1. INTRODUCTION
Water deficit is one of the most important abiotic stress restricting plant production. As a consequence of climate change, drought events are projected to increase in intensity, duration, and frequency (Lesk et al., 2016). Thus, the improvement of drought-resistance of crops represents an urgent need that demands the identification of key regulators and pathways as potential targets for drought-resistance improvement. Reproductive stage drought stress causes more severe damage and yield loss than any other stage of development of crop plants (Dorion et al., 1996; Sheoran and Saini, 1996). Therefore, an effective way to improve drought-resistance of crops is to select for yield and its components during reproductive development under drought stress. Reduction in grain yield due to drought has mainly been attributed to male sterility because the male organ is more drought sensitive than the female organ which remains fertile under stress condition that causes sterility in the male (Bingham, 1966; Saini and Aspinall, 1981). Hence, the male development under drought has attracted greater research attention than the female. Commercially, tomato is the most important vegetable crop. However, it is susceptible to abiotic stresses including drought. An earlier study examined the effects of drought on tomato utilizing vegetative tissues (Seng, 2014). However, for a fruit vegetable crop like tomato, fruit set is the most important trait for evaluating drought tolerance, thus evaluating drought tolerance of the reproductive organs especially the male is of enormous importance.
Anther, the male organ of flowering plants comprises of concentric cell layers including the epidermis, endothecium, middle layers and tapetum, the innermost layer surrounding a central locule that contains sporogenous cells. Sporogenous cells develop into pollen grains, the male gametophytes within the locule. During pollen development, anther wall layers play important roles in nutrition, protection, and pollen release. The tapetum serves as a source of energy for the developing microspores, secrete enzymes e.g. callase that releases the tetrads from the callose wall (Izhar and Frankel, 1971) and precursors for exine wall formation. Initially, tapetum development proceeds normally and at later stage, undergoes program cell death (PCD) and disintegrates (Parish and Li, 2010). The timely tapetum-specific PCD and disintegration are necessary for normal pollen development. Precocious or postponed tapetum degeneration results in male sterility (Bhadula and Sawhney, 1988; Graybosch and Palmer, 1985). The modified epidermal cells, the stomia modulate pollen release process. Failure of stomia cells to degenerate leads to male sterility due to anther indehiscence (Cecchetti et al., 2013). Abnormalities in anther and pollen development leading to induction of male sterility as a result of drought have been extensively investigated in cereal crops (Ji et al., 2010; Saini and Westgate, 1999). However, there are limited or no information on the impacts of drought on anther and pollen development in tomato.
Carbohydrate metabolism and sugar movement from source organs to sink tissues such as anthers are important processes for pollen development. Studies with cereals have shown that disturbances in these processes lead to male sterility associated mainly with abnormal starch accumulation in the pollen (Koonjul et al., 2005; Oliver et al., 2005). Sucrose accumulation, inadequate starch build-up in pollen grains and subsequent abortion of pollen development are attributed to repression of genes involved in sucrose and starch metabolism in drought and cold-stress anthers (Koonjul et al., 2005; Oliver et al., 2005). Additionally, drought-stressed rice anthers accumulate more starch granules in connective tissues than in pollen grains (Jin et al., 2013; Lalonde et al., 1997). However, studies on changes in starch and soluble sugar accumulation and the underlying molecular mechanisms in developing anthers under drought stress are scarce in fruit vegetable crops and for tomato in particular, there are no reports.
Phytohormones are important endogenous chemical messengers that modulate plant growth and development and responses to adverse stress factors. Among the phytohormones, abscisic acid (ABA) is the key plant hormone known to mediate responses to abiotic stresses such as drought and temperature (Zhang et al., 2006). In reproductive tissues, there is increasing evidence of a strong correlation between ABA level increase and pollen sterility during abiotic stresses. Higher pollen abortion and greater reduction in grain set with concomitant higher ABA accumulation in anthers of drought-susceptible than drought-tolerant wheat cultivars have been reported (Dong et al., 2017; Ji et al., 2011). Besides, there is proof of crosstalk between ABA and sugar signaling. The expression ofOSINV4 , a cell wall invertase gene, was repressed by ABA in cold stress rice anthers (Oliver et al., 2007). Recent study implicated auxin (IAA) in the control of plant response to abiotic stress during reproductive development. Reduction in pollen fertility and grain yield was associated with reduced accumulation of endogenous IAA in rice spikelets due to drought-induced repression of YUCCA genes that are involved in IAA biosynthesis (Sharma et al., 2018). Jasmonic acid (JA) has been demonstrated to participate in drought stress responses in vegetative tissues (De Ollas Valverde et al., 2015; Du et al., 2013) but there are no reports on JA mediation of drought responses in reproductive organs. However, induction of stigma exsertion as a result of reduction in endogenous JA in tomato anthers under high temperature has been reported (Pan et al., 2019). Although phytohormones play critical roles in regulating crops responses to drought stress, the dynamics of endogenous hormone metabolisms and the relationships with behavioural patterns of anthers and pollen at different stages of development under drought stress are not well understood.
In this study, we have examined the effects of drought stress on anther morpho-physiological and molecular responses in tomato. The major objectives of the study were to determine: (a) the consequences of subjecting anthers of varied developmental stages to water deficit on flowering phenology, flower development anther morphology and fruit set/yield (b) whether different stages of anther development response differently to drought stress (c) the effect of drought stress on male gametophyte fertility (d) the histological and cytological changes in anthers at different stages under drought stress (e) effects of drought stress on the levels of endogenous IAA, ABA, and JA; the contents of sucrose, glucose and fructose; and starch accumulation at different stages of anther development (f) the underlying molecular mechanisms determining the physio-morphological changes in the male organ at different stages of development under stress conditions. Findings in this study will contribute to the understanding of the behavioural patterns and defects in the anther, tapetum, and pollen at different stages of development and the associated physiological and molecular mechanisms in response to drought stress in tomato.