Germinal and somatic cell fate acquisition in early maize anther development [electronic resource] : morphogenetic mechanisms underlying pre-meiotic differentiation

One fundamental difference between plants and animals is the existence of a germ-line in animals and its absence in plants. Despite the central importance of sexual reproduction, the mechanism(s) by which germinal cells differentiate from somatic precursors in plants is unknown. Current models invoke simultaneous specification of germinal and supporting somatic niche cells from division of a singular, positionally defined precursor cell. Through confocal reconstruction of fertile, mac1 (encoding a secreted signaling protein; absence results in excess germinal and fewer somatic cells), and msca1 (encoding a glutaredoxin; absence results in loss of both somatic and germinal anther cell types) maize anthers I established that germinal cells have a multiclonal origin within a field of pluripotent progenitors and that these cells subsequently utilize a MAC1-dependent pathway to direct cell fate setting in neighbors that differentiate as somatic support tissues. I demonstrated that cellular redox status determines germinal fate by manipulating the gas and chemical environment of immature anthers. Treatments that decreased oxygen and/or H2O2 significantly increased germinal cell numbers with ectopic germinal cell formation nearer the anther surface. Conversely, oxidizing environments significantly inhibited germinal specification, delayed somatic development, and caused germinal differentiation in deeper tissues. Remarkably, I was able to correct the msca1 phenotype chemically, restoring germinal differentiation and development of anatomically normal anthers. This led to a new model in which a field of equivalent, pluripotent progenitors proliferates until the reductive environment activates MSCA1, which in turn induces germinal cell differentiation and increases Mac1 expression to direct somatic differentiation in neighboring cells. This model includes two novel features: (1) a physiological trigger generated by the growth of the developing tissue, and (2) the involvement of hypoxia in germinal fate specification, which may permit germinal cells to limit levels of DNA-damaging reactive oxygen species (ROS) accumulation in reproductive cells. To develop these insights further, I isolated pre-meiotic germinal cells via laser microdissection and compared them to the enveloping somatic tissues and to whole anthers at the same stage by microarray hybridization. I repeated this at two stages -- one and four days post-germinal specification. These and other comparisons involving the mac1 and msca1 mutants led to the identification of cell-type specific markers in the somatic and germinal cells at early and late stages of pre-meiotic development. These arrays were the first set of transcriptomic profiles of staged pre-meiotic cells in any plant or animal, and were by far the earliest germinal to somatic comparison ever done in any organism. For confirmation I used RNA in situ hybridization to identify eight germinal cell markers, the first found in monocots and eight of the first nine in flowering plants identified to date, along with many somatic markers including one secondary parietal layer marker. These arrays are informative not just for maize anther development, but for the biology of germinal cells in general. Because of the shared parentage of the germinal cells and their somatic neighbors, differences likely represented rapid changes that may be key to setting or reinforcing the germinal / somatic cell fate boundary. The germinal-specific set was highly represented by translational components, RNAi machinery, and redox maintenance genes. Meanwhile there was a high representation of receptor-like kinases in the somatic set, along with an RNA-directed RNA polymerase responsible for the generation of siRNAs. I also found germinal enrichment for two uncharacterized ARGONAUTES related to AtAGO4 involved in RNA-directed DNA methylation. These results suggested the existence of previously uncovered roles for small RNAs in anther development. Furthermore, mapping the metabolic genes onto known pathways indicated that germinal cells utilize alternative metabolism that bypasses the electron transport chain. This metabolic switch in the germinal cells may enable cell proliferation in a hypoxic environment thus minimizing ROS. These findings not only provide insight into the mechanism of germinal fate acquisition in plants, but also open up intriguing new areas of research into stem cell metabolism under hypoxic conditions and the utilization of post-transcriptional and translational control over cell fate during development.

Keywords: Biology.
Advisor: Walbot Virginia
Mudgett Mary Beth
Nelson W. J. (W. James) advisor. Thesis advisor