Zahide Neslihan ÖZTÜRK, Steffen GREINER, Thomas RAUSCH

Subcellular localization and developmental regulation of cytosolic, soluble pyrophosphatase isoforms in Arabidopsis thaliana

Pyrophosphate (PPI) is the by-product of several reversible key reactions of primary metabolism. Thus, generated PPi should be removed by hydrolysis via pyrophosphatases to prevent accumulation and to drive anabolism. In plastids, a plastidic, soluble pyrophosphatase splits PPi released by ADPG pyrophosphorylase. The cytosolic PPi pool is believed to be hydrolyzed by tonoplast- and/or Golgi-integral H+-translocating pyrophosphatases. The Arabidopsis thaliana (L.) Heynh. genome encodes 6 soluble pyrophosphatase isoforms (PPas), 1 of which was shown to be localized in plastids. The remaining 5 of those PPas (PPas 1-5) are more similar to each other with highly conserved protein sequences. They all lack known targeting sequences and are thought to reside in the cytosol. To address their role and redundancy in plants, we performed (i) subcellular targeting of C-terminal PPa:EGFP fusions, (ii) analysis of transgenic promoter Beta-glucuronidase (GUS) lines to depict differential expression during plant development, and (iii) transcript quantification via real-time PCR to corroborate promoter-GUS data. The results revealed pronounced tissue specificity and developmental regulation for each PPa isoform, but also partial redundancy with coexpression of several PPa isoforms in all plant tissues.

Anahtar Kelimeler:

Brassicaceae, Cruciferae, soluble pyrophosphatase family, in silico subcellular localization, EGFP chimeras, histochemical GUS staining, real-time PCR

Subcellular localization and developmental regulation of cytosolic, soluble pyrophosphatase isoforms in Arabidopsis thaliana

Keywords:

Brassicaceae, Cruciferae, soluble pyrophosphatase family, in silico subcellular localization, EGFP chimeras, histochemical GUS staining, real-time PCR,

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Aydın S, Büyük İ, Aras ES (2014). Expression of SOD gene and evaluating its role in stress tolerance in NaCl and PEG stressed Lycopersicum esculentum. Turk J Bot 38: 89–98.
Baltscheffsky M, Schultz A, Baltscheffsky H (1999). H(+)-PPases: a tightly membrane-bound family. FEBS Lett 457: 527–533.
Casolo V, Micolini S, Macri F, Vianello A (2002). Pyrophosphate import and synthesis by plant mitochondria. Physiol Plantarum 114: 516–523.
Claros MG, Vincens P (1996). Computational method to predict mitochondrially imported proteins and their target sequences. Eur J Biochem 241: 779–786.
Clough SJ, Bent AF (1998). Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735–743.
Cokol M, Nair R, Rost B (2000). Finding nuclear localization signals. EMBO Rep 1: 411–415.
Dennis DT, Blakeley SD (2000). Carbohydrate metabolism. In: Buchanan BB, Gruissem W, Jones RL, editors. Biochemistry and Molecular Biology of Plants. Rockville, MD, USA: American Society of Plant Physiologists, pp. 630–675.
du Jardin P, Rojas-Beltran J, Gebhardt C, Brasseur R (1995). Molecular cloning and characterization of a soluble inorganic pyrophosphatase in potato. Plant Physiol 109: 853–860.
Emanuelsson O, Nielsen H, Brunak S, von Heijne G (2000). Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol 300: 1005–1016.
ExPASy:SIB Bioinformatics Resource Portal. Website: http://web. expasy.org/compute_pi/ [accessed 01 December 2013].
Ferjani A, Segami S, Horiguchi G, Muto Y, Maeshima M, Tsukaya H (2011). Keep an eye on PPi: the vacuolar-type H+- pyrophosphatase regulates postgerminative development in Arabidopsis. Plant Cell 23: 2895–2908.
Franceschi VR, Nakata PA (2005). Calcium oxalate in plants: formation and function. Annu Rev Plant Biol 56: 41–71.
George GM, van der Merwe MJ, Nunes-Nesi A, Bauer R, Fernie AR, Kossmann J, Lloyd JR (2010). Virus-induced gene silencing of plastidial soluble inorganic pyrophosphatase impairs essential leaf anabolic pathways and reduced drought stress tolerance in Nicotiana benthamiana. Plant Physiol 154: 55–66.
Gharari Z, Khavari Nejad R, Shekaste Band R, Najafi F, Nabiuni M, Irian S (2014). The role of Mn-SOD and Fe-SOD genes in the response to low temperature in chs mutants of Arabidopsis. Turk J Bot 38: 80–88.
Haasen D, Kohler C, Neuhaus G, Merkle T (1999). Nuclear export of proteins in plants: AtXPO1 is the export receptor for leucine- rich nuclear export signals in Arabidopsis thaliana. Plant J 20: 695–705.
Jelitto T, Sonnewald U, Willmitzer L, Hajirezaei M, Stitt M (1992). Inorganic pyrophosphate content and metabolites in potato and tobacco plants expressing E. coli pyrophosphatase in their cytosol. Planta 188: 238–244.
Kamiri M, Inzé D, Depicker A (2002). GATEWAY™ vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7: 193–195.
Koroleva OA, Tomlinson ML, Leader D, Shaw P, Doonan JH (2005). High-throughput protein localization in Arabidopsis using Agrobacterium-mediated transient expression of GFP-ORF fusions. Plant J 41: 162–174.
Lerchl J, Geigenberger P, Stitt M, Sonnewald U (1995). Impaired photoassimilate partitioning caused by phloem-specific removal of pyrophosphate can be complemented by a phloem- specific cytosolic yeast-derived invertase in transgenic plants. Plant Cell 7: 259–270.
Maeshima M (2000). Vacuolar H(+)-pyrophosphatase. Biochim Biophys Acta 1465: 37–51.
Maeshima M, Mimura T, Sato T (1994). Distribution of vacuolar H+- pyrophosphatase and a membrane integral protein in a variety of green plants. Plant Cell Physiol 35: 323–328.
Meyer K, Stecca KL, Ewell-Hicks K, Allen SM, Everard JD (2012). Oil and protein accumulation in developing seeds is influenced by the expression of a cytosolic pyrophosphatase in Arabidopsis. Plant Physiol 159: 1221–1234.
Muller PY, Janovjak H, Miserez AR, Dobbie Z (2002). Processing of gene expression data generated by quantitative real-time RT- PCR. Biotechniques 32: 1372–1379.
Nair R, Rost B (2005). Mimicking cellular sorting improves prediction of subcellular localization. J Mol Biol 348: 85–100.
Nakai K, Horton P (1999). PSORT: a program for detecting the sorting signals of proteins and predicting their subcellular localization. Trends Biochem Sci 24: 34–35.
Navarro-De la Sancha E, Coello-Coutino MP, Valencia-Turcotte LG, Hernandez-Dominguez EE, Trejo-Yepes G, Rodriguez- Sotres R (2007). Characterization of two soluble inorganic pyrophosphatases from Arabidopsis thaliana. Plant Sci 172: 796–807.
Noren H, Suensson P, Anderson B (2004). A convenient and versatile hydroponic cultivation system for Arabidopsis thaliana. Physiol Plantarum 121: 343–348.
Perez-Castineira JR, Gomez-Garcia R, Lopez-Marques RL, Losada M, Serrano A (2001). Enzymatic systems of inorganic pyrophosphate bioenergetics in photosynthetic and heterotrophic protists: remnants or metabolic cornerstones? Int Microbiol 4: 135–142.
Schulze S, Mant A, Kossmann J, Lloyd JR (2004). Identification of an Arabidopsis inorganic pyrophosphatase capable of being imported into chloroplasts. FEBS Lett 565: 101–105.
Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J et al (2011). Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7: 539.
Sivula T, Salminen A, Parfenyev AN, Pohjanjoki P, Goldman A, Cooperman BS, Baykov AA, Lahti R (1999). Evolutionary aspects of inorganic pyrophosphatase. FEBS Lett 454: 75–80.
Smart LB, Vojdani F, Maeshima M, Wilkins TA (1998). Genes involved in osmoregulation during turgor-driven cell expansion of developing cotton fibers are differentially regulated. Plant Physiol 116: 1539–1549.
Sonnewald U (1992). Expression of E. coli inorganic pyrophosphatase in transgenic plants alters photoassimilate partitioning. Plant J 2: 571–581.
Stitt M (1998). Pyrophosphate as an energy donor in the cytosol of plant cells: an enigmatic alternative to ATP. Bot Acta 111: 167–175.
The Arabidopsis Information Resource (TAIR). Website: http:// www.arabidopsis.org [accessed 01 December 2013].
Vianello A, Macri F (1999). Proton pumping pyrophosphatase from higher plant mitochondria. Physiol Plantarum 105: 763–768.
Webb MA (1999). Cell-mediated crystallization of calcium oxalate in plant. Plant Cell 11: 751–761.
Weiner H, Stitt M, Heldt HW (1987). Subcellular compartmentation of pyrophosphate and alkaline pyrophosphatase in leaves. Biochim Biophys Acta 893: 13–21.
Wroblewski T, Tomczak A, Michelmore R (2005). Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce, tomato and Arabidopsis. Plant Biotechnol J 3: 259–273.
Zancani M, Macri F, Dal Belin Peruffo A, Vianello A (1995). Isolation of the catalytic subunit of a membrane-bound H+- pyrophosphatase from pea stem mitochondria. Eur J Biochem 228: 138–143.