Stanley J. Roux

Extracellular ATP Induces the Accumulation of Superoxide via NADPH Oxidases in Arabidopsis

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.

Evidence of a Novel Cell Signaling Role for Extracellular Adenosine Triphosphates and Diphosphates in Arabidopsis

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 ([Ca2+]cyt). We have evaluated the ability of exogenously applied adenosine 5′-[γ-thio]triphosphate (ATPγS), adenosine 5′-[β-thio]diphosphate (ADPβS), and adenosine 5′-O-thiomonophosphate to alter [Ca2+]cyt in intact apoaequorin transgenic Arabidopsis thaliana seedlings. ATPγS and ADPβS increase [Ca2+]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 [Ca2+]cyt induced by nucleotide derivatives can be ablated by Ca2+-channel blocking agents and by the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-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.

A Role for Ectophosphatase in Xenobiotic Resistance

Xenobiotic resistance in animals, plants, yeast, and bacteria is known to involve ATP binding cassette transporters that efflux invading toxins. We present data from yeast and a higher plant indicating that xenobiotic resistance also involves extracellular ATP degradation. Transgenic upregulation of ecto-ATPase alone confers resistance to organisms that have had no previous exposure to toxins. Similarly, cells that are deficient in extracellular ATPase activity are more sensitive to xenobiotics. On the basis of these and other supporting data, we hypothesize that the hydrolysis of extracellular ATP by phosphatases and ATPases may be necessary for the resistance conferred by P-glycoprotein.

The Role of Annexin 1 in Drought Stress 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 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 Ca2+ 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.

Disruption of Apyrases Inhibits Pollen Germination in Arabidopsis

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 β-glucuronidase staining in the pollen of apyrase promoter:β-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.

Apyrases (Nucleoside Triphosphate-Diphosphohydrolases) Play a Key Role in Growth Control in Arabidopsis

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 β-glucuronidase fusion constructs with the promoters of both genes. As evaluated by β-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 double-knockout 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.

Extracellular ATP Inhibits Root Gravitropism at Concentrations That Inhibit Polar Auxin Transport

Raising the level of extracellular ATP to mmconcentrations similar to those found inside cells can block gravitropism of Arabidopsis roots. When plants are grown in Murashige and Skoog medium supplied with 1 mm ATP, their roots grow horizontally instead of growing straight down. Medium with 2 mm ATP induces root curling, and 3 mm ATP stimulates lateral root growth. When plants are transferred to medium containing exogenous ATP, the gravity response is reduced or in some cases completely blocked by ATP. Equivalent concentrations of ADP or inorganic phosphate have slight but usually statistically insignificant effects, suggesting the specificity of ATP in these responses. The ATP effects may be attributable to the disturbance of auxin distribution in roots by exogenously applied ATP, because extracellular ATP can alter the pattern of auxin-induced gene expression in DR5-β-glucuronidase transgenic plants and increase the response sensitivity of plant roots to exogenously added auxin. The presence of extracellular ATP also decreases basipetal auxin transport in a dose-dependent fashion in both maize (Zea mays) and Arabidopsis roots and increases the retention of [3H]indole-3-acetic acid in root tips of maize. Taken together, these results suggest that the inhibitory effects of extracellular ATP on auxin distribution may happen at the level of auxin export. The potential role of the trans-plasma membrane ATP gradient in auxin export and plant root gravitropism is discussed.

An improved method for the subcellular localization of calcium using a modification of the antimonate precipitation technique.

A new variation of the antimonate precipitation technique, employing tannic acid in the primary aldehyde-antimonate fixative, is described for use in the subcellular localization of calcium in various tissues. Chelation studies and electron microscopic, X-ray microanalytical studies of antimonate precipitates in etiolated oat tissues indicate that calcium is the major cation localized using the present experimental protocol. Preservation of ultrastructural morphology in these tissues is greatly improved over that observed in tissues fixed with conventional antimonate-aldehyde or antimonate-osmium fixatives. The regularity and reproducibility of tissue precipitate patterns suggests that 1) penetration of the tissue by the fixative, and subsequent precipitation of calcium, is rapid and uniform and 2) ion displacement during sample preparation is negligible. Calcium appears to be immobilized efficiently in situ, with greater than 90% 45Ca retention in radiolabeled tissues prepared for electron microscopy. Quantitative aspects of calcium precipitation by antimonate in 45Ca-labeled CaCl2 solutions were examined over a wide range of calcium concentrations. Precipitation was essentially linear over the expected range of biological concentrations of calcium. Furthermore, the 3:1 antimonate to calcium ratio estimated for test tube precipitates was also established for Sb/Ca in tissue precipitates analyzed using energy dispersive x-ray microanalytical (EDX) techniques. These observations suggest that the present technique is potentially useful in the semiquantitative estimation of tissue calcium levels.

Multiherbicide tolerance conferred by AtPgp1 and apyrase overexpression in Arabidopsis thaliana

Herbicide resistance is an important trait often introduced into crop plants. Mechanisms of resistance can involve a mutant target protein that is unaffected by the herbicide, or metabolic detoxification or degradation of the herbicide. Recently, we showed that overexpression in Arabidopsis thaliana of either psNTP9, the garden pea apyrase gene, or AtPgp1, the A. thaliana homolog of the plant multidrug resistance (MDR) gene, enabled A. thaliana to germinate on the toxin cycloheximide and to grow better on toxic levels of the plant hormone N6-[2-isopentyl]adenine (2iP). Here we report that overexpression of either MDR or apyrase proteins resulted in increased resistance to herbicides from different chemical classes. Apyrase inhibition by small molecule inhibitors reversed this resistance. Treatment of untransformed plants with an apyrase inhibitor increased their sensitivity to the same herbicides. These results indicate that the genes may be involved in a resistance mechanism relating to decreased retention or increased active efflux of herbicide from the plant cell.

Structure, function, and mechanism of action of Calmodulin

The structure, function, and mechanism of action of plant calmodulins have been the subject of intense study for plant scientists during the past several years. While precedents in animal biochemistry and physiology have suggested logical starting points for studies of how calmodulin is involved in higher plant and algal cell function, recent biochemical studies have demonstrated unique structural characteristics for this highly conserved protein. Enzym‐ological analyses have demonstrated novel functional properties and provided limited insight into the molecular mechanisms of calmodulin action. This review will summarize much of the early work, but will concern itself mainly with the methods and approaches that are used to study plant and animal calmodulins as well as models for how calmodulin may be involved in plant cell function.