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.