Pollen Development at High Temperature: From Acclimation to Collapse

The seeds and fruits derived from the sexual reproduction of flowering plants constitute the major part of the human diet. Our capacity to generate sufficient crop yield is increasingly compromised by human population expansion, competition for land use, biodiversity loss, and global climate change. Hot days and heat waves are predicted to increase in frequency and intensity in many temperate regions in the coming decades as a consequence of global warming (Pachauri et al., 2014). Exposure to high temperature episodes often coincides with the reproductive phase of the plant life cycle. As pollen development and functioning are among the most heat-sensitive processes that impact upon plant fertility, it is crucial to understand the mechanisms and processes underlying heat-related male sterility in order to maintain food security. Sexual plant reproduction in flowering plants involves two central processes: meiosis, which rearranges the genes and reduces the number of chromosomes; and fertilization, which restores the diploid chromosome number. In between these two, haploid spores develop into multicellular gametophytes, which produce the male or female gametes. Development of the male gametophyte (pollen) has been shown to be sensitive to environmental fluctuations and suboptimal conditions, thereby limiting sexual reproduction (Iwahori, 1965; Schoper et al., 1987; Peet et al., 1998; Dupuis and Dumas, 1990; Ahmed et al., 1992; Kim et al., 2001). Pollen is formed inside the anther locules from diploid pollen mother cells that undergo meiosis to give rise to a tetrad of four haploid microspores surrounded by locular fluid. After release from the tetrad, the free microspores enlarge and divide asymmetrically (pollen mitosis I) to form a larger vegetative cell and a smaller generative cell. The generative cell is then engulfed by the vegetative cell and undergoes a second mitosis (pollen mitosis II), either before pollen is released from the anther or during pollen tube growth, to form two sperm cells (McCormick, 2004). During the differentiation of the pollen mother cells, the innermost anther wall layer forms the tapetum (Goldberg et al., 1993). This tissue is metabolically active, especially at early microspore stage, providing the developing microspores with carbohydrates, nutrients, enzymes, and compounds required for the synthesis of the outer pollen wall (exine). Development of the tapetum is tightly coordinated with microspore development and its degeneration begins shortly after microspores are released from the tetrad. The correct functioning of the tapetum and its timely degradation are essential for pollen development (Parish and Li, 2010). One of the abiotic conditions with the most impact on pollen development is high temperature. Plants are sessile organisms, inevitably exposed to ambient temperatures throughout their life cycle and can overheat relative to their environment due to direct absorbance of solar radiation. High temperatures can change the structure of biomolecules, such as DNA, RNA, proteins, and lipids, which in turn can affect basic metabolic processes like translation, photosynthesis, respiration, and redox regulation (Bokszczanin et al., 2013). At organismal level, this leads to disturbance of growth and development, with harmful effects often manifesting themselves only after transition to the reproductive phase. Pollen heat sensitivity is a conserved feature among diverse plant species, including monocots and dicots, and occurs under various high temperature regimes, for example, short heat shock or prolonged growth under mildly elevated day and/or night temperatures (Mesihovic et al., 2016). The sensitivity varies over the course of pollen development, with later stages, after pollen mitosis, being relatively heat tolerant. Medium high-temperature stress may disturb meiosis, but peak sensitivity occurs from meiosis to pollen mitosis I (i.e. at microspore stage), which is the focus of this work. The development of pollen after exposure to heat stress at the microspore stage has not been described in detail but includes abortion of microspores as well as failure at later stages, leading to a reduction in the number of pollen grains at anthesis and the proportion of mature pollen grains that is viable and able to germinate (Mesihovic et al., 2016). The fact that high-temperature stress may result in a mixed population of both dead and perfectly viable pollen within the same anther locule has recently been explained with a model where initially small differences between microspores in metabolic performance or developmental progression are amplified by competition for nutrients in the locular fluid (Carrizo García et al., 2017). Notably, pollen injury is often accompanied by aberrations in tapetum development (hypertrophy) or morphology and alterations in the timing of tapetum degeneration (Iwahori, 1965; Saini et al., 1984; Ahmed et al., 1992; Kim et al., 2001; Suzuki et al., 2001; Abiko et al., 2005; Oshino et al., 2007; Djanaguiraman et al., 2013; Harsant et al., 2013). While elevated temperatures may elicit acclimation responses that permit pollen development under restricted heat stress conditions, physiological injury leading to failure of pollen development and functioning occurs at higher temperature stress. The balance between acclimation and collapse thus depends on the heat regime experienced as well as the levels of basal and acquired thermotolerance. Here, we discuss the current understanding of heat acclimation responses and heat injury during microspore development. Thermotolerance mechanisms of mature and germinating pollen as well as vegetative tissues have been discussed elsewhere (Mittler et al., 2012; Bokszczanin et al., 2013; Tunc-Ozdemir et al., 2013; Burke and Chen, 2015). Data accumulated over the past few decades indicate that pollen and the surrounding anther tissues respond to an increase in temperature at the transcriptome, proteome, and metabolome levels, similar to other plant cell types. Several heat responses that have been characterized as adaptive in vegetative tissues were also found to occur in these reproductive tissues. In recent years, experimental evidence for a role of these responses in maintaining physiological homeostasis during pollen development at high temperature has emerged. One of the main damaging effects of high temperature results from changes in protein structure. This may interfere with protein function, and if more hydrophobic regions are exposed, proteins can aggregate and become cytotoxic. Failure to prevent the accumulation and aggregation of misfolded proteins may eventually lead to cell death. To counteract these effects, the expression of heat shock protein (HSP) chaperones is induced at high temperature in a process known as the heat shock response (HSR). HSPs accumulate in the cytoplasm and organelles to stabilize, resolubilize, and refold proteins (Vierling, 1991; Hartl et al., 2011). Recently, it was demonstrated that small HSPs are important for heat tolerance in Arabidopsis. Two of the most abundant classes of sHSPs (C1 and CII) were shown to interact with and to protect an overlapping set of heat sensitive proteins involved in translation initiation (eIF4A) and elongation (eEF1B), together with HSP101 (McLoughlin et al., 2016). Underlying the HSR is a network of heat stress transcription factors (HSFs) that bind to a palindromic DNA sequence, the heat shock element, to induce the expression of heat-responsive genes (Scharf et al., 2012; Ohama et al., 2017). High-temperature induction of HSF and HSP genes in developing anthers, microspores, and pollen has been reported for different species (Frank et al., 2009; Giorno et al., 2010; Bita et al., 2011; Zhang et al., 2014; Li et al., 2015). Furthermore, proteins of different types of HSPs (i.e. belonging to diverse families, like sHSP, HSP70, HSP90, and HSP100) accumulate in developing anthers and pollen grains after a short period of high-temperature stress (Frova et al., 1989, 1991; Jagadish et al., 2010; Chaturvedi et al., 2015), which points to a capacity for both tissues to activate “classical” thermotolerance mechanisms. A recent study in tomato (Solanum lycopersicum) has shown that induction of HSR protects microspores from high temperature (Fragkostefanakis et al., 2016b). One of the main HSFs regulating the HSR is HSFA2, which forms a “superactivator complex” with HSFA1 proteins (Scharf et al., 2012). Knockdown of HSFA2 resulted in increased sensitivity of developing tomato pollen to a short period of high temperature (Fragkostefanakis et al., 2016b). The effect of reduced levels of HSPs on pollen thermotolerance has not been tested, but members of the Bcl-2-associated athanogene (BAG) family have been shown to be involved in pollen thermotolerance (Doukhanina et al., 2006). BAG proteins are cochaperones involved in recruiting HSPs to client proteins; they are expressed in developing tomato pollen under heat stress, and their expression may be under control of HSFA2 (Frank et al., 2009; Fragkostefanakis et al., 2015). Taken together, various HSFs and HSPs are induced in anthers and microspores by high temperature, and genetic studies confirm that they play an active role in protecting developing pollen against heat stress. Upon exposure to elevated temperatures, a second set of genes, including ones that encode chaperones, is induced to protect cells against toxic levels of unfolded proteins in the endoplasmic reticulum (ER) (Howell, 2013). Protein folding is an essential function of the ER, involving the guidance of polypeptides through several modification steps to reach their desired conformation. The folded protein may remain in the endomembrane system, be targeted to the cell membrane, or be secreted. A polypeptide that is not glycosylated is folded by the luminal binding protein (BiP)-Hsp70/DnaJ and Hsp90 chaperone machineries, which are favored by the highly oxidizing environment of the ER. Protein disulfide isomerases facilitate formation of disulfide bonds, which confer structural stability to the protein. Alternatively, polypeptides are glycosylated at ER entry through N-linked glycosylation, followed by disulfide bond formation. N-linked glycosylation provides the sugar molecules that form the key ligand for the lectin-like chaperones calreticulin and calnexin. An excess of misfolded proteins in the ER leads to enhanced expression of a number of the components of the ER protein folding machinery, in a process known as the unfolded protein response (UPR). Two pathways are involved in eliciting this response; one is dependent on release of bZIP28/bZIP17 from the ER membrane and the other on alternative splicing of bZIP60 by IRE1 (Deng et al., 2011; Srivastava et al., 2013). However, bZIP60 is able to heterodimerize with bZIP28 and bZIP17, indicating that the two arms of the UPR signaling pathways merge. Several of the UPR genes are induced upon heat and seedlings of a bZIP28 knockout mutant were shown to be sensitive to high temperatures, suggesting an essential role of the UPR in general heat stress response and thermotolerance (Fragkostefanakis et al., 2016a). The main components of the ER protein folding machinery, such as calreticulin, calnexin, and BiP, are present throughout microspore and pollen development (Honys and Twell, 2004; Sheoran et al., 2006; Chaturvedi et al., 2013). Moreover, the UPR is essential for regular pollen development, given that several mutants in the pathway have male gametophyte defective or lethal phenotypes (Fragkostefanakis et al., 2016a). During or shortly after a heat shock or long-term exposure to mild heat, the UPR is up-regulated in male reproductive tissues at transcript and protein level (Frank et al., 2009; Bita et al., 2011; Chaturvedi et al., 2015; Li et al., 2015; Fragkostefanakis et al., 2016b), suggesting the UPR has a function in acclimation of developing pollen to heat. Experimental support for this hypothesis was recently delivered, through the analysis of an ire1a ire1b double knockout mutant, which inactivates the RNA-splicing arm of the UPR signaling pathway. The mutant was found to be fertile at room temperature, but male sterile at slightly elevated temperatures, showing reduced viability of mature pollen and altered pollen coat composition (Deng et al., 2016). Interestingly, conditional male sterility in the mutant was a sporophytic and the double mutant was at elevated temperature, in the structure of the is that increased protein folding capacity of the ER is essential for functioning at high temperature, given the high of the of the by high temperature is to accumulation of species are during from different including and the (Mittler et al., 2004). In to being toxic metabolic also as signaling molecules stress responses et al., 2014). and machinery, which of such as and as well as like and (Mittler et al., 2004). of the genes are to levels, which results in a balance between and under Exposure to high temperature can disturb this and a number of studies in vegetative tissues and cell types that heat leads to accumulation of in a stress. To with the excess of upon heat, the expression of and levels of are up-regulated by heat et al., 2015). The fact that increased vegetative thermotolerance in different plant species that this response is adaptive et al., and et al., 2013). play an important role in the formation of viable The cell of cells during microspore development involves levels in anthers were shown to peak during tapetum degeneration and at pollen in and et al., 2011; et al., 2014; et al., 2016). a study and in developing pollen et al., 2013). Pollen and tapetum cells have also been shown to accumulate of and high of and and 2014). high temperatures, this be to a increase in long-term mild heat has been shown to increase levels in pollen et al., 2014). In heat induced the expression of several genes in et al., 2012; Zhang et al., and and genes were in developing tomato anthers and pollen (Frank et al., 2009; Bita et al., 2011; Fragkostefanakis et al., 2016b). The of and genes in response to heat is by increased levels of the proteins et al., 2015). the accumulation of in anthers and pollen upon heat, genes are also to heat in a due to the of heat shock in their et al., 2015). is direct evidence that enhanced pollen development under high temperature However, at the microspore stage in to and pollen abortion et al., 2011; et al., 2013). and mutants with reduced of to activate the timely of tapetum cell leading to pollen failure et al., 2014; et al., 2016). the regulation of is essential for the of viable it that increased to heat support for this hypothesis is by in and which increased pollen viability upon heat after of et al., 2014; et al., 2016). studies have shown that high temperature a of and changes in developing anthers and many of which are not with the heat acclimation responses described Abiko et al., 2005; Oshino et al., 2007; et al., 2009; et al., 2009; Bita et al., 2011; Zhang et al., 2012; et al., 2014; Zhang et al., 2014; Li et al., 2015; Fragkostefanakis et al., Jagadish et al., 2010; Chaturvedi et al., 2015; Li et al., 2015; Fragkostefanakis et al., 2016b). of these changes may be of heat injury and play role in it that have adaptive differentiation of the two example, to and signaling accumulate in developing tomato pollen and in after a short heat (Frank et al., 2009; Bita et al., 2011; Zhang et al., 2012; Zhang et al., 2014). 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Fragkostefanakis et found that most heat-responsive genes in anthers the HSF targeted heat shock in their A number of these responses were shown to on HSFA2 and the same was for such as accumulation of the which is to have a role under stress and a number of responses may to heat their are as discussed in the the acclimation pollen development is at temperatures at which most other plant tissues and processes In developing pollen and high temperature but the is more sensitive to heat and Peet et al., 1998; Oshino et al., The of that lead to pollen failure under high temperature to be and the that are have not been in high temperature affect developing microspores, the sporophytic or The of under stress conditions has been et al., 2010). 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At of pollen development, HSFs and HSPs are induced by heat, but to a in vegetative tissue (Frova et al., et al., et al., 2006; Fragkostefanakis et al., 2016b), which may to higher heat stress sensitivity of developing has been that microspores for nutrients in the anther especially under suboptimal growth conditions (Carrizo García et al., 2017). While in vegetative plant HSPs accumulate to become among the most abundant proteins upon high-temperature exposure (Vierling, prevent microspores from in Furthermore, the relatively high of in developing pollen and cells produce levels of species in response to heat, as in pollen et al., 2014). 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