Manual Isolation of Living Cells from the Arabidopsis thaliana Female Gametophyte by Micromanipulation.

The few-celled female gametophyte, or embryo sac, of flowering plants is not easily accessible as it is buried within the sporophytic tissues of the ovule. Nevertheless, it has become an attractive model system to study the molecular mechanisms underlying patterning and cell type specification, as well as fertilization of the two female gametes, the egg and the central cell. While female gametes, zygotes, and early embryos can be manually isolated from the embryo sacs in maize, wheat, tobacco, and rice by micromanipulation, this approach had been considered impossible for the much smaller embryo sac of the model plant Arabidopsis thaliana. Here, we describe a method to isolate living cells from the Arabidopsis female gametophyte by micromanipulation. The manual isolation of egg cells, central cells, and synergid cells is a technique that enables a number of important studies such as cell-type-specific transcriptional profiling or the analysis of DNA methylation profiles. It also offers the possibility to use isolated female gametes for in vitro fertilization studies.

Brassinosteroids promote Arabidopsis pollen germination and growth.

Pollen tubes are among the fastest tip-growing plant cells and represent an excellent experimental system for studying the dynamics and spatiotemporal control of polarized cell growth. However, investigating pollen tube tip growth in the model plant Arabidopsis remains difficult because in vitro pollen germination and pollen tube growth rates are highly variable and largely different from those observed in pistils, most likely due to growth-promoting properties of the female reproductive tract. We found that in vitro grown Arabidopsis pollen respond to brassinosteroid (BR) in a dose-dependent manner. Pollen germination and pollen tube growth increased nine- and fivefold, respectively, when media were supplemented with 10 µM epibrassinolide (epiBL), resulting in growth kinetics more similar to growth in vivo. Expression analyses show that the promoter of one of the key enzymes in BR biosynthesis, CYP90A1/CPD, is highly active in the cells of the reproductive tract that form the pathway for pollen tubes from the stigma to the ovules. Pollen tubes grew significantly shorter through the reproductive tract of a cyp90a1 mutant compared to the wild type, or to a BR perception mutant. Our results show that epiBL promotes pollen germination and tube growth in vitro and suggest that the cells of the reproductive tract provide BR compounds to stimulate pollen tube growth.

Same same but different: sperm-activating EC1 and ECA1 gametogenesis-related family proteins.

During double fertilization in Arabidopsis thaliana, the egg cell secretes small cysteine-rich EC1 (egg cell 1) proteins, which enable the arriving sperm pair to rapidly interact with the two female gametes. EC1 proteins are members of the large and unexplored group of ECA1 (early culture abundant 1) gametogenesis-related family proteins, characterized by a prolamin-like domain with six conserved cysteine residues that may form three pairs of disulfide bonds. The distinguishing marks of egg-cell-expressed EC1 proteins are, however, two short amino acid sequence motifs present in all EC1-like proteins. EC1 genes appear to encode the major CRPs (cysteine-rich proteins) expressed by the plant egg cell, and they are restricted to flowering plants, including the most basal extant flowering plant Amborella trichopoda. Many other ECA1 gametogenesis-related family genes are preferentially expressed in the synergid cell. Functional diversification among the ECA1 gametogenesis-related family is suggested by the different patterns of expression in the female gametophyte and the low primary sequence conservation.