Differentiation of gametes is a crucial step in the life cycle of all organisms with sexual reproduction. In animals, gametes are formed directly after meiosis. In plants, the haploid cells produced by meiosis undergo mitotic divisions to generate specialized structures in which the gametes are specified. These structures are called gametophytes. The male gametophyte is the pollen grain, and the female gametophyte is the embryo sac, or megagametophyte. Even though in most flowering plants the embryo sac contains only seven cells of four distinct cell types, megagametogenesis involves complex, tightly orchestrated developmental mechanisms.
Embryo sac development starts with the differentiation of the megaspore mother cell (MMC) in the developing wild-type ovule. The MMC undergoes meiosis to generate four spores, three of which undergo programmed cell death. The functional megaspore then undergoes three sequential mitotic divisions to generate an embryo sac containing eight nuclei. Subsequent nuclei migration, cellularization, and nuclei fusion result in the formation of the seven cells constituting the megagametophyte. The mature embryo sac contains two gametes: the egg cell that will form the embryo upon fertilization and the central cell that after fertilization will give rise to the endosperm, a nursing tissue. The embryo sac also has accessory cells, the synergids, located at each side of the egg cell, that attract the pollen tube toward the micropyle of the ovule, and three cells of undetermined function called the antipodals that undergo cell death before fertilization.
In our lab, we are interested in studying the molecular mechanisms underlying cell specification during female gametogenesis, as well as the signaling events that direct fertilization and early embryogenesis. Our model system is Arabidopsis thaliana, a small flowering plant related to the mustard plant that is very convenient for molecular and genetic approaches. We first became intrigued about a high number of gametophytic mutants with insertions that disrupt genes related to reactive oxygen species (ROS) detoxification identified through genomic approaches. When we studied ROS production along embryo sac development, we found that very localized and transient oxidative bursts accompany cell death events during gametogenesis. To know more about how ROS homeostasis is regulated during female gametogenesis, we are looking at different mutant lines with insertions in genes that might be related to ROS control during gametogenesis. Using genetic, biochemical, and molecular tools, we aim to understand the function of ROS, not only as cell death triggers but also as signaling molecules regulating different aspects of embryo sac development.
A second line of research in our lab involves the study of the signals implicated in the fertilization process. After a pollen grain germinates on the surface of the stigma, the pollen tube grows through the transmitting track between the walls of the pistil toward the ovules. The pollen tube follows a funiculus (the structure that supports and connects the ovules with the placental tissue) to reach the micropyle of an ovule, an opening that it is used by the pollen tube to reach the embryo sac. At the micropylar end of the embryo sac, the tip of the pollen tube bursts to release two sperm cells inside a synergid cell. One of the sperm cells will fertilize the egg cell, forming the zygote, and the remaining sperm cell fertilizes the central cell to originate the endosperm. Thus, at the final stage of the sperm cell's journey toward the female gametes, the ability of the embryo sac to attract a pollen tube to the micropyle becomes crucial.
We are interested in understanding how pollen tubes are guided to the embryo sacs. Typically, only one pollen tube reaches the micropylar end of an embryo sac, suggesting that after a pollen tube reaches a gametophyte, the ovule or the embryo sac releases repellent substances that prevent other pollen tubes to reach the same ovule. In addition, pollen tube growth stops at the micropyle of the embryo sac, where it ruptures. Using genetic tools, we identified several mutants with defects in pollen tube attraction. Specifically, pollen tubes invade the embryo sacs as if they are not able to "see" the synergid cells inside the gametophytes. Furthermore, we have mutants in which the ovules have more than one pollen tube at the micropyle, suggesting that the repulsion signal is impaired. We are currently characterizing those mutants at the genetic, molecular, and biochemical levels with the purpose of identifying the guidance signals generated by the embryo sac that are responsible for pollen tube attraction and repulsion.
We are discovering not only new signaling molecules involved in these events but also regulatory mechanisms that mediate fertilization and early embryogenesis. Because almost all major agricultural crops rely on this system for seed production, it is critical to understand the developmental biology of plant fertility and reproduction.
This research is also supported by a grant from the National Agency for the Promotion of Science and Technology (ANPCyT) and a grant from the National University of Mar del Plata, Argentina.
As of January 17, 2012