AtAPY1 and AtAPY2 Function as Golgi localized Nucleoside Diphosphatases in Arabidopsis thaliana.
ABSTRACT NTPDases (Apyrases) (EC 220.127.116.11) hydrolyze di- and triphosphate nucleotides, but not monophosphate nucleotides. They are categorized as E-type ATPases, have a broad divalent cation (Mg(2+), Ca(2+)) requirement for activation, and are insensitive to inhibitors of F-type, P-type, and V-type ATPases. Among the seven NTPDases identified in Arabidopsis, only APYRASE 1 (AtAPY1) and APYRASE 2 (AtAPY2) have been previously characterized. In this work, either AtAPY1 or AtAPY2 tagged with C-terminal green fluorescence protein (GFP) driven by their respective native promoter can rescue the apy1 apy2 double knockout (apy1 apy2 dKO) successfully, and confocal microscopy reveals that these two Arabidopsis apyrases reside in the Golgi apparatus. In Saccharomyces cerevisiae, both AtAPY1 and AtAPY2 can complement the Golgi-localized GDA1 mutant rescuing its aberrant protein glycosylation phenotype. In Arabidopsis, microsomes of wildtype show higher substrate preferences toward UDP compared to other NDP substrates. Loss-of-function Arabidopsis AtAPY1 mutants exhibit reduced microsomal UDPase activity, and this activity is even more significantly reduced in the loss-of-function AtAPY2 mutant and in the AtAPY1/AtAPY2 RNAi technology repressor lines. Microsomes from wildtype plants also have detectable GDPase activity, which is significantly reduced in apy2 but not apy1 mutants. The GFP tagged AtAPY1 or AtAPY2 constructs in the apy1 apy2 dKO plants can restore microsomal UDP/GDPase activity confirming that they both also have functional competency. The cell walls of apy1, apy2 and the RNAi silenced lines all have increased composition of galactose, but the transport efficiency of UDP-galactose across microsomal membranes was not altered. Taken together these results reveal that AtAPY1 and AtAPY2 are Golgi localized nucleotide diphosphatases and are likely to have roles in regulating UDP/GDP concentrations in the Golgi lumen.
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ABSTRACT: Apyrase (ATP diphosphohydrolase, EC 18.104.22.168) catalyzes hydrolysis of nucleoside tri- and di-phosphates to nucleoside monophosphates and orthophosphates. In the present study, the spatio-temporal expression of an apyrase gene (PsAPY1) in pea (Pisum sativum L. var. Alaska), was investigated during early stages of apical hook development using nonradioactive mRNA in-situ hybridization. During the formation of apical hook; at 45 hours after sowing (HAS), expression of PsAPY1 was obvious in epidermis and vascular bundle. By 60 HAS, the apical hook was completely formed. At this stage, transcript accumulation became higher than at the previous stage and expression was also visible in the cortex tissues of the developing hook. However, at 78 HAS, the curvature of the hook was reduced and hook was in the process of opening. At this time, expression of PsAPY1 was visible in all the above-mentioned tissues although the level of expression was slightly lower than at the previous stage (60 HAS). Apical hook formation provides a unique mechanism of protection for delicate shoot meristem in dicot plants. Its establishment is orchestrated by differential elongation rates of cells within the structure. The expression pattern of a gene provides essential information concerning the likely appearance and localization of its encoded protein and this helps to understand the mechanism of development of plant cells and tissues. Higher expression of PsAPY1 during the process of hook development indicates its essential role in the process of formation and maintenance of hook curvature and thus aids in protection of delicate shoot meristem.Biologia 03/2014; 69(3). DOI:10.2478/s11756-013-0325-9 · 0.70 Impact Factor
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ABSTRACT: Recent evidence indicates that extracellular nucleotides regulate plant growth. Exogenous ATP has been shown to block auxin transport and gravitropic growth in primary roots of Arabidopsis. Cells limit the concentration of extracellular ATP in part through the activity of ectoapyrases (ecto-nucleoside triphosphate diphosphohydrolases), and two nearly identical Arabidopsis apyrases, APY1 and APY2, appear to share this function. These findings, plus the fact that suppression of APY1 and APY2 blocks growth in Arabidopsis, suggested that the expression of these apyrases could influence auxin transport. This report tests that hypothesis. The polar movement of [3H]IAA in both hypocotyl sections and primary roots of Arabidopsis seedlings was measured. In both tissues polar auxin transport was significantly reduced in apy2 null mutants when they were induced by estradiol to suppress the expression of APY1 by RNAi. In the hypocotyl assays the basal halves of APY-suppressed hypocotyls contained considerably lower free IAA levels when compared to wild-type plants, and disrupted auxin transport in the APY-suppressed roots was reflected by their significant morphological abnormalities. When a GFP fluorescence signal encoded by a DR5:GFP construct was measured in primary roots whose apyrase expression was suppressed either genetically or chemically, the roots showed no signal asymmetry following gravistimulation, and both their growth and gravitropic curvature were inhibited. Chemicals that suppress apyrase activity also inhibit gravitropic curvature and, to a lesser extent, growth. Taken together these results indicate that a critical step connecting apyrase suppression to growth suppression is the inhibition of polar auxin transport.Plant physiology 10/2012; DOI:10.1104/pp.112.202887 · 7.39 Impact Factor
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ABSTRACT: A mutation in the ALTERED XYLOGLUCAN 9 (AXY9) gene was found to be causative for the decreased xyloglucan acetylation phenotype of the axy9.1 mutant, which was identified in a forward genetic screen for Arabidopsis thaliana mutants. The axy9.1 mutant also exhibits decreased O-acetylation of xylan, implying the AXY9 protein has a broad role in polysaccharide acetylation. An axy9 T-DNA mutant exhibits severe growth defects and collapsed xylem, demonstrating the importance of wall polysaccharide O-acetylation for normal plant growth and development. Localization and topological experiments indicate that the active site of the AXY9 protein resides within the Golgi lumen. The AXY9 protein appears to be a component of the plant cell wall polysaccharide acetylation pathway, which also includes the RWA and TBL proteins. The AXY9 protein is distinct from the TBLs, reported to be polysaccharide acetyltransferases, but does share homology with them and other acetyltransferases suggesting that the AXY9 protein may act to produce an acetylated intermediate that is part of the O-acetylation pathway. Copyright © 2015, American Society of Plant Biologists.Plant physiology 02/2015; 167(4). DOI:10.1104/pp.114.256479 · 7.39 Impact Factor