Thomas J. Bushart

Profile and Analysis of Gene Expression Changes during Early Development in Germinating Spores of Ceratopteris richardii

Analysis of an expressed sequence tag library with more than 5,000 sequences from spores of the fern Ceratopteris richardii reveals that more than 3,900 of them represent distinct genes, and almost 70% of these have significant similarity to Arabidopsis (Arabidopsis thaliana) genes. Eight genes are common between three very different dormant plant systems, Ceratopteris spores, Arabidopsis seeds, and Arabidopsis pollen. We evaluated the pattern of mRNA abundance over the first 48 h of spore development using a microarray of cDNAs representing 3,207 distinct genes of C. richardii and determined the relative levels of RNA abundance for 3,143 of these genes using a Bayesian method of statistical analysis. More than 900 of them (29%) show a significant change between any of the five time points analyzed, and these have been annotated based on their sequence similarity with the Arabidopsis proteome. Novel data arising from these analyses identify genes likely to be critical for the germination and subsequent early development of diverse cells and tissues emerging from dormancy.

RNA-seq analysis identifies potential modulators of gravity response in spores of Ceratopteris (Parkeriaceae): evidence for modulation by calcium pumps and apyrase activity.

PREMISE OF THE STUDY Gravity regulates the magnitude and direction of a trans-cell calcium current in germinating spores of Ceratopteris richardii. Blocking this current with nifedipine blocks the spores downward polarity alignment, a polarization that is fixed by gravity ∼10 h after light induces the spores to germinate. RNA-seq analysis at 10 h was used to identify genes potentially important for the gravity response. The data set will be valuable for other developmental and phylogenetic studies. METHODS De novo Newbler assembly of 958 527 reads from Roche 454 sequencing was executed. The sequences were identified and analyzed using in silico methods. The roles of endomembrane Ca(2+)-ATPase pumps and apyrases in the gravity response were further tested using pharmacological agents. KEY RESULTS Transcripts related to calcium signaling and ethylene biosynthesis were identified as notable constituents of the transcriptome. Inhibiting the activity of endomembrane Ca(2+)-ATPase pumps with 2,5-di-(t-butyl)-1,4-hydroquinone diminished the trans-cell current, but increased the orientation of the polar axis to gravity. The effects of applied nucleotides and purinoceptor antagonists gave novel evidence implicating extracellular nucleotides as regulators of the gravity response in these fern spores. CONCLUSIONS In addition to revealing general features of the transcriptome of germinating spores, the results highlight a number of calcium-responsive and light-receptive transcripts. Pharmacologic assays indicate endomembrane Ca(2+)-ATPases and extracellular nucleotides may play regulatory roles in the gravity response of Ceratopteris spores.

Studying Molecular Changes During Gravity Perception and Response in a Single Cell

Early studies revealed a highly predictable pattern of gravity-directed growth and development in Ceratopteris richardii spores. This makes the spores a valuable model system for the study of how a single cell senses and responds to the force of gravity. Gravity regulates both the direction and magnitude of a trans-cell calcium current in germinating spores, and the orientation of this current predicts the polarization of spore development. Molecular techniques have been developed to evaluate the transcriptomic and proteomic profiles of spores before and after gravity establishes the polarity of their development. Here we describe these techniques, along with protocols for sterilizing the spores, sowing them in a solid or liquid growth media, and evaluating germination.