Developmental biology

Gamete Production in Angiosperms

Like those of mosses and ferns, angiosperm gametes are produced by the gametophyte

generation. Angiosperm gametophytes are associated with flowers. The gametes they produce

join to form the sporophyte. The study of embryonic development in plants is therefore the study

of early sporophyte development. In angiosperms, the sporophyte is what is commonly seen as

the plant body. The shoot meristem of the sporophyte produces a series of vegetative structures.

At a certain point in development, internal and external signals trigger a shift from vegetative to

reproductive (flower-producing) development (see reviews by McDaniel et al. 1992 and Levy and

Dean 1998). Once the meristem becomes floral, it initiates the development of floral parts

sequentially in whorls of organs modified from leaves . The first and second whorls

become sepals and petals, respectively; these organs are sterile. The pollen-producing stamens

are initiated in the third whorl of the flower. The carpel in the fourth whorl contains the female

gametophyte. The stamens contain four groups of cells, called the microsporangia (pollen sacs),

within an anther. The microsporangia undergo meiosis to produce microspores. Unlike most

ferns, angiosperms are heterosporous, so the prefix micro is used to identify the spores that

mitotically yield the male gametophytes pollen grains. The inner wall of the pollen sac, the

tapetum, provides nourishment for the developing pollen.

Pollen

The pollen grain is an extremely simple multicellular structure. The outer wall of the pollen grain,

the exine, is composed of resistant material provided by both the tapetum (sporophyte generation)

and the microspore (gametophyte generation). The inner wall, the intine, is produced by the

microspore. A mature pollen grain consists of two cells, one within the other  The tube cell contains a generative cell within it. The generative cell divides to produce two sperm.

The tube cell nucleus guides pollen germination and the growth of the pollen tube after the pollen

lands on the stigma of a female gametophyte. One of the two sperm will fuse with the egg cell to

produce the next sporophyte generation. The second sperm will participate in the formation of the

endosperm, a structure that provides nourishment for the embryo.

The ovary

The fourth whorl of organs within the flower forms the carpel, which gives rise to the female

gametophyte  The carpel consists of the stigma (where the pollen lands), the style,

and the ovary. Following fertilization, the ovary wall will develop into the fruit. This unique

angiosperm structure provides further protection for the developing embryo and also enhances

seed dispersal by frugivores (fruit-eating animals). Within the ovary are one or more ovules

attached by a placenta to the ovary wall. Fully developed ovules are called seeds. The ovule has

one or two outer layers of cells called the integuments. These enclose the megasporangium,

which contains sporophyte cells that undergo meiosis to produce megaspores 

There is a small opening in the integuments, called the micropyle, through which the pollen tube

will grow. The integuments an innovation first appearing in the gymnosperms develop into

the seed coat, which protects the embryo by providing a waterproof physical barrier. When the

mature embryo disperses from the parent plant, diploid sporophyte tissue accompanies the

embryo in the form of the seed coat and the fruit.

Within the ovule, meiosis and unequal cytokinesis yield four megaspores. The largest of these

megaspores undergoes three mitotic divisions to produce a seven-celled embryo sac with eight

nuclei. One of these cells is the egg. The two synergid cells surrounding the egg

may be evolutionary remnants of the archegonium (the female sex organ seen in mosses and

ferns). The central cell contains two or more polar nuclei, which will fuse with the second sperm

nucleus and develop into the polyploid endosperm. Three antipodal cells form at the opposite

end of the embryo sac from the synergids and degenerate before or during embryonic

development. There is no known function for the antipodals. Genetic analyses of female

gametophyte development in maize and Arabidopsis* are providing insight into the regulation of

the specific steps in this process (Drews et al. 1998).

*A small weed in the mustard family, Arabidopsis is used as a model system because of its very

small genome.

Pollination

Pollination refers to the landing and subsequent germination of the pollen on the stigma. Hence it

involves an interaction between the gametophytic generation of the male (the pollen) and the

sporophytic generation of the female (the stigmatic surface of the carpel). Pollination can occur

within a single flower (self-fertilization), or pollen can land on a different flower on the same or a

different plant. About 96% of flowering plant species produce male and female gametophytes on

the same plant. However, about 25% of these produce two different types of flowers on the same

plant, rather than perfect flowers containing both male and female gametophytes. Staminate

flowers lack carpels, while carpellate flowers lack stamens. Maize plants, for example, have

staminate (tassel) and carpellate (ear) flowers on the same plant. Such species are considered to

be monoecious (Greek mono, "one"; oecos, "house"). The remaining 4% of species (e.g.,

willows) produce staminate and carpellate flowers on separate plants . These species are

considered to be dioecious ("two houses"). Only a few plant species have true sex chromosomes.

The terms "male" and "female" are most correctly applied only to the gametophyte generation of

heterosporous plants, not to the sporophyte (Cruden and Lloyd 1995).

The arrival of a viable pollen grain on a receptive stigma does not guarantee fertilization.

Interspecific incompatibility refers to the failure of pollen from one species to germinate and/or

grow on the stigma of another species (for a review, see Taylor 1996). Intraspecific

incompatibility is incompatibility that occurs within a species. Self-incompatibility

incompatibility between the pollen and the stigmas of the same individual is an example of

intraspecific incompatibility. Self-incompatibility blocks fertilization between two genetically

similar gametes, increasing the probability of new gene combinations by promoting outcrossing

(pollination by a different individual of the same species). Groups of closely related plants can

contain a mix of self-compatible and self-incompatible species.

Several different self-incompatibility systems have evolved . Recognition of self

depends on the multiallelic self-incompatibility (S) locus (Nasrallah et al. 1994; Dodds et al.

1996; Gaude and McCormick 1999). Gametophytic self-incompatibility occurs when the S allele

of the pollen grain matches either of the S alleles of the stigma (remember that the stigma is part

of the diploid sporophyte generation, which has two S alleles). In this case, the pollen tube begins

developing, but stops before reaching the micropyle. Sporophytic self-incompatibility occurs

when one of the two S alleles of the pollen-producing sporophyte (not the gametophyte) matches

one of the S alleles of the stigma. Most likely, sporophyte contributions to the exine are

responsible.

The S locus consists of several physically linked genes that regulate recognition and rejection of

pollen. An S gene has been cloned that codes for an RNase called S RNase, which is sufficient, in

the gametophytically self-incompatible petunia pistil, to recognize and reject self-pollen (Lee et

al. 1994). The pollen component recognized is most likely a different gene in the S locus, but has

not yet been identified in either gametophytically or sporophytically self-incompatible plants. In

sporophytic self-incompatibility, a ligand on the pollen is thought to bind to a membrane-bound

kinase receptor in the stigma that starts a signaling process leading to pollen rejection. The

mechanism of pollen degradation is unclear, but appears to be highly specific.

If the pollen and the stigma are compatible, the pollen takes up water (hydrates) and the pollen

tube emerges. The pollen tube grows down the style of the carpel toward the micropyle (Figure

20.10). The tube nucleus and the sperm cells are kept at the growing tip by bands of callose (a

complex carbohydrate). It is possible that this may be an exception to the "plant cells do not

move" rule, as the generative cell(s) appear to move ahead via adhesive molecules (Lord et al.

1996). Pollen tube growth is quite slow in gymnosperms (up to a year), while in some

angiosperms the tube can grow as rapidly as 1 cm/hour.

Calcium has long been known to play an essential role in pollen tube growth (Brewbaker and

Kwack 1963). Calcium accumulates in the tip of the pollen tubes, where open calcium channels

are concentrated (Jaffe et al. 1975; Trewavas and Malho 1998). There is direct evidence that

pollen tube growth in the field poppy is regulated by a slow-moving calcium wave controlled by

the phosphoinositide signaling pathway (Figure 20.11; Franklin-Tong et al. 1996). Cytoskeletal

investigations show that organelle positioning during pollen tube growth depends on interactions

with cytoskeletal components. This must link to signaling, but the specifics are still unknown (Cai

and Cresti 1999).

Genetic approaches have been useful in investigating how the growing pollen tube is guided

toward unfertilized ovules. In Arabidopsis, the pollen tube appears to be guided by a longdistance

signal from the ovule (Hulskamp et al. 1995; Wilhelmi and Preuss 1999). Analysis of

pollen tube growth in ovule mutants of Arabidopsis indicates that the haploid embryo sac is

particularly important in the long-range guidance of pollen tube growth. Mutants with defective

sporophyte tissue in the ovule but a normal haploid embryo sac appear to stimulate normal pollen

tube development.

While the evidence points primarily to the role of the gametophyte generation in pollen tube

guidance, diploid cells may make some contribution. Two Arabidopsis genes, POP2 and POP3,

have been identified that specifically guide pollen tubes to the ovule with no other apparent effect

on the plant (Wilhelmi and Preuss 1996, 1999). These genes function in both the pollen and the

pistil, thus implicating the sporophyte generation in the guidance system

Fertilization

The growing pollen tube enters the embryo sac through the micropyle and grows through one of

the synergids. The two sperm cells are released, and a double fertilization event occurs (see

review by Southworth 1996). One sperm cell fuses with the egg, producing the zygote that will

develop into the sporophyte. The second sperm cell fuses with the bi- or multinucleate central

cell, giving rise to the endosperm, which nourishes the developing embryo. This second event is

not true fertilization in the sense of male and female gametes undergoing syngamy (fusion). That

is, it does not result in a zygote, but in nutritionally supportive tissue. (When you eat popcorn,

you are eating "popped" endosperm.) The other accessory cells in the embryo sac degenerate after

fertilization.

The zygote of the angiosperm produces only a single embryo; the zygote of the gymnosperm, on

the other hand, produces two or more embryos after cell division begins, by a process known as

cleavage embryogenesis. Double fertilization, first identified a century ago, is generally restricted

to the angiosperms, but it has also been found in the gymnosperms Ephedra and Gnetum,

although no endosperm forms. Friedman (1998) has suggested that endosperm may have evolved

from a second zygote "sacrificed" as a food supply in a gymnosperm with double fertilization.

Investigations of the most closely related extant relative of the basal angiosperm, Amborella,

should provide information on the evolutionary origin of the endosperm 

Fertilization is not an absolute prerequisite for angiosperm embryonic development (Mogie

1992). Embryos can form within embryo sacs from haploid eggs and from cells that did not

divide meiotically. This phenomenon is called apomixis (Greek, "without mixing"), and results in

viable seeds. The viability of the resulting haploid sporophytes indicates that ploidy alone does

not account for the morphological distinctions between the gametophyte and the sporophyte.

Embryos can also develop from cultured sporophytic tissue. These embryos develop with no

associated endosperm, and they lack a seed coat

Embryonic Development

Experimental studies

The angiosperm zygote is embedded within the ovule and ovary and thus is not readily accessible

for experimental manipulation. The following approaches, however, can yield information on the

formation of the plant embryo:

Histological studies of embryos at different stages show how carefully regulated cell

division results in the construction of an organism, even without the ability to move cells and

tissues to shape the embryo.

Culture experiments using embryos isolated from ovules and embryos developing de novo

from cultured sporophytic tissue provide information on the interactions between the embryo and

surrounding sporophytic and endosperm tissue.

In vitro fertilization experiments provide information on gamete interactions.

Biochemical analyses of embryos at different stages of development provides information

on such things as the stage-specific gene products necessary for patterning and establishing food

reserves.

Genetic and molecular analyses of developmental mutants characterized using the above

approaches have greatly enhanced our understanding of embryonic development.

Clonal analysis involves marking individual cells and following their fate in development

(see Poethig 1987 for details on the methodology). For example, seeds heterozygous for a

pigmentation gene may be irradiated so that a certain cell loses the ability to produce pigment. Its

descendants will form a colorless sector that can be identified and related to the overall body

pattern.

References

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