Abstract
Apyrases hydrolyze nucleoside triphosphates and diphosphates and are found in all eukaryotes and a few prokaryotes. Although their enzymatic properties have been well characterized, relatively little is known regarding their subcellular localization and physiological function in plants. In this study, we used reverse genetic and biochemical approaches to investigate the role of potato (Solanum tuberosum)-specific apyrase. Silencing of the apyrase gene family with RNA interference constructs under the control of the constitutive 35S promoter led to a strong decrease in apyrase activity to below 10% of the wild-type level. This decreased activity led to phenotypic changes in the transgenic lines, including a general retardation in growth, an increase in tuber number per plant, and differences in tuber morphology. Silencing of apyrase under the control of a tuber-specific promoter led to similar changes in tuber morphology; however, there were no direct effects of apyrase inhibition on tuber metabolism. DNA microarrays revealed that decreased expression of apyrase leads to increased levels of transcripts coding for cell wall proteins involved in growth and genes involved in energy transfer and starch synthesis. To place these results in context, we determined the subcellular localization of the potato-specific apyrase. Using a combination of approaches, we were able to demonstrate that this enzyme is localized to the apoplast. We describe the evidence that underlies both this fact and that potato-specific apyrase has a crucial role in regulating growth and development.
Highlights
Apyrases hydrolyze nucleoside triphosphates and diphosphates and are found in all eukaryotes and a few prokaryotes
A comprehensive database search revealed tentative consensus (TC) sequences generated from ESTs for all three apyrases in the potato gene indices database provided by the Computational Biology and Functional Genomics Laboratory (Supplemental Table S1)
Complete or partial protein sequences were derived from these TC sequences and analyzed with regard to identity toward StAPY1 to StAPY3 and AtAPY1 and AtAPY2
Summary
Apyrases hydrolyze nucleoside triphosphates and diphosphates and are found in all eukaryotes and a few prokaryotes. DNA microarrays revealed that decreased expression of apyrase leads to increased levels of transcripts coding for cell wall proteins involved in growth and genes involved in energy transfer and starch synthesis. To place these results in context, we determined the subcellular localization of the potato-specific apyrase. On the basis of their proteomic analyses of Golgi preparations, Dunkley et al (2004) suggested a Golgi localization of AtAPY2 While these previous studies revealed a number of possible functions of apyrase, the general importance of this enzyme in plants has not been fully resolved. The combined results of these studies indicate that potato apyrase is an apoplastic enzyme of major importance for growth and development
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