Developmental defects and seedling lethality in apyrase AtAPY1 and AtAPY2 double knockout mutants
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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.
KeywordsApyrase Cell division Cell expansion Stomatal patterning Pollen-specific promoter Seedling lethality
Arabidopsis thaliana apyrase 1
Arabidopsis thaliana apyrase 2
Murashige and Skoog basal salt mixture
Open reading frame
Shaker pollen inward K+ channel
Wild type or wild-type
We thank A. A. Very and K. Mouline, both from H. Sentenac’s lab, for providing SPIK:GUS seeds and the plasmid containing the SPIK promoter, respectively. We are also grateful to Y. Sun from S. J. Roux’s lab for seeds from the AtAPY1:GUS and AtAPY2:GUS lines, to M. Curtis for the plant transformation vector pMDC123 and to J. Siemens for the A. tumefaciens strain GV3103. We gratefully acknowledge C. Seidel for training of C.W. on microsectioning, A. Lloyd and S. J. Roux for discussion of the electron micrographs and T. Graber for critical reading of the manuscript. Portions of this research by M.H. and I.S. were made possible with financial support from the German Academic Exchange Service (DAAD).
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