Developmental defects and seedling lethality in apyrase AtAPY1 and AtAPY2 double knockout mutants

Abstract

Previously it was shown that the Arabidopsis apyrase genes AtAPY1 and AtAPY2 are crucial for male fertility because mutant pollen (apy1-1; apy2-1) with T-DNA insertions in both genes could not germinate (Steinebrunner et al. (2003) Plant Physiol. 131: 1638-1647). In this study, pollen germination was restored and apyrase T-DNA double knockouts (DKO) apy1-1/apy1-1; apy2-1/apy2-1 were generated by complementation with AtAPY2 under the control of a pollen-specific promoter. The DKO phenotype displayed developmental defects including the lack of functional root and shoot meristems. In cotyledons, morphogenetic and patterning abnormalities were apparent, e.g., unlobed pavement cells and stomatal clusters. Another set of lines was created which carried either AtAPY1 or AtAPY2 under a dexamethasone-(DEX)-inducible promoter as an additional transgene to the pollen-specific gene construct. Application of DEX did not reverse the DKO phenotype to wild-type, but some inducible lines exhibited less severe defects even in the absence of the inducer, probably due to some background expression. However, even these DKO mutants were seedling-lethal and shared other defects regarding cell division, cell expansion and stomatal patterning. Taken together, the defects in the DKO mutants demonstrate that AtAPY1 and AtAPY2 are essential for normal plant development.

Extracellular Nucleotides and Apyrases Regulate Stomatal Aperture in Arabidopsis

“…During the day, when stomates are generally open, APY1 and APY2 promoter activity was observed in guard cells ( Fig. 2A, top left panel), as published previously (Wolf et al, 2007). Higher humidity levels of 85% relative air humidity (RH), which increase stomata opening, also increased the GUS staining of the guard cells ( Fig.…”

“…Total suppression of both APY1 and APY2 expression is lethal (Steinebrunner et al, 2003;Wolf et al, 2007), so partial suppression of these genes by RNAi has been the preferred genetic approach to see how a reduction in apyrase expression affects plant growth and development (Wu et al, 2007;Clark et al, 2010b). These RNAi-suppressed plants are apy2 knockouts and are conditionally suppressed in APY1 expression via RNAi under the inducer estradiol (Wu et al, 2007).…”

“…An additional rationale for testing the role of eATP in stomata function was the evidence of Wolf et al (2007) that the apyrase (nucleoside triphosphate diphosphohydrolase) enzymes APY1 and APY2 were strongly expressed in guard cells of Arabidopsis (Arabidopsis thaliana). These observations led us to test whether the heightened presence of APY1 and APY2 in guard cells reflected a role for eATP in guard cell function and whether eATP signaling could be upstream of the NO and ROS signals known to regulate stomatal aperture.…”

Isolation and Proteomic Characterization of the Arabidopsis Golgi Defines Functional and Novel Components Involved in Plant Cell Wall Biosynthesis

“…Both ATAPY2 and ATAPY1 proteins share high sequence identity (Steinebrunner et al, 2000), indicating that ATAPY2 may also function as a Golgi-resident NDPase. The expression of both ATAPY1 and ATAPY2 is fairly ubiquitous throughout the plant, with increased expression associated with dividing cells, an expression pattern consistent with their roles as NDPases (Wolf et al, 2007;Wu et al, 2007).…”

Extracellular Nucleotides Elicit Cytosolic Free Calcium Oscillations in Arabidopsis

“…A number of reports suggest that plant extracellular ATP levels could be modulated through the action of ectoapyrases (Steinebrunner et al, 2003;Wolf et al, 2007;Wu et al, 2007;Govindarajulu et al, 2009); that is, membrane-associated nucleoside triphosphohydrolases and diphosphohydrolases with the predicted catalytic domain in the apoplast. Hence, it is possible that the differential response to ADP and ADPbS was due to the hydrolysis of nucleotides by ectoapyrase.…”