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SUMMARYPlants are foundational to global ecological and economic systems, yet 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. Using co-fractionation mass spectrometry, we recovered known complexes, confirmed complexes predicted to occur in plants, and identified novel interactions conserved over 1.1 billion years of green plant evolution. Several novel complexes are involved in vernalization and pathogen defense, traits critical to agriculture. We also uncovered plant analogs of animal complexes with distinct molecular assemblies, including a megadalton-scale tRNA multi-synthetase complex. The resulting map offers the first 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.

Recent spaceflight experiments have provided many new insights into the role of gravity in plant growth and development. Scientists have been taking seeds and plants into space for decades in an effort to understand how the stressful environment of space affects them. The resultant data have yielded significant advances in the development of advanced life-support systems for long-duration spaceflight and a better understanding of the fundamental role of gravity in directing the growth and development of plants. Experiments have improved as new spaceflight hardware and technology paved the way for progressively more insightful and rigorous plant research in space. The International Space Station (ISS) has provided an opportunity for scientists to both monitor and control their experiments in real-time. Experiments on the ISS have provided valuable insights into endogenous growth responses, light responses, and transcriptomic and proteomic changes that occur in the microgravity environment. In recent years most studies of plants in space have used Arabidopsis thaliana, but the single-celled, Ceratopteris richardii spore is also a valuable model system that has been used to understand plant gravity response. Experiments using these fern spores have revealed a dynamic and gravity-responsive trans-cell Ca2+ current that directs polarization of these spores and a possible role of extracellular nucleotides in establishing or contributing to this current. As technology continues to improve, spaceflight experiments will provide many new insights into the role and effects of gravity on plant growth and development.

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.

This report describes the development of lab-on-a-chip device designed to measure changes in cellular ion gradients that are induced by changes in gravitational (g) forces. The bioCD presented here detects differential calcium ion concentrations outside of individual cells. The device includes sufficient replicates for statistical analysis of the gradients around multiple single cells and around control wells that are empty or include dead cells. In the data presented, the degree of the cellular response correlates with the magnitude of the g-force applied via rotation of the bioCD. The experiments recorded the longest continuous observation of a cellular response to hypergravity made to date, and they demonstrate the potential utility of this device for assaying the threshold of cells' g-force responses in spaceflight conditions.

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.

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.

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.

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.

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.

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.

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.

SporeSat – a lab-on-a-chip (LOC) centrifuge platform designed for integration as the payload of a small (5.5 kg), free-flying satellite – has been developed to determine the gravitational thresholds for calcium-ion channel activation of a single-cell spore from the fern Ceratopteris richardii. This fern is an important model system for gravity-directed plant-cell development during variable-gravity conditions attainable only in space flight. Calcium-ion channel activity is measured by photolithographically defined calcium ion–selective electrodes (ISEs) at opposite ends of each spore. Artificial gravity is created by rotating a disk-like platform that contains the spores in wells along with the calcium ISEs. Ground experiments reveal a maximum calcium concentration ratio at 2.2xg, between micro-ion-selective electrodes near the “top” and “bottom” ends of the spore, indicating an increasing calcium concentration at one “end” of the fern spore with respect to the other. Confocal micrographs of rhizoid formation confirm the light-induced germination. SporeSat is a spaceflight experiment that will take ~ 4 days; data will be telemetered to Earth over ~ 100 days.

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.

Ceratopteris richardii is an aquatic fern that has proven to be a ­productive model system for studies in the genetics, biochemistry, and cell ­biology of early gametophytic development. Here we review and discuss the recent literature that documents the cellular, molecular, and gene expression changes that occur in gametophytes during polarity development and sexual differentiation, and in response to the environmental stimuli of light and gravity. Different approaches to achieve genetic transformation in this fern will also be discussed.

The methyl-D-erythritol 4-phosphate pathway is responsible for the biosynthesis of a substantial number of natural compounds of biological and biotechnological importance. In recent years, this pathway has become an obvious target to develop new herbicides and antimicrobial drugs. In addition, the production of a variety of compounds of medical and agricultural interest may be possible through the genetic manipulation of this pathway. To this end, a complete understanding of the molecular mechanisms that regulate this pathway is of tremendous importance. Recent data have accumulated that show some of the multiple mechanisms that regulate the methyl-D-erythritol 4-phosphate pathway in plants. In this review we will describe some of these and discuss their implications. It has been demonstrated that 1-deoxy-D-xylulose-5-phosphate synthase (DXS), the first enzyme of this route, plays a major role in the overall regulation of the pathway. A small gene family codes for this enzyme in most of the plants which have been analysed so far, and the members of these gene families belong to different phylogenetic groups. Each of these genes exhibits a distinct expression pattern, suggesting unique functions. One of the most interesting regulatory mechanisms recently described for this pathway is the post-transcriptional regulation of the level of DXS and DXR proteins. In the case of DXS, this regulation appears conserved among plants, supporting its importance. The evidence accumulated suggests that this regulation might link the activity of this pathway with the plant's physiological conditions and the metabolic demand for the final products of this route.

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. © 2008 Yao et al; licensee BioMed Central Ltd., published_or_final_version

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.