A Pan-plant Protein Complex Map Reveals Deep Conservation and Novel Assemblies.

Plants are foundational for global ecological and economic systems, but most plant proteins remain uncharacterized. Protein interaction networks often suggest protein functions and open new avenues to characterize genes and proteins. We therefore systematically determined protein complexes from 13 plant species of scientific and agricultural importance, greatly expanding the known repertoire of stable protein complexes in plants. By using co-fractionation mass spectrometry, we recovered known complexes, confirmed complexes predicted to occur in plants, and identified previously unknown interactions conserved over 1.1 billion years of green plant evolution. Several novel complexes are involved in vernalization and pathogen defense, traits critical for agriculture. We also observed plant analogs of animal complexes with distinct molecular assemblies, including a megadalton-scale tRNA multi-synthetase complex. The resulting map offers a cross-species view of conserved, stable protein assemblies shared across plant cells and provides a mechanistic, biochemical framework for interpreting plant genetics and mutant phenotypes.

Apyrase suppression raises extracellular ATP levels and induces gene expression and cell wall changes characteristic of stress responses.

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.

Two distinct light-induced reactions are needed to promote germination in spores of Ceratopteris richardii.

Germination of Ceratopteris richardii spores is initiated by light and terminates 3-4 days later with the emergence of a rhizoid. Early studies documented that the photoreceptor for initiating this response is phytochrome. However, completion of germination requires additional light input. If no further light stimulus is given after phytochrome photoactivation, the spores do not germinate. Here we show that a crucial second light reaction is required, and its function is to activate and sustain photosynthesis. Even in the presence of light, blocking photosynthesis with DCMU after phytochrome photoactivation blocks germination. In addition, RT-PCR showed that transcripts for different phytochromes are expressed in spores in darkness, and the photoactivation of these phytochromes results in the increased transcription of messages encoding chlorophyll a/b binding proteins. The lack of chlorophyll-binding protein transcripts in unirradiated spores and their slow accumulation makes it unlikely that photosynthesis is required for the initial light reaction. This conclusion is supported by the observation that the transient presence of DCMU, only during the initial light reaction, had no effect on germination. Additionally, the [ATP] in Ceratopteris richardii spores increased coincidentally with the length of light treatment during germination. Overall, these results support the conclusion that two distinct light reactions are required for the germination of Ceratopteris richardii spores.

Polarized distribution of extracellular nucleotides promotes gravity-directed polarization of development in spores of Ceratopteris richardii.

Gravity directs the polarization of Ceratopteris fern spores. This process begins with the uptake of calcium through channels at the bottom of the spore, a step necessary for the gravity response. Data showing that extracellular ATP (eATP) regulates calcium channels led to the hypothesis that extracellular nucleotides could play a role in the gravity-directed polarization of Ceratopteris spores. In animal and plant cells ATP can be released from mechanosensitive channels. This report tests the hypothesis that the polarized release of ATP from spores could be activated by gravity, preferentially along the bottom of the spore, leading to an asymmetrical accumulation of eATP. In order to carry out this test, an ATP biosensor was used to measure the [eATP] at the bottom and top of germinating spores during gravity-directed polarization. The [eATP] along the bottom of the spore averaged 7-fold higher than the concentration at the top. All treatments that disrupted eATP signaling resulted in a statistically significant decrease in the gravity response. In order to investigate the source of ATP release, spores were treated with Brefeldin A (BFA) and gadolinium trichloride (GdCl3). These treatments resulted in a significant decrease in gravity-directed polarization. An ATP biosensor was also used to measure ATP release after treatment with both BFA and GdCl3. Both of these treatments caused a significant decrease in [ATP] measured around spores. These results support the hypothesis that ATP could be released from mechanosensitive channels and secretory vesicles during the gravity-directed polarization of Ceratopteris spores.

Changes in gravity rapidly alter the magnitude and direction of a cellular calcium current.

In single-celled spores of the fern Ceratopteris richardii, gravity directs polarity of development and induces a directional, trans-cellular calcium (Ca(2+)) current. To clarify how gravity polarizes this electrophysiological process, we measured the kinetics of the cellular response to changes in the gravity vector, which we initially estimated using the self-referencing calcium microsensor. In order to generate more precise and detailed data, we developed a silicon microfabricated sensor array which facilitated a lab-on-a-chip approach to simultaneously measure calcium currents from multiple cells in real time. These experiments revealed that the direction of the gravity-dependent polar calcium current is reversed in less than 25 s when the cells are inverted, and that changes in the magnitude of the calcium current parallel rapidly changing g-forces during parabolic flight on the NASA C-9 aircraft. The data also revealed a hysteresis in the response of cells in the transition from 2g to micro-g in comparison to cells in the micro-g to 2-g transition, a result consistent with a role for mechanosensitive ion channels in the gravity response. The calcium current is suppressed by either nifedipine (calcium-channel blocker) or eosin yellow (plasma membrane calcium pump inhibitor). Nifedipine disrupts gravity-directed cell polarity, but not spore germination. These results indicate that gravity perception in single plant cells may be mediated by mechanosensitive calcium channels, an idea consistent with some previously proposed models of plant gravity perception.

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

BACKGROUND: Currently, 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. RESULTS: In 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. CONCLUSION: This 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.

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.

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.

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.

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 Ca(2+), 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.

Gene expression changes induced by space flight in single-cells of the fern Ceratopteris richardii.

This work describes a rare high-throughput evaluation of gene expression changes induced by space flight in a single plant cell. The cell evaluated is the spore of the fern Ceratopteris richardii, which exhibits both perception and response to gravity. cDNA microarray and Q RT-PCR analysis of spores germinating in microgravity onboard NASA space shuttle flight STS-93 revealed changes in the mRNA expression of roughly 5% of genes analyzed. These gene expression changes were compared with gene expression changes that occur during gravity perception and response in animal cells and multicellular plants. Our data contribute to a better understanding of the impact of space flight conditions, including microgravity, on cellular growth and development, and provide insights into the adaptive strategies of individual cells in response to these conditions.

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. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers BE 640669 to BE 643506, BQ 086920 to BQ 087668, and CV 734654 to CV 736151.

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.