Extracellular ATP can serve as a signaling agent in animal cells, and, as suggested by recent reports, may also do so in plant cells. In animal cells it induces the production of reactive oxygen species through the mediation of NADPH oxidase. Similarly, here we report that in leaves of Arabidopsis (Arabidopsis thaliana), applied ATP, but not AMP or phosphate, induces the accumulation of superoxide (O2−) in a biphasic, dose-dependent manner, with a threshold at 500 nm ATP. This effect did not require ATP hydrolysis for it was mimicked by ATPγS. ATP also induced increased levels of Arabidopsis respiratory burst oxidase homolog D (AtrbohD) mRNA, but ATP-treated plants that had disrupted AtrbohD and AtrbohF genes did not accumulate O2−, indicating that NADPH oxidases are responsible for the induced O2− accumulation. Inhibitors of mammalian P2-type ATP receptors abolished ATP-induced O2− production, suggesting that the ATP effects may be mediated through P2-like receptors in plants. Cytosolic Ca2+ and calmodulin are likely to help transduce the ATP responses, as they do in animal cells, because a Ca2+ channel blocker, a Ca2+ chelator, and calmodulin antagonist all reduced ATP-induced O2− accumulation. Furthermore, ATP treatment enhanced the expression of genes that are induced by wounds and other stresses. The ATP measured at wound sites averaged 40 μm, well above the level needed to induce O2− accumulation and gene expression changes. Transgenic plants overexpressing an apyrase gene had reduced O2− production in response to applied ATP and wounding. Together, these data suggest a possible role for extracellular ATP as a signal potentially in wound and stress responses.

Extracellular ATP is a known receptor agonist in animals and was previously shown to alter plant growth, and so we investigated whether ATP derivatives could function outside plant cells as signaling agents. Signaling responses induced by exogenous nucleotides in animal cells typically include increases in free cytoplasmic calcium concentration ([Ca 2þ ] cyt ). We have evaluated the ability of exogenously applied adenosine 59-[g-thio]triphosphate (ATPgS), adenosine 59-[b-thio]diphosphate (ADPbS), and adenosine 59-O-thiomonophosphate to alter [Ca 2þ ] cyt in intact apoaequorin transgenic Arabidopsis thaliana seedlings. ATPgS and ADPbS increase [Ca 2þ ] cyt , and this increase is enhanced further when the nucleotides are added with the elicitor oligogalacturonic acid. Exogenous treatment with ATP also increases the level of transcripts encoding mitogen-activated protein kinases and proteins involved in ethylene biosynthesis and signal transduction. The increase in [Ca 2þ ] cyt induced by nucleotide derivatives can be ablated by Ca 2þ -channel blocking agents and by the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N9,N9-tetraacetic acid (BAPTA), and the changes in gene expression can be partially blocked by these agents. These observations suggest that extracellular ATP can activate calcium-mediated cell-signaling pathways in plants, potentially playing a physiological role in transducing stress and wound responses.

Expression of two Arabidopsis (Arabidopsis thaliana) apyrase (nucleoside triphosphate-diphosphohydrolase) genes with high similarity, APY1 and APY2, was analyzed during seedling development and under different light treatments using b-glucuronidase fusion constructs with the promoters of both genes. As evaluated by b-glucuronidase staining and independently confirmed by other methods, the highest expression of both apyrases was in rapidly growing tissues and/or tissues that accumulate high auxin levels. Red-light treatment of etiolated seedlings suppressed the protein and message level of both apyrases at least as rapidly as it inhibited hypocotyl growth. Adult apy1 and apy2 single mutants had near-normal growth, but apy1apy2 doubleknockout plants were dwarf, due primarily to reduced cell elongation. Pollen tubes and etiolated hypocotyls overexpressing an apyrase had faster growth rates than wild-type plants. Growing pollen tubes released ATP into the growth medium and suppression of apyrase activity by antiapyrase antibodies or by inhibitors simultaneously increased medium ATP levels and inhibited pollen tube growth. These results imply that APY1 and APY2, like their homologs in animals, act to reduce the concentration of extracellular nucleotides, and that this function is important for the regulation of growth in Arabidopsis.

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 upregulated 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.

Highlights d A global snapshot of protein organization in plants from deep proteomics profiling d Biochemical fractionation reveals stable protein complexes conserved across plants d Many observed complexes have previously only been inferred in plants by gene content d Known molecular modules are elaborated in plants with novel subunits and organization

Although in most plant species no more than two annexin genes have been reported to date, seven annexin homologs have been identified in Arabidopsis, Annexin Arabidopsis 1–7 (AnnAt1–AnnAt7). This establishes that annexins can be a diverse, multigene protein family in a single plant species. Here we compare and analyze these seven annexin gene sequences and present the in situ RNA localization patterns of two of these genes, AnnAt1 andAnnAt2, during different stages of Arabidopsis development. Sequence analysis of AnnAt1–AnnAt7 reveals that they contain the characteristic four structural repeats including the more highly conserved 17-amino acid endonexin fold region found in vertebrate annexins. Alignment comparisons show that there are differences within the repeat regions that may have functional importance. To assess the relative level of expression in various tissues, reverse transcription-PCR was carried out using gene-specific primers for each of the Arabidopsis annexin genes. In addition, northern blot analysis using gene-specific probes indicates differences in AnnAt1 and AnnAt2expression levels in different tissues. AnnAt1 is expressed in all tissues examined and is most abundant in stems, whereas AnnAt2 is expressed mainly in root tissue and to a lesser extent in stems and flowers. In situ RNA localization demonstrates that these two annexin genes display developmentally regulated tissue-specific and cell-specific expression patterns. These patterns are both distinct and overlapping. The developmental expression patterns for both annexins provide further support for the hypothesis that annexins are involved in the Golgi-mediated secretion of polysaccharides.

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In Arabidopsis, we previously identified two highly similar apyrases, AtAPY1 and AtAPY2. Here, T-DNA knockout (KO) mutations of each gene were isolated in a reverse genetic approach. The single KO mutants lacked a discernible phenotype. The double KO mutants, however, exhibited a complete inhibition of pollen germination, and this correlated with positive ␤-glucuronidase staining in the pollen of apyrase promoter:␤-glucuronidase fusion transgenic lines. The vast majority of the pollen grains of these mutants were identical to wild type in size, shape, and nuclear state and were viable as assayed by metabolic activity and plasma membrane integrity. Complementation with either AtAPY1 or AtAPY2 cDNA rescued pollen germination, confirming that the phenotype was apyrase specific. Despite the redundancy of the two apyrases in rescue potential, transmission analyses suggested a greater role for AtAPY2 in male gamete success. The effect of mutant apyrase on the transmission through the female gametophyte was only marginal, and embryo development appeared normal in the absence of apyrases. The male-specific double KO mutation is fully penetrant and shows that apyrases play a crucial role in pollen germination.Pollen germination represents a short, yet very critical event in a series of steps leading to the fertilization of the ovule. Mature pollen is shed from the anther as desiccated grains and rehydrates upon contact with the stigma. During hydration, the pollen shape changes from elongated to round, and Ca 2ϩ enters the pollen grain, leading to rearrangement of the cytoskeleton and establishment of a Ca 2ϩ gradient (Heslop-Harrison and Heslop-Harrison, 1992a, 1992b; for schematic illustration, see Johnson and McCormick, 2001). Germination follows rapidly, usually within minutes in Arabidopsis (Pruitt and Hü lskamp, 1994), and is visually manifested by the emergence of a pollen tube. Although pollen germination in vivo is a tightly regulated process dependent on multiple cues from the stigma (Johnson and Preuss, 2002), pollen grains will germinate in vitro in aqueous medium minimally containing Suc, boric acid, and Ca 2ϩ (Taylor and Hepler, 1997). Few genes have been identified so far that are specific for the germination stage (for reviews, see Franklin-Tong, 2002; Johnson and Preuss, 2002). Here, we introduce the enzyme apyrase as an essential player in this early gametophytic phase.Apyrases hydrolyze nucleoside tri-and diphosphates and are highly active and most likely ubiquitous, because they have been found in all pro-and eukaryotic organisms examined for their presence. In animals, these enzymes have been shown to play important regulatory roles in the mediation of signaling events, for example the quenching of the neurotransmitter ATP (Plesner, 1995;Komoszynski and Wojtczak, 1996).The function of plant apyrases has been studied most intensively in legumes, namely Dolichos biflorus, wild soybean (Glycine soja), Medicago truncatula, and pea (Pisum sativum). In wild soybean and D. biflorus, two apyrases each we...