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.

An NCBI nucleotide blast keyed to apyrase (ATP-diphosphohydrolases, EC 3.6.1.5) conserved regions revealed five apyrases, AtAPYs (3-7), in addition to the previously identified AtAPY1 and 2. Here we report the functional analyses of two of the newly defined apyrases, AtAPY6 and AtAPY7. We analyzed tissue specificity of AtAPY6 and 7 expression by qRT-PCR and promoter:GUS fusion assays. We characterized the phenotypes of single and double knockout mutants for AtAPY6 and 7 in anther and pollen by light microscopy and electron microscopy. The transcripts of both AtAPY6 and 7 are expressed in mature pollen grains. Single knockout mutants of AtAPY6 and 7 displayed a minor change in pollen exine pattern under scanning electron microscopy without obvious change in fertility. Double knockout mutants of AtAPY6 and 7 (apy6apy7) displayed severe defects in pollen exine pattern, deformed pollen shape and reduced male fertility. An analysis of pollen from heterozygous apy6apy7 plants suggests that the defects in pollen exine wall are determined by the diploid genome. Our findings demonstrate that AtAPY6 and AtAPY7 are enzymes that play an important role in exine development of pollen grains, possibly through regulating the production of key polysaccharides needed for proper assembly of the exine layer.

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 spore's 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.

Annexins are an homologous, structurally related superfamily of proteins known to associate with membrane lipid and cytoskeletal components. Their involvement in membrane organization, vesicle trafficking and signaling is fundamental to cellular processes such as growth, differentiation, secretion and repair. Annexins exist in some prokaryotes and all eukaryotic phyla within which plant annexins represent a monophyletic clade of homologs descended from green algae. Genomic, proteomic and transcriptomic approaches have provided data on the diversity, cellular localization and expression patterns of different plant annexins. The availability of 35 complete plant genomes has enabled systematic comparative analysis to determine phylogenetic relationships, characterize structures and observe functional specificity between and within individual subfamilies. Short amino termini and selective erosion of the canonical type 2 calcium coordinating sites in domains 2 and 3 are typical of plant annexins. The convergent evolution of alternate functional motifs such as 'KGD', redox-sensitive Cys and hydrophobic Trp/Phe residues argues for their functional relevance and contribution to mechanistic diversity in plant annexins. This review examines recent findings and advances in plant annexin research with special focus on their structural diversity, cellular and molecular interactions and their potential integrated functions in the broader context of physiological responses.

Nucleoside triphosphate diphosphohydrolases (NTPDases; apyrases) (EC 3.6.1.5) hydrolyze di- and triphosphate nucleotides, but not monophosphate nucleotides. They are categorized as E-type ATPases, have a broad divalent cation (Mg(2+), Ca(2+)) requirement for activation and are insensitive to inhibitors of F-type, P-type and V-type ATPases. Among the seven NTPDases identified in Arabidopsis, only APYRASE 1 (AtAPY1) and APYRASE 2 (AtAPY2) have been previously characterized. In this work, either AtAPY1 or AtAPY2 tagged with C-terminal green fluorescent protein (GFP) driven by their respective native promoter can rescue the apy1 apy2 double knockout (apy1 apy2 dKO) successfully, and confocal microscopy reveals that these two Arabidopsis apyrases reside in the Golgi apparatus. In Saccharomyces cerevisiae, both AtAPY1 and AtAPY2 can complement the Golgi-localized GDA1 mutant, rescuing its aberrant protein glycosylation phenotype. In Arabidopsis, microsomes of the wild type show higher substrate preferences toward UDP compared with other NDP substrates. Loss-of-function Arabidopsis AtAPY1 mutants exhibit reduced microsomal UDPase activity, and this activity is even more significantly reduced in the loss-of-function AtAPY2 mutant and in the AtAPY1/AtAPY2 RNA interference (RNAi) technology repressor lines. Microsomes from wild-type plants also have detectable GDPase activity, which is significantly reduced in apy2 but not apy1 mutants. The GFP-tagged AtAPY1 or AtAPY2 constructs in the apy1 apy2 dKO plants can restore microsomal UDP/GDPase activity, confirming that they both also have functional competency. The cell walls of apy1, apy2 and the RNAi-silenced lines all have an increased composition of galactose, but the transport efficiency of UDP-galactose across microsomal membranes was not altered. Taken together, these results reveal that AtAPY1 and AtAPY2 are Golgi-localized nucleotide diphosphatases and are likely to have roles in regulating UDP/GDP concentrations in the Golgi lumen.

Gravity has major effects on both the form and overall length of root growth. Numerous papers have documented these effects (over 300 publications in the last 5 years), the most well-studied being gravitropism, which is a growth re-orientation directed by gravity toward the earth's center. Less studied effects of gravity are undulations due to the regular periodic change in the direction root tips grow, called waving, and the slanted angle of growth roots exhibit when they are growing along a nearly-vertical surface, called skewing. Although diverse studies have led to the conclusion that a gravity stimulus is needed for plant roots to show waving and skewing, the novel results just published by Paul et al. (2012) reveal that this conclusion is not correct. In studies carried out in microgravity on the International Space Station, the authors used a new imaging system to collect digital photographs of plants every six hours during 15 days of spaceflight. The imaging system allowed them to observe how roots grew when their orientation was directed not by gravity but by overhead LED lights, which roots grew away from because they are negatively phototropic. Surprisingly, the authors observed both skewing and waving in spaceflight plants, thus demonstrating that both growth phenomena were gravity independent. Touch responses and differential auxin transport would be common features of root waving and skewing at 1-g and micro-g, and the novel results of Paul et al. will focus the attention of cell and molecular biologists more on these features as they try to decipher the signaling pathways that regulate root skewing and waving.

Plant annexins are Ca(2+)-dependent phospholipid-binding proteins and are encoded by multigene families. They are implicated in the regulation of plant development as well as protection from drought and other stresses. They are well characterized in Arabidopsis, however no such characterization of rice annexin gene family has been reported thus far. With the availability of the rice genome sequence information, we have identified ten members of the rice annexin gene family. At the protein level, they share 16-64% identity with predicted molecular masses ranging from 32 to 40 kDa. Phylogenetic analysis of rice annexins together with annexins from other monocots led to their classification into five different orthologous groups and share similar motif patterns in their protein sequences. Expression analysis by real-time RT-PCR revealed differential temporal and spatial regulation of these genes. The rice annexin genes are also found to be regulated in seedling stage by various abiotic stressors including salinity, drought, heat and cold. Additionally, in silico analysis of the putative upstream sequences was analyzed for the presence of stress-responsive cis-elements. These results provide a basis for further functional characterization of specific rice annexin genes at the tissue/developmental level and in response to abiotic stresses.

Recent evidence indicates that extracellular nucleotides regulate plant growth. Exogenous ATP has been shown to block auxin transport and gravitropic growth in primary roots of Arabidopsis (Arabidopsis thaliana). Cells limit the concentration of extracellular ATP in part through the activity of ectoapyrases (ectonucleoside triphosphate diphosphohydrolases), and two nearly identical Arabidopsis apyrases, APY1 and APY2, appear to share this function. These findings, plus the fact that suppression of APY1 and APY2 blocks growth in Arabidopsis, suggested that the expression of these apyrases could influence auxin transport. This report tests that hypothesis. The polar movement of [(3)H]indole-3-acetic acid in both hypocotyl sections and primary roots of Arabidopsis seedlings was measured. In both tissues, polar auxin transport was significantly reduced in apy2 null mutants when they were induced by estradiol to suppress the expression of APY1 by RNA interference. In the hypocotyl assays, the basal halves of APY-suppressed hypocotyls contained considerably lower free indole-3-acetic acid levels when compared with wild-type plants, and disrupted auxin transport in the APY-suppressed roots was reflected by their significant morphological abnormalities. When a green fluorescent protein fluorescence signal encoded by a DR5:green fluorescent protein construct was measured in primary roots whose apyrase expression was suppressed either genetically or chemically, the roots showed no signal asymmetry following gravistimulation, and both their growth and gravitropic curvature were inhibited. Chemicals that suppress apyrase activity also inhibit gravitropic curvature and, to a lesser extent, growth. Taken together, these results indicate that a critical step connecting apyrase suppression to growth suppression is the inhibition of polar auxin transport.

Although no definitive receptor for extracellular ATP (eATP) has been identified in plants, there is now stronger physiological evidence that the effects of eATP on plant growth are mediated by a receptor, or, as in animals, by multiple receptors. Recent papers clarify how extracellular nucleotides induce changes in [Ca(2+)](cyt), and the production of nitric oxide (NO) and reactive oxygen species. They document links between eATP signaling and the synthesis or transport of hormones, and they reveal that applied nucleotides can regulate the aperture of stomates, which release ATP when stimulated by light and hormones. Ectoapyrases (ecto-nucleoside triphosphate-diphosphohydrolase) help control both the diverse signaling changes and downstream growth changes induced by extracellular nucleotides by limiting their concentration in the extracellular matrix (ECM).

This study investigates the role of extracellular nucleotides and apyrase enzymes in regulating stomatal aperture. Prior data indicate that the expression of two apyrases in Arabidopsis (Arabidopsis thaliana), APY1 and APY2, is strongly correlated with cell growth and secretory activity. Both are expressed strongly in guard cell protoplasts, as determined by reverse transcription-polymerase chain reaction and immunoblot analyses. Promoter activity assays for APY1 and APY2 show that expression of both apyrases correlates with conditions that favor stomatal opening. Correspondingly, immunoblot data indicate that APY expression in guard cell protoplasts rises quickly when these cells are moved from darkness into light. Both short-term inhibition of ectoapyrase activity by polyclonal antibodies and long-term suppression of APY1 and APY2 transcript levels significantly disrupt normal stomatal behavior in light. Stomatal aperture shows a biphasic response to applied adenosine 5'-[γ-thio]triphosphate (ATPγS) or adenosine 5'-[β-thio] diphosphate, with lower concentrations inducing stomatal opening and higher concentrations inducing closure. Equivalent concentrations of adenosine 5'-O-thiomonophosphate have no effect on aperture. Two mammalian purinoceptor inhibitors block ATPγS- and adenosine 5'-[β-thio] diphosphate-induced opening and closing and also partially block the ability of abscisic acid to induce stomatal closure and of light to induce stomatal opening. Treatment of epidermal peels with ATPγS induces increased levels of nitric oxide and reactive oxygen species, and genetically suppressing the synthesis of these agents blocks the effects of nucleotides on stomatal aperture. A luciferase assay indicates that treatments that induce either the closing or opening of stomates also induce the release of ATP from guard cells. These data favor the novel conclusion that ectoapyrases and extracellular nucleotides play key roles in regulating stomatal functions.

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.

Root hairs secrete ATP as they grow, and extracellular ATP and ADP can trigger signaling pathways that regulate plant cell growth. In several plant tissues the level of extracellular nucleotides is limited in part by ectoapyrases (ecto-NTPDases), and the growth of these tissues is strongly influenced by their level of ectoapyrase expression. Both chemical inhibition of ectoapyrase activity and suppression of the expression of two ectoapyrase enzymes by RNAi in Arabidopsis resulted in inhibition of root hair growth. As assayed by a dose-response curve, different concentrations of the poorly hydrolysable nucleotides, ATPγS and ADPβS, could either stimulate (at 7.5-25 μM) or inhibit (at ≥ 150 μM) the growth rate of root hairs in less than an hour. Equal amounts of AMPS, used as a control, had no effect on root hair growth. Root hairs of nia1nia2 mutants, which are suppressed in nitric oxide (NO) production, and of atrbohD/F mutants, which are suppressed in the production of H(2)O(2), did not show growth responses to applied nucleotides, indicating that the growth changes induced by these nucleotides in wild-type plants were likely transduced via NO and H(2)O(2) signals. Consistent with this interpretation, treatment of root hairs with different concentrations of ATPγS induced different accumulations of NO and H(2)O(2) in root hair tips. Two mammalian purinoceptor antagonists also blocked the growth responses induced by extracellular nucleotides, suggesting that they were initiated by a receptor-based mechanism.

Accumulating evidence suggest that certain annexins can play a role in abiotic stress responses in plants. We found that for one member of the Arabidopsis thaliana annexin gene family, annexin 1 (AnnAt1), loss-of-function mutants are more sensitive to drought stress and gain-of-function mutants are more tolerant. We also found that AnnAt1 is able to regulate accumulation of H(2)O(2) in vivo in Arabidopsis cells based on the observation that the level of ROS accumulation following induction by ABA correlates with the level of AnnAt1 protein in transgenic Arabidopsis plants. Here we provide more commentary on the antioxidant activity of AnnAt1, critically assess the evidence that AnnAt1 and other annexins possess peroxidase activity, emphasize a redox-induced post-translational modification which occurs to AnnAt1 during ABA signaling, and discuss ways this annexin's membrane associations could mediate stress signaling while addressing the potential that AnnAt1 is a multifunctional protein in plants.

Ectoapyrase enzymes remove the terminal phosphate from extracellular nucleoside tri- and diphosphates. In Arabidopsis (Arabidopsis thaliana), two ectoapyrases, AtAPY1 and AtAPY2, have been implicated as key modulators of growth. In fibers of cotton (Gossypium hirsutum), transcript levels for GhAPY1 and GhAPY2, two closely related ectoapyrases that have high sequence similarity to AtAPY1 and AtAPY2, are up-regulated when fibers enter their rapid growth phase. In an ovule culture system, fibers release ATP as they grow, and when their ectoapyrase activity is blocked by the addition of polyclonal anti-apyrase antibodies or by two different small molecule inhibitors, the medium ATP level rises and fiber growth is suppressed. High concentrations of the poorly hydrolyzable nucleotides ATPgammaS and ADPbetaS applied to the medium inhibit fiber growth, and low concentrations of them stimulate growth, but treatment with adenosine 5'-O-thiomonophosphate causes no change in the growth rate. Both the inhibition and stimulation of growth by applied nucleotides can be blocked by an antagonist that blocks purinoceptors in animal cells, and by adenosine. Treatment of cotton ovule cultures with ATPgammaS induces increased levels of ethylene, and two ethylene antagonists, aminovinylglycine and silver nitrate, block both the growth stimulatory and growth inhibitory effects of applied nucleotides. In addition, the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid, lowers the concentration of nucleotide needed to promote fiber growth. These data indicate that ectoapyrases and extracellular nucleotides play a significant role in regulating cotton fiber growth and that ethylene is a likely downstream component of the signaling pathway.

Annexins act as targets of calcium signals in eukaryotic cells, and recent results suggest that they play an important role in plant stress responses. We found that in Arabidopsis (Arabidopsis thaliana), AnnAt1 (for annexin 1) mRNA levels were up-regulated in leaves by most of the stress treatments applied. Plants overexpressing AnnAt1 protein were more drought tolerant and knockout plants were more drought sensitive than ecotype Columbia plants. We also observed that hydrogen peroxide accumulation in guard cells was reduced in overexpressing plants and increased in knockout plants both before and after treatment with abscisic acid. Oxidative protection resulting from AnnAt1 overexpression could be due to the low level of intrinsic peroxidase activity exhibited by this protein in vitro, previously linked to a conserved histidine residue found in a peroxidase-like motif. However, analyses of a mutant H40A AnnAt1 protein in a bacterial complementation test and in peroxidase activity assays indicate that this residue is not critical to the ability of AnnAt1 to confer oxidative protection. To further examine the mechanism(s) linking AnnAt1 expression to stress resistance, we analyzed the reactive S3 cluster to determine if it plays a role in AnnAt1 oligomerization and/or is the site for posttranslational modification. We found that the two cysteine residues in this cluster do not form intramolecular or intermolecular bonds but are highly susceptible to oxidation-driven S-glutathionylation, which decreases the Ca(2+) affinity of AnnAt1 in vitro. Moreover, S-glutathionylation of AnnAt1 occurs in planta after abscisic acid treatment, which suggests that this modification could be important in regulating the cellular function of AnnAt1 during stress responses.

Plant and animal cells release or secrete ATP by various mechanisms, and this activity allows extracellular ATP to serve as a signalling molecule. Recent reports suggest that extracellular ATP induces plant responses ranging from increased cytosolic calcium to changes in auxin transport, xenobiotic resistance, pollen germination, and growth. Although calcium has been identified as a secondary messenger for the extracellular ATP signal, other parts of this signal transduction chain remain unknown. Increasing the extracellular concentration of ATPgammaS, a poorly-hydrolysable ATP analogue, inhibited both pollen germination and pollen tube elongation, while the addition of AMPS had no effect. Because pollen tube elongation is also sensitive to nitric oxide, this raised the possibility that a connection exists between the two pathways. Four approaches were used to test whether the germination and growth effects of extracellular ATPgammaS were transduced via nitric oxide. The results showed that increases in extracellular ATPgammaS induced increases in cellular nitric oxide, chemical agonists of the nitric oxide signalling pathway lowered the threshold of extracellular ATPgammaS that inhibits pollen germination, an antagonist of guanylate cyclase, which can inhibit some nitric oxide signalling pathways, blocked the ATPgammaS-induced inhibition of both pollen germination and pollen tube elongation, and the effects of applied ATPgammaS were blocked in nia1nia2 mutants, which have diminished NO production. The concurrence of these four data sets support the conclusion that the suppression of pollen germination and pollen tube elongation by extracellular nucleotides is mediated in part via the nitric oxide signalling pathway.

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.

Plant annexins belong to a multigene family and are suggested to play a role in stress responses. A full-length cDNA for a gene encoding an annexin protein was isolated and characterized from Brassica juncea (AnnBj1). AnnBj1 message levels were regulated by abscisic acid, ethephon, salicylic acid, and methyl jasmonate as well as chemicals that induce osmotic stress (NaCl, Mannitol or PEG), heavy metal stress (CdCl(2)) and oxidative stress (methyl viologen or H(2)O(2)). In order to determine if AnnBj1 functions in protection against stress, we generated transgenic tobacco plants ectopically expressing AnnBj1 under the control of constitutive CaMV 35S promoter. The transgenic tobacco plants showed significant tolerance to dehydration (mannitol), salt (NaCl), heavy metal (CdCl(2)) and oxidative stress (H(2)O(2)) at the seedling stage and retained higher chlorophyll levels in response to the above stresses as determined in detached leaf senescence assays. The transgenic plants also showed decreased accumulation of thiobarbituric acid-reactive substances (TBARS) compared to wild-type plants in response to mannitol treatments in leaf disc assays. AnnBj1 recombinant protein exhibited low levels of peroxidase activity in vitro and transgenic plants showed increased total peroxidase activity. Additionally, the transgenic plants showed enhanced resistance to the oomycete pathogen, Phytophthora parasitica var. nicotianae, and increased message levels for several pathogenesis-related proteins. Our results demonstrate that ectopic expression of AnnBj1 in tobacco provides tolerance to a variety of abiotic and biotic stresses.

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.