Mari L. Salmi

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.

Between Two Fern Genomes

Ferns are the only major lineage of vascular plants not represented by a sequenced nuclear genome. This lack of genome sequence information significantly impedes our ability to understand and reconstruct genome evolution not only in ferns, but across all land plants. Azolla and Ceratopteris are ideal and complementary candidates to be the first ferns to have their nuclear genomes sequenced. They differ dramatically in genome size, life history, and habit, and thus represent the immense diversity of extant ferns. Together, this pair of genomes will facilitate myriad large-scale comparative analyses across ferns and all land plants. Here we review the unique biological characteristics of ferns and describe a number of outstanding questions in plant biology that will benefit from the addition of ferns to the set of taxa with sequenced nuclear genomes. We explain why the fern clade is pivotal for understanding genome evolution across land plants, and we provide a rationale for how knowledge of fern genomes will enable progress in research beyond the ferns themselves.

Nitric Oxide and cGMP Signaling in Calcium-Dependent Development of Cell Polarity in Ceratopteris richardii

Single-celled spores of the fern Ceratopteris richardii undergo gravity-directed cell polarity development that is driven by polar calcium currents. Here we present results that establish a role for nitric oxide (NO)/cGMP signaling in transducing the stimulus of gravity to directed polarization of the spores. Application of specific NO donors and scavengers inhibited the calcium-dependent gravity response in a dose-dependent manner. The effects of NO donor exposure were antagonized by application of NO scavenger compounds. Similarly, the guanylate cyclase inhibitors 6-anilino-5,8-quinolinedione and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin, and the phosphodiesterase inhibitor Viagra, which modulate NO-dependent cGMP levels in the cells, disrupted gravity-directed cell polarity in a dose-dependent manner. Viagra effects were antagonized by application of NO scavengers, consistent with the postulate that NO and cGMP are linked in the signaling pathway. To identify other components of the signaling system we analyzed gene expression changes induced by Viagra treatment using microarrays and quantitative real-time reverse transcription-polymerase chain reaction. Preliminary microarray analysis revealed several genes whose expression was significantly altered by Viagra treatment. Three of these genes had strong sequence similarity to key signal transduction or stress response genes and quantitative real-time reverse transcription-polymerase chain reaction was used to more rigorously quantify the effects of Viagra on their expression in spores and to test how closely these effects could be mimicked by treatment with dibutyryl cGMP. Taken together our results implicate NO and cGMP as downstream effectors that help link the gravity stimulus to polarized growth in C. richardii spores.

Apyrase Suppression Raises Extracellular ATP Levels and Induces Gene Expression and Cell Wall Changes Characteristic of Stress Responses

Suppressing the expression of two apyrase genes raises extracellular ATP levels and induces gene expression, growth, and cell wall changes characteristic of stress responses, thus implicating extracellular nucleotides as early signals linking biotic and abiotic stresses to growth inhibition. Plant cells release ATP into their extracellular matrix as they grow, and extracellular ATP (eATP) can modulate the rate of cell growth in diverse tissues. Two closely related apyrases (APYs) in Arabidopsis (Arabidopsis thaliana), APY1 and APY2, function, in part, to control the concentration of eATP. The expression of APY1/APY2 can be inhibited by RNA interference, and this suppression leads to an increase in the concentration of eATP in the extracellular medium and severely reduces growth. To clarify how the suppression of APY1 and APY2 is linked to growth inhibition, the gene expression changes that occur in seedlings when apyrase expression is suppressed were assayed by microarray and quantitative real-time-PCR analyses. The most significant gene expression changes induced by APY suppression were in genes involved in biotic stress responses, which include those genes regulating wall composition and extensibility. These expression changes predicted specific chemical changes in the walls of mutant seedlings, and two of these changes, wall lignification and decreased methyl ester bonds, were verified by direct analyses. Taken together, the results are consistent with the hypothesis that APY1, APY2, and eATP play important roles in the signaling steps that link biotic stresses to plant defense responses and growth changes.

Genome-scale cluster analysis of replicated microarrays using shrinkage correlation coefficient

BackgroundCurrently, clustering with some form of correlation coefficient as the gene similarity metric has become a popular method for profiling genomic data. The Pearson correlation coefficient and the standard deviation (SD)-weighted correlation coefficient are the two most widely-used correlations as the similarity metrics in clustering microarray data. However, these two correlations are not optimal for analyzing replicated microarray data generated by most laboratories. An effective correlation coefficient is needed to provide statistically sufficient analysis of replicated microarray data.ResultsIn this study, we describe a novel correlation coefficient, shrinkage correlation coefficient (SCC), that fully exploits the similarity between the replicated microarray experimental samples. The methodology considers both the number of replicates and the variance within each experimental group in clustering expression data, and provides a robust statistical estimation of the error of replicated microarray data. The value of SCC is revealed by its comparison with two other correlation coefficients that are currently the most widely-used (Pearson correlation coefficient and SD-weighted correlation coefficient) using statistical measures on both synthetic expression data as well as real gene expression data from Saccharomyces cerevisiae. Two leading clustering methods, hierarchical and k-means clustering were applied for the comparison. The comparison indicated that using SCC achieves better clustering performance. Applying SCC-based hierarchical clustering to the replicated microarray data obtained from germinating spores of the fern Ceratopteris richardii, we discovered two clusters of genes with shared expression patterns during spore germination. Functional analysis suggested that some of the genetic mechanisms that control germination in such diverse plant lineages as mosses and angiosperms are also conserved among ferns.ConclusionThis study shows that SCC is an alternative to the Pearson correlation coefficient and the SD-weighted correlation coefficient, and is particularly useful for clustering replicated microarray data. This computational approach should be generally useful for proteomic data or other high-throughput analysis methodology.

Breakthroughs spotlighting roles for extracellular nucleotides and apyrases in stress responses and growth and development.

Animal and plant cells release nucleotides into their extracellular matrix when touched, wounded, and when their plasma membranes are stretched during delivery of secretory vesicles and growth. These released nucleotides then function as signaling agents that induce rapid increases in the concentration of cytosolic calcium, nitric oxide and superoxide. These, in turn, are transduced into downstream physiological changes. These changes in plants include changes in the growth of diverse tissues, in gravitropism, and in the opening and closing of stomates. The concentration of extracellular nucleotides is controlled by various phosphatases, prominent among which are apyrases EC 3.6.1.5 (nucleoside triphosphate diphosphohydrolases, NTPDases). This review provides phylogenetic and pHMM analyses of plant apyrases as well as analysis of predicted post-translational modifications for Arabidopsis apyrases. This review also summarizes and discusses recent advances in research on the roles of apyrases and extracellular nucleotides in controlling plant growth and development. These include new findings that document how apyrases and extracellular nucleotides control auxin transport, modulate stomatal aperture, and mediate biotic and abiotic stress responses, and on how apyrase suppression leads to growth inhibition.

Current status and proposed roles for nitric oxide as a key mediator of the effects of extracellular nucleotides on plant growth

Recent data indicate that nucleotides are released into the extracellular matrix during plant cell growth, and that these extracellular nucleotides induce signaling changes that can, in a dose-dependent manner, increase or decrease the cell growth. After activation of a presumed receptor, the earliest signaling change induced by extracellular nucleotides is an increase in the concentration of cytosolic Ca2+, but rapidly following this change is an increase in the cellular level of nitric oxide (NO). In Arabidopsis, mutants deficient in nitrate reductase activity (nia1nia2) have drastically reduced nitric oxide production and cannot transduce the effects of applied nucleotides into growth changes. Both increased levels of extracellular nucleotides and increased NO production inhibit auxin transport and inhibit growth, and these effects are potentially due to disruption of the localization and/or function of auxin transport facilitators. However, because NO- and auxin-induced signaling pathways can intersect at multiple points, there may be diverse ways by which the induction of NO by extracellular ATP could modulate auxin signaling and thus influence growth. This review will discuss these optional mechanisms and suggest possible regulatory routes based on current experimental data and predictive computational analyses.

Generation of Transgenic Spores of the Fern Ceratopteris richardii to Analyze Ca 2+ Transport Dynamics During Gravity-Directed Polarization

Spores from the fern, Ceratopteris richardii, have been used to study gravity-directed cell polarization for over three decades. This system is ideal for these studies because it has a highly predictable growth and developmental pattern and primarily responds to the mechanical force of gravity during polarization. Early studies on the development in this system showed that during the first 24 h of germination, Ceratopteris spores establish a Ca2+ concentration differential along the outer periphery of the cell that is defined by the uptake of Ca2+ through channels at the bottom of the spore and an efflux of Ca2+ through pumps at the top. This 100-fold [Ca2+] differential is sensitive to the direction and magnitude of the gravitational force. In a low-gravity environment or when the uptake of Ca2+ is blocked, spore polarization becomes random. These results support the hypothesis that the uptake of Ca2+ is necessary for gravity-directed polarization in Ceratopteris spores. For many years, studies of Ceratopteris were limited by the inability to produce stable transformants. However, recent protocols have led to the first stably transformed lines in Ceratopteris richardii. This work reviews and discusses the latest studies of gravity-induced changes in Ca2+ transport dynamics in Ceratopteris spores, the use of transformation protocols to overexpress or knock out genes relevant to the transport of Ca2+ in Ceratopteris, and the potential value of mutants to more thoroughly understand the role of Ca2+ transport dynamics in gravity-directed polarization of 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.

Gene-ontology-based analysis of gene expression changes in early development of Ceratopteris spores

Following exposure to light, single-celled spores of the aquatic fern Ceratopteris richardii undergo a series of rapid developmental changes similar to germination in higher plants. To investigate this process, Salmi, et al. tracked gene expression changes over 48 hours post-light exposure using a microarray printed with partially-sequenced Ceratopteris clones. Here, we extend this work by analyzing over-representation of gene ontology terms among differentially up-regulated genes from each time point.