in higher sucrose levels in agreement with past reports
(Dorion et al., 1996;
Nguyen et al., 2010). In contrast,β-fructofuranosidase and SlSUS6 were induced which
correlated with lower sucrose levels at TED-UM stage.
Committing glucose to participate in starch biosynthesis and other
metabolic processes requires its phosphorylation by hexokinase which is
specifically expressed in the tapetum and developing pollen
(Granot et al., 2013;
Suwabe et al., 2008). We observed two
hexokinase genes, Slhxk1 and Slhxk2 generally repressed at
all three stages of development (Figure 6A) resulting increased levels
of fructose and glucose, in agreement with
Nguyen et (al., 2010). In addition,
drought repressed the expression of ADGPase gene, the rate
limiting enzyme in starch biosynthesis, consistent with
Lalonde et al., (1997). Interestingly,β-amylase 8 , the starch hydrolyzing enzyme was induced at BIN-MP
stage. These results suggest that reduction pollen viability due to
reduced starch accumulation under drought stress is highly associated
with diminished sugar utilization and hydrolysis of de novo and/or
existing starch in maturing anthers.
Previous studies provided evidence of IAA involvement in abiotic stress
responses in vegetative organ (Min et al.,
2014; Sakata et al., 2010). In
reproductive organs, drought- reduction of IAA biosynthesis genes
expression and contents reduces pollen and spikelet fertility in rice
(Sharma et al., 2018). In our study, gene
expression analysis showed that the tryptophan aminotransferase
of Arabidopsis -related (TAR) family member, SlTAR2was largely repressed at PMC-MEI stage whereas two TAR genes, includingSlTAR2 and SlTAR1-like genes , and two YUC genes, ToFZY1 and
ToFZY2 were significantly induced at TED-VUM stage (Figure 7C), which
were in agreement with endogenous IAA levels (Figure 7B). Intriguingly,
the TED-VUM stage, that had IAA biosynthesis genes and content
significantly induced and increased respectively under drought stress,
exhibited severe pollen abnormalities (Figure 3B, DS (4 d)) and anther
buds abortion (Figure 1C). On the contrary, the PMC-MEI stage with
conspicuous attenuation in IAA biosynthesis and amount had a good number
of its anthers (type-1) producing viable pollen (Figure 2A) and
excellent fruit set (Figure S1C), inconsistent with
Sharma et al., (2018).
JA plays roles in multiple abiotic stresses including drought stress
(de Ollas et al., 2013;
De Ollas Valverde et al., 2015). We
demonstrated that, with exception of SlLOX5 which was moderately
increased at PMC-MEI stage, drought repressed the expression of JA
biosynthesis genes at all stages of anther development with concomitant
significant decrease in JA levels in anthers at TED-VUM and BIN-MP
stages (Figure 7) similar to Pan et al.,
(2019) under high-temperature stress. In ArabidopsisJA-deficient mutants lines exhibit anther indehiscence (Cecchetti et
al., 2013). Interestingly, JA reduction at BIN-MP stage occurred
concurrently with extreme reduction in fruit set (Figure S1C) which
might be due to male sterility as a result of impaired anther dehiscence
since a large number of pollen grains were retained in mature anthers
(Figure 3A, DS (4 d). It is speculated that anther indehiscence
exhibited by type-2 and type-3 anthers at anthesis (Figure 3A) is
correlated with low JA levels.
Drought stress triggers ABA biosynthesis and increases its content in
reproductive organs with a lower and higher level increase in ABA
concentrations associated with abiotic stress tolerance and
susceptibility respectively (Zhang et al.,
2006). In this study, many ABA metabolic and signaling genes were
differentially expressed at different stages of anther development
(Table S10). Expression analysis revealed that thedehydrogenase/reductase (SDR ) genes: 3-oxoacyl-CoA
reductase 1and SlSDR12-like, involved in ABA biosynthesis were
significantly induced at PMC-MEI stage and SlSDR12-like was
induced at TED-VUM stage while Slcyp707a, that inactivates
bioactive ABA was highly and moderately induced at PMC-MEI and BIN-MP
stages respectively (Figure 7C). It appears that the regulation of ABA
homeostasis rests on the coordinated expression of ABA biosynthesis and
catabolic genes. At PMC-MEI stage (drought tolerant), higher expression
of ABA biosynthesis coincided with higher expression of Slcyp707aand lower ABA accumulation. On the contrary, at TED-VUM stage (drought
susceptible) ABA overproduction coincided with higher and reduced
expression of ABA biosynthesis and catabolic genes respectively (Figure
7B, C), consistent with a study in which distinct drought tolerant and
susceptible wheat cultivars were used (Ji
et al., 2011). Thus, up-regulation of ABA inactivation pathway appears
as a core mechanism for modulating ABA level and conferring drought
tolerance in tomato anthers consistent with
Ji et al., (2011) but inconsistent with
Jin et al., (2013). The inconsistency
might be related to differences in crop species, stress level and
duration, and technique used to analyze the transcriptomes. Put
together, our results suggest that ABA and its catabolic pathway play a
decisive role in regulating ABA homeostasis and drought tolerance in
tomato anthers. Further, it is speculated that antagonistic interactions
between ABA and IAA confers drought tolerance in pre-meiotic anthers,
and between ABA and JA, in controlling anther dehiscence in meiotic
anthers in tomato.
To sum up, it is explicit that increase bud and flower abortions and
subsequent reduction in fruit yield of DS plants were largely attributed
to drought-induced male sterility caused by abnormalities in anther,
tapetum and pollen development resulting in attenuation of pollen
fertility, anther indehiscence and stigma exsertion. Under drought
stress, different stages of anther development exhibit differential
responses with the sensitivity of anthers to drought stress spanning the
period from meiotic mother cell stage to binucleate stage with TED-VUM
stage, the most sensitive to drought, whereas PMC-MEI stage the most
drought tolerant. The drought tolerance exhibited by the PMC-MEI stage
is probably the result of moderate increase in ABA level due to the
high-level expression of its catabolic enzymes which maintains a level
of ABA optimum to trigger signaling and activating ABA-dependent drought
adaptive gene expression and repression of IAA signaling (Figure 8). Our
findings provide insight into the behavioural patterns and defects in
the anther, tapetum and pollen at different development stages and
associated physiological and molecular mechanisms in response to drought
stress and give a novel insight into potential drought tolerance
mechanism which can be engineered for improvement of drought tolerance
in tomato.