Plants play an essential role in the maintenance of today’s atmosphere through the production of oxygen and simultaneous consumption of carbon dioxide (Igamberdiev and Lea, 2006). Further, flowering plants are essential for the food supply of the growing world population and therefore of great interest (Hill and Li, 2016). In general, flowering plants undergo a repetitive transition between a haploid gametophyte and a diploid sporophyte generation during their life cycle (Borg et al., 2009).
Main function of the gametophyte generation is the development of female and male haploid gametophytes, which give rise to a diploid zygote after successful fertilization, marking the beginning of a new sporophyte generation (Yadegari and Drews, 2004). The development of the male gametophyte, better known as pollen, takes place in the anthers of stamina, which are specialized reproductive organs of the sporophyte (Scott et al., 2004; Yadegari and Drews, 2004). Pollen provides a model system for the analysis of cell growth and differentiation (Becker et al., 2003; Honys and Twell, 2004) and was focus of multiple studies in a variety of plant species, such as A. thaliana (Becker et al., 2003; Honys and Twell, 2003; Honys and Twell, 2004) and Solanum lycopersicum (hereinafter referred to as tomato; Chaturvedi et al., 2013; Giorno et al., 2013; Keller et al., 2017).
The development of pollen can be separated in two consecutive phases, namely microsporogenesis and microgametogenesis (Figure 2). During microsporogenesis, the diploid pollen mother cell, also known as microsporocyte, undergoes meiotic division to give rise to a tetrad of four haploid microspores. The phase is completed when the post-meiotic microspores are released from the tetrads (Borg et al., 2009). The release of the microspores is achieved by the activity of enzymes
secreted by the tapetum, which is the inner layer of the stamina and in addition responsible for the nutrient supply of the developing pollen (Scott et al., 2004). During microgametogenesis, the post-meiotic microspores enlarge and produce a large vacuole, which leads to the migration of the nucleus towards the cell wall. A final asymmetric cell division, better known as pollen mitosis I, leads to the characteristic cell-within-a-cell structure of the pollen grain with a generative cell that is embedded in the cytoplasm of a larger vegetative cell (Yamamoto et al., 2003).
Figure 2: Development of pollen in tomato
During microsporogenesis, the diploid pollen mother cell undergoes meiotic division, which produces a tetrad of four haploid microspores. After release, the post-meiotic microspores enlarge and undergo an asymmetric mitotic division, which leads to a cell-within-a-cell structure. The mature pollen is bicellular and composed of a generative cell engulfed within the cytoplasm of a larger vegetative cell.
The engulfed generative cell, which is representing the male germline, and the surrounding vegetative cell have distinct fates (Borg et al., 2009). The vegetative cell is responsible for the nutrient supply of the generative cell and gives rise to the pollen tube after successful pollination. In contrast, the generative cell gives rise to a pair of sperm cells by a second mitotic division, also known as pollen mitosis II (Giorno et al., 2013; Rutley and Twell, 2015). In plant species with mature bicellular pollen grains (e.g. tomato) pollen mitosis II occurs during pollen tube growth, whereas in species with mature tricellular pollen grains (e.g. A. thaliana) it already occurs before the end of maturation (Giorno et al., 2013). After maturation, pollen grains dehydrate, followed by the opening of the anthers, which allows the dispersal of the pollen grains in the environment. Upon adhesion to the stigma, pollen grains rehydrate, start germination and produce the pollen tube. The pollen tube then intrudes into the stigma and grows towards the ovary, where it comes to a double fertilization of the female gametophyte with the two sperm cells (Firon et al., 2012).
1.2.1 Transcriptome and proteome dynamics during pollen development
The development of pollen is accompanied by dynamic changes in the composition and quantity of mRNAs and proteins. Most of the pollen studies published so far are based on mature pollen, as it is one of the most accessible stages (Ischebeck et al., 2014). Initial large-scale transcriptomic studies were performed in 2003 by Honys and Twell (2003) and Becker et al. (2003). In both studies, the authors used Arabidopsis GeneChip arrays, which allowed the detection of up to 8,000 of the 27,000
annotated protein-encoding genes. In total, the authors were able to identify 992 (Honys and Twell, 2003) and 1,584 (Becker et al., 2003) genes as expressed in mature pollen. Based on their findings, Honys and Twell (2003) estimated the number of expressed genes in mature A. thaliana pollen to be higher than 3,500. Subsequent functional classifications of genes exclusively expressed in pollen revealed an enrichment of signal transduction, cell wall metabolism, metabolic processes and cytoskeleton in both studies. Only one year later Honys and Twell (2004) expanded their analyses to four pollen developmental stages, namely microspores, bicellular pollen, immature tricellular pollen and mature pollen grains. Further, they utilized with the ATH1 GeneChip an array that carried probe sets for around 22,500 annotated genes, which increased the amount of detectable genes by a factor of about three. The transcriptome diversity of the developmental stages revealed a decrease from earlier to later developmental stages with 11,565 detected genes in microspores and only 7,235 genes in mature pollen grains. A similar decrease in transcriptome diversity was observed in rice, where the number of expressed genes decreased from 14,590 in microspores to only 5,945 in mature pollen grains (Wei et al., 2010). Today, the estimated number of expressed genes in mature A. thaliana pollen ranges from 3,945 to 7,235 across different studies with an average of 6,044 expressed genes (Rutley and Twell, 2015).
In contrast to the relatively well-characterized transcriptome of developing pollen, information about the pollen proteome is much more limited. Early proteomic pollen studies were based on 2-DE and rather limited in their output with only 110 to 135 detected proteins (Holmes-Davis et al., 2005; Noir et al., 2005; Sheoran et al., 2006). The first large-scale analysis in A. thaliana revealed the presence of at least 3,465 proteins in mature pollen grains (Grobei et al., 2009). The authors could further show an overrepresentation of proteins related to metabolism, energy, protein fate, protein synthesis, cellular transport and development.
Changes of the proteome along the course of pollen development was so far analyzed in tomato and tobacco based on five and eight developmental stages, respectively (Chaturvedi et al., 2013;
Ischebeck et al., 2014). In total, in developing tomato and tobacco pollen 1,821 and 3,817 proteins, respectively, could be identified. The authors of both studies could further show that early developmental stages tend to accumulate proteins related to heat stress (HS), such as heat shock proteins (Hsps). In contrast, late developmental stages accumulate proteins required for germination and pollen tube growth, like those of cell wall and lipid metabolism as well as vesicle trafficking and the tricarboxylic acid (TCA) cycle.
When comparing the transcriptome and proteome of pollen, it turns out that the overrepresentation of processes like cell wall metabolism and signal transduction in the transcriptome of mature pollen (Becker et al., 2003; Honys and Twell, 2003) is also apparent in the proteome (Dai et al., 2006; Grobei et al., 2009; Chaturvedi et al., 2013). However, other processes like carbon and energy metabolism, which are overrepresented in the proteome of mature pollen (Dai et al., 2006) showed no overrepresentation in one of the transcriptomic studies. Further, a comparison of tobacco mRNA and A. thaliana protein levels of a phosphoglycerate kinase and a pyruvate decarboxylase revealed different abundance patterns along the course of pollen development. Both enzymes had most abundant protein levels in dehydrated pollen grains, whereas transcript levels peaked in earlier stages and were strongly diminished in mature pollen (Ischebeck et al., 2014).