Following microsporogenesis, the anther locules contain free microspores.

These become heavily

vacuolized, pushing the nucleus to the edge of the cell. The microspores then undergo a stereotyped set
of cell divisions (one asymmetric, one symmetric), to produce the three nuclei of the microgametophyte.
The first mitosis is asymmetric because the nucleus is at the periphery of the cell. The large daughter cell
is the vegetative cell, and contains most of the cytoplasm from the microspore. The smaller cell, the
generative cell, is entirely engulfed by the vegetative cell. It then undergoes a further,symmetric division
to produce two sperm cells. This final division occurs within the developing pollen grain in some species
(those with trinucleate pollen), but in other species (those with binucleate pollen) it occurs during pollen
tube growth after pollination (Ma 2005). The developing pollen grain is coated with two protective
layers, the exine and the intine. The exine is made of highly elaborate sporopollenin, which mediates
interactions with stigmatic cells, while the intine is made from cellulose, pectin, and proteins. Both layers
appear to be mainly derived from the anther tapetum. The mature pollen grain is then dehydrated ready
for release into the outside world. As with megagametogenesis, microgametogenesis is regulated by
genes expressed by the gametophyte nuclei themselves. However, because the tapetum contributes to
pollen coat development, there is also a clear role for sporophytically expressed genes in
microgametogenesis. For example, the genes described earlier which are necessary to correctly specify
tapetal and microspore fate also influence gametophyte development. Those microspores which survive
in mutants defective in these genes fail to undergo correct gametogenesis due to the abnormal tapetal
layer surrounding them. Similarly, the sporophyte expresses many genes involved in building the pollen
coat. Plants carrying mutations in these genes are usually male sterile, as a result of perturbed exine and
intine development. For example, the male sterile 2 mutant of Arabidopsis carries a lesion in a gene
encoding a protein that promotes the synthesis of long-chain molecules, such as sporopollenin (cross-
linked carotenoids). The mutant produces non-viable pollen grains with thin cell walls lacking an exine
(Aarts et al. 1997). A number of such mutants are reviewed by Ma (2005). Male gametophytic mutants
influence segregation ratios in the progeny of plants, usually resulting in abortion of 50% of pollen
grains. However, unlike the situation with female gametophytic mutants, where this results in 50%
reduction in seed set, the vast excess of pollen relative to egg cells means that male gametophytic
mutants can be difficult to identify using conventional screens. Instead, perturbations in the segregation
ratios of other markers are used to pick out male gametophyte mutations. Since many thousands of
genes are likely to be expressed in the male gametophyte, it is not surprising that many mutants have
been described. Indeed, recent microarray analysis of genes expressed in the pollen grain has revealed
that 992 genes of the 7792 on the array were expressed, suggesting a likely total of around 4000 pollen-
expressed genes when the whole genome is considered. The expressed genes included a high proportion
of essential genes with roles in signal transduction and cell wall biogenesis (Honys and Twell 2003). Many
of the mutants that have been described so far underline the importance of the asymmetry of the first
mitotic division. For example, the gemini pollen 1 mutant produces pollen grains with apparently
randomly orientated first divisions, which then arrest after that first division (Park et al. 1998). The cells
produced by this first division express vegetative cell markers, indicating that the vegetative cell is the
default state in the absence of asymmetry. The GEM1 protein binds to microtubules and appears to be
involved in microtubule positioning, apparently essential for the appropriate asymmetry of the first
mitotic division (Twell et al. 2002). Other mutants are affected in the number of mitotic divisions that
occur, the positioning of the vegetative and generative cells with respect to one another, and the
positioning of the different cells within the pollen grain itself. It is clear that many genes are involved in
microgametogenesis, and it should therefore be noted that the reduced size and duration of the male
gametophyte stage of the angiosperm life cycle is still quite sufficient to ensure that few lethal mutations
are retained within the genome.