AtAPY1 and AtAPY2 Function as Golgi localized Nucleoside Diphosphatases in Arabidopsis thaliana.
ABSTRACT NTPDases (Apyrases) (EC 184.108.40.206) 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.
SourceAvailable from: Shunnosuke Abe[Show abstract] [Hide abstract]
ABSTRACT: Apyrase (ATP diphosphohydrolase, EC 220.127.116.11) 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: Animal and plant cells release nucleotides into their extracellular matrix when touched, wounded, and when their plasma membranes are stretched during delivery of secretory vesicles and growth. These released nucleotides then function as signaling agents that induce rapid increases in the concentration of cytosolic calcium, nitric oxide and superoxide. These, in turn, are transduced into downstream physiological changes. These changes in plants include changes in the growth of diverse tissues, in gravitropism, and in the opening and closing of stomates. The concentration of extracellular nucleotides is controlled by various phosphatases, prominent among which are apyrases EC 18.104.22.168 (nucleoside triphosphate diphosphohydrolases, NTPDases). This review provides phylogenetic and pHMM analyses of plant apyrases as well as analysis of predicted post-translational modifications for Arabidopsis apyrases. This review also summarizes and discusses recent advances in research on the roles of apyrases and extracellular nucleotides in controlling plant growth and development. These include new findings that document how apyrases and extracellular nucleotides control auxin transport, modulate stomatal aperture, and mediate biotic and abiotic stress responses, and on how apyrase suppression leads to growth inhibition.Plant Science 08/2014; 225:107–116. DOI:10.1016/j.plantsci.2014.06.002 · 4.11 Impact Factor
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ABSTRACT: The glycosyltransferases (GTs) are an important and functionally diverse family of enzymes involved in glycan and glycoside biosynthesis. Plants have evolved large families of GTs which undertake the array of glycosylation reactions that occur during plant development and growth. Based on the Carbohydrate-Active enZymes (CAZy) database, the genome of the reference plant Arabidopsis thaliana codes for over 450 GTs, while the rice genome (Oryza sativa) contains over 600 members. Collectively, GTs from these reference plants can be classified into over 40 distinct GT families. Although these enzymes are involved in many important plant specific processes such as cell wall and secondary metabolite biosynthesis, few have been functionally characterized. We have sought to develop a plant GTs clone resource that will enable functional genomic approaches to be undertaken by the plant research community. A total of 403 (88%) of CAZy defined Arabidopsis GTs have been cloned, while 96 (15%) of the GTs coded by rice have been cloned. The collection resulted in the update of a number of Arabidopsis GT gene models. The clones represent full-length coding sequences without termination codons and are Gateway® compatible. To demonstrate the utility of this JBEI GT collection, a set of efficient particle bombardment plasmids (pBullet) was also constructed with markers for the endomembrane. The utility of the pBullet collection was demonstrated by localizing all members of the Arabidopsis GT14 family to the Golgi apparatus or the ER. Updates to these resources are available at the JBEI GT Collection website http://gt.jbei.org/.The Plant Journal 08/2014; DOI:10.1111/tpj.12577 · 6.82 Impact Factor