PLS 622, Plant Physiology I, Wednesday, October 18, 2006

Reproductive development:

Objectives for this lecture are to learn and understand:

- The stages of pollen development in the anther.

- The progressive alteration of anther tissues leading to dehiscence.

- The four main facets of anther dehiscence.

- Aspects of pollen adherence and rehydration.

- Pollen tube growth and guidance.

The male gametes are produced in the anthers, usually found, in almost all angiosperms, at the end of the filament. The anthers (collectively known as the androecium) are comprised of microsporangia (pollen sacs). The anthers and the filaments together are referred to as the stamens.

In arabidopsis, there are six stamens, two short and four long, arising from the floral meristem after the formation of the floral buttress and sepal primordia. These stamen primordia invaginate towards the base and effectively define the lower portion that will become the filament from the upper portion that will become the anther. The filament next elongates at the same rate as the gynoecium so that upon attaining maturity, the anthers are positioned to dust the stigma with prolific amounts of pollen.

Pollen development, pollination, and fertilization:

There are three main phases of pollen development. The first is the development of the sporophytic cells and meiosis; the second, the formation of free microspores; the third, microspore mitosis up to and including the formation of the two generative cells and one vegetative cell.

Stage I and II:

The sporophytic cells responsible for the generation of the male gametes divides to produce one tapetal initial and one sporogenous initial (pollen mother cell). Subsequent sporogenous cell meioses produce a pollen tetrad of haploid cells surrounded by a cell wall comprised of callose. This callose cell wall is degraded by enzymes released from the tapetal layer, primarily callase that is necessary but not sufficient to release the individual cells of the tetrad from each other to generate free microspores. At least two other enzymes are required for tetrad release as evinced by the phenotype of the quartet mutants qrt1-1, qrt2-1, and qrt3-1. These mutants produce normal amounts of callase and yet do not separate. Recent evidence suggests that the quartet mutants may be/are deficient in pectin degradation. Biochemical evidence suggests that the qrt1-1 mutant is deficient in a pectin methylesterase while the qrt2-1 is deficient in a polygalacturonase because the pectin cementing the tetrad together is not degraded as it is in wild type pollen of Arabidopsis. Neither QRT1 nor QRT2 are cloned to date but QRT3 has been identified and it is indeed a gene that encodes a protein capable of pectin degradation, a polygalacturonase.

While the pollen tetrad is still surrounded by the callose wall, each pollen grain is surrounded by two layers, an inner intine and an outer exine. The intine is comprised primarily of pectin and cellulose while the exine is comprised of a complex, very resilient material, sporopollenin. The exine is laid down in a species specific pattern in an as yet undetermined fashion by the sporophyte. Pores develop on the maturing pollen grain where the exine is reduced or absent leaving only the intine. It is through the pores that the pollen tube germinates. Upon the generation of free microspores the rate of deposition of the exine increases and the circumference of the microspores increases.

Stage III:

The uninucleate microspores undergo asymmetric mitotic division, producing a large vegetative cell and a smaller generative cell enclosed within the vegetative cell, a so-called bicellular (previously named binucleate) pollen grain. This division can be considered a determinative division in that the two cells thus produced undergo very different fates. If the asymmetry of the mitotic division to produce bicellular pollen is altered, the microspores do not develop as gametophytes but rather as sporophytes that form a haploid callus and/or haploid plants. In most plants, the bicellular pollen grain is released upon dehiscence and the second mitotic division, to produce two generative cells, occurs after pollen germination as the pollen tube grows through the style. In some plants however, a third cell, formed from a second mitotic division of the generative cell, is produced forming a tricellular (formerly trinucleate) pollen grain prior to anther dehiscence. Typically, these tricellular pollens are very short lived.

Pollination:

Upon attaining maturity, the anther bursts apart (dehisces), along a thin region of the anther where the inner endothecium cells fail to develop leaving only the epidermis. A specialized cell group referred to as the stomium (portion of the anther where the endothecium does not develop, leaving the epidermal cells only) occupies this position and it is their programmed destruction that permits normal dehiscence, exposing the mature pollen grains. There are four main facets to anther dehiscence. These are; 1) the appearance of thickened regions of fibrous bands of unknown constituents in the endothecial cell wall; 2) destruction of the circular cell cluster which permits the joining of the theca in adjoining anthers; 3) destruction of the connective and tapetum; and 4) the destruction of the stomium allowing anther dehiscence and the release of the mature pollen grains. This series of events parallels pollen maturation events commencing at the time of tetrad formation but it is not dependent on signals from pollen to progress since male-sterile plants follow the same dehiscence schedule as normal plants. Depending on the species, the pollen is either displayed for attachment to visiting insects and/or birds and/or mammals or released for wind dispersal.

Dehiscence of the anther is the first stage leading to pollination and is under genetic control. Numerous mutants have been isolated that do not permit the release of otherwise viable pollen from the anthers usually due to a defective stomium. These include the msH mutant in arabidopsis, and ps mutants in tomato.

Pollen germination:

Upon landing on a compatible stigma, the pollen must often re-hydrate, the pollen tube protrude through one of the pores on the pollen exine, extend through the stylar tissue, locate the ovary and deliver its two generative nuclei to the ovule completing double fertilization in the angiosperms. In most plants all of the proteins necessary for the pollen grain to complete germination are already present in the mature pollen grain when it lands on a stigma. Hence, pollen germination in most plant species can occur without protein synthesis, although there are exceptions. Additionally, although pollen grains can be germinated in vitro, this occurs only by mimicking the chemical environment of the female stigma. Hence, as one plant developmental geneticist recently put it, the development of the male gametophyte from pollination to fertilization is dependent on/controlled by the female reproductive system.

Adhesion of the pollen grain to the stigma is a controlled event. Pollen grains from non-crucifer species do not adhere as tightly to the stigmatic surface of Brassica as do grains from species of the Cruciferae.

The very act of rehydration is one that is tightly controlled by the maternal plant tissue. It has been demonstrated that aquaporins, water-conducting channels, in conjunction with a receptor protein kinase are activated when compatible pollen lands on a stigma, thereby ensuring that the pollen grain has sufficient water to re-hydrate and complete germination. Pollen grains from non-compatible plants are not capable of inducing the aquaporins and therefore remain dehydrated and do not complete germination. This system, which operates in the crucifer family, will be discussed in more detail in the next lecture on self-incompatibility.

Pollen may need exogenous, diffusible signal molecules, supplied by the stigma, to complete germination. One of the most convincing arguments supporting this contention is that mutants in pollen function that do not complete germination in vitro, can be induced to do so by the addition of wild type pollen, which presumably supply a signal molecule necessary for pollen germination. In other mutants deficient in tryphine, a lipid and protein extracellular coat covering the sporopollenin exine, the stigma cells in contact with the mutant pollen are induced to form callose plugs, inhibiting pollen germination.

Tip growth:

Upon completing germination, the pollen tube elongates through the style toward the ovary using a unique form of cell elongation (shared by root hairs only) whereby the components for the tube cell wall are deposited at the end of the growing tip and which get incorporated and modified as the tip grows beyond them. This mode of elongation is called tip growth and is in marked contrast to the usual diffuse elongation found in almost all other plant cells. In tip growth, the cell wall components required for wall synthesis to permit continued elongation are deposited at the very tip of the pollen tube by fusion of dictyosome vesicles with the plasmamembrane. The wall components subsequently undergo modification such as desterification of pectin as new waves of wall material deposition displace the former tip wall back along the tube. Although there is but a single wall at the tip, further back along the tube there are at least two distinct layers of cell wall.

As the pollen tube extends into the transmitting style, the cytoplasmic contents become increasingly diffuse. To maintain appropriate cytoplasmic concentration, the pollen tube is plugged at intervals with a callose plug. It is important to note that this is not cytokinesis. Presumably the callose plug allows the limited cytoplasm to continue to exert positive pressure for tube elongation within the every increasing length of the tube.

Pollen tubes are required to make many sharp turns as they grow down the transmitting tract toward the ovules. Especially when they are approaching the micropyle, the route out of the transmitting tract, along the funiculus, into the micropyle is tortuous. Add to this the fact that many tubes are physically bound to the transmitting tract epidermal cells (see below) and incapable of altering their position once so bound, and it is easy to see the advantages tip growth has to offer in getting the tube to the micropyle (see figure on page 9). Should a change of direction be required, the Golgi apparati realign to commence deposition of wall material on the face of the tube perpendicular to the preferred direction of growth.

Pollen tube guidance systems:

The nature of the chemical thought to provide guidance to the pollen tube has recently been the topic of much investigation. In tobacco, a transmitting tract-specific glycoprotein (TTS protein, an arabinogalactan protein) was discovered and has been proven to function at least for pollen adhesion. In lily, a species with a hollow transmitting tract, two other molecules have been discovered that are produced by the stigma and style and assist in binding the pollen tube to the transmitting tract epidermis. These molecules have been identified as a small cysteine-rich protein similar to lipid transfer proteins and now called the Stigma/Style Cysteine-Rich Adhesin (SCA). It has a remarkable ability to bind to pectin and the second molecule produced by the transmitting tract epidermis was a pectin. Not only can SCA bind to the stylar pectin, it can also, simultaneously bind to the pectin present on the pollen tube wall, thereby tightly binding the tube to the transmitting tract epidermis. In one scenario, a pollen produced arabinogalactan protein (AGP) similar to the TTS protein discovered in tobacco (see above), is released by the pollen tube and aids the retention of pollen tube pectin in the tube wall (see handout figure).

A very elegant guidance system has been elucidated by Daphne Preuss’s Lab. They found that the small amino acid gamma amino butyric acid (GABA) is produced in large quantities throughout the stigma, style, and the integument cells at the micropylar end of the ovules. There is an enzyme (GABA transaminase) that is also produced in increasing amounts by these same cells the further away from the ovule one proceeds. Hence, the integument cells produce a lot of GABA but little GABA transaminase, so GABA concentration remains high, while the stigma produces a lot of GABA but also a lot of GABA transaminase which degrades GABA to Succinic semialdehyde and GABA concentrations are low. Thus, a concentration gradient of GABA is set up that has the most GABA present in the integuments of the micropyle and the least present in the stigma. Not only that, but the pollen tube itself synthesizes the same GABA transaminase. Hence, the GABA that enters the pollen tube as a signal is rapidly degraded, keeping the pollen tube responsive to GABA because GABA does not build up in the tube and overwhelm the sensory mechanism responsive to it. The loss of the GABA transaminase due to mutations in the gene encoding this protein result in decreased fertility. These so called POllen-Pistal Incompatible (pop) mutants produce pollen that are inhibited in tube growth through pop styles (due to excessively high GABA concentrations) and which cannot ‘find’ the micropyle again due to overwhelming GABA concentrations at the ovule and the tubes inability to degrade this GABA.

There are two basic forms of style in angiosperms. One is a solid stigma and style (such as in arabidopsis) the other is a hollow stigma and style (as in the lily). In the former case, the pollen tubes must grow through the transmitting stylar tissue, through the ovary and find their way to the ovules and through the micropyle. In the latter case, although the tube need not force its way though tissue it grows along the inside wall of the style and is guided by molecules comprising this wall. In either case, directional growth is thought to occur by a combination of chemotropism and pistil structure. Flavanoids, responsible for imparting the yellow color to pollen, are one of the chemicals necessary for normal pollen growth. Mutants deficient in chalcone synthase CHS are normal in all aspects except that they are white in color and are not capable of fertilization. In maize, white pollen is produced by plants carrying two recessive mutations c2 and whp, which both code for chalcone synthase. Additionally, the flavonids necessary for proper pollen function can be supplied by either the stigma or the pollen grain itself. It appears that the flavanol necessary for pollen germination is kaempferol.

Guidance into the ovule:

There appears to be considerable evidence that the guidance of the pollen tube to the micropyle of the embryo sac is based on a chemical signal (GABA is one)! This chemical signal is sufficient to attract most pollen tubes to the micropyle from as far away as 75 mm. Additionally, experiments with arabidopsis have demonstrated that usually only one pollen tube visits each ovule. Hence the nature of the chemical signal from the ovules is complex in that is must be sufficient to attract a pollen tube to achieve fertilization (the raison d’etre for this whole elaborate mechanism) while being ephemeral so that, once utilized by a pollen tube it quickly dissipates so that no more pollen tubes are attracted to the now fertilized zygote. This fits well with what we know of GABA. Once a pollen tube has traversed through an area, it has absorbed and degraded much of the GABA that had been in the transmitting tract or coming from the micropyle. This lower GABA amount will not attract additional pollen tubes which are following greater GABA concentrations elsewhere in the transmitting tract.