title: NO₃⁻/H⁺ antiport in the tonoplast of cucumber root cells is stimulated by nitrate supply: evidence for a reversible nitrate-induced phosphorylation of vacuolar NO₃⁻/H⁺ antiport.
abstract: Studies in the last few years have shed light on the process of nitrate accumulation within plant cells, achieving molecular identification and partial characterization of the genes and proteins involved in this process. However, contrary to the plasma membrane-localized nitrate transport activities, the kinetics of active nitrate influx into the vacuole and its adaptation to external nitrate availability remain poorly understood. In this work, we have investigated the activity and regulation of the tonoplast-localized H(+)/NO₃(-) antiport in cucumber roots in response to N starvation and NO₃(-) induction. The time course of nitrate availability strongly influenced H(+)/NO₃(-) antiport activity at the tonoplast of root cells. However, under N starvation active nitrate accumulation within the vacuole still occurred. Hence, either a constitutive H(+)-coupled transport system specific for nitrate operates at the tonoplast, or nitrate uses another transport protein of broader specificity to different anions to enter the vacuole via a proton-dependent process. H(+)/NO₃(-) antiport in cucumber was significantly stimulated in NO₃(-)-induced plants that were supplied with nitrate for 24 hours following 6-day-long N starvation. The cytosolic fraction isolated from the roots of NO₃(-)-induced plants significantly stimulated H(+)/NO₃(-) antiport in tonoplast membranes isolated from cucumbers growing on nitrate. The stimulatory effect of the cytosolic fraction was completely abolished by EGTA and the protein kinase inhibitor staurosporine and slightly enhanced by the phosphatase inhibitors okadaic acid and cantharidin. Hence, we conclude that stimulation of H(+)/NO₃(-) antiport at the tonoplast of cucumber roots in response to nitrate provision may occur through the phosphorylation of a membrane antiporter involving Ca-dependent, staurosporine-sensitive protein kinase.
authors.affiliation: Wrocław University, Institute of Experimental Biology, Department of Plant Molecular Physiology, Wrocław, Poland.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Migocka
authors.forename: Anna
authors.initials: A
authors.lastname: Warzybok
authors.forename: Anna
authors.initials: A
authors.lastname: Papierniak
authors.forename: Grażyna
authors.initials: G
authors.lastname: Kłobus
mesh_terms: D017920:Antiporters / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D018553:Cucumis sativus / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D006859:Hydrogen / Q000378:metabolism*; D006868:Hydrolysis; D009566:Nitrates / Q000378:metabolism* / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D059828:Plant Cells / Q000187:drug effects / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D018517:Plant Roots / Q000187:drug effects / Q000378:metabolism*; D011522:Protons; D013431:Sulfates / Q000378:metabolism; D022161:Transport Vesicles / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism
chemical_list: D017920:Antiporters; D002712:Chlorides; D009566:Nitrates; D010940:Plant Proteins; D011522:Protons; D013431:Sulfates; D006859:Hydrogen; D025262:Vacuolar Proton-Translocating ATPases
references.citation: Physiol Plant. 2004 May;121(1):84-92
references.pmid: 15086821
references.citation: Proc Natl Acad Sci U S A. 2008 Mar 18;105(11):4524-9
references.pmid: 18334638
references.citation: Plant J. 2009 Jan;57(2):264-78
references.pmid: 18798873
references.citation: Biochim Biophys Acta. 2008 Jul-Aug;1777(7-8):599-604
references.pmid: 18423392
references.citation: Plant Physiol. 1998 Feb;116(2):589-97
references.pmid: 9489011
references.citation: Plant Cell. 2004 Dec;16(12):3285-303
references.pmid: 15539469
references.citation: Plant Physiol. 1989 Jul;90(3):806-13
references.pmid: 16666881
references.citation: Philos Trans R Soc Lond B Biol Sci. 2009 Jan 27;364(1514):195-201
references.pmid: 18957376
references.citation: Curr Opin Plant Biol. 2010 Jun;13(3):266-73
references.pmid: 20093067
references.citation: Biophys J. 2003 Dec;85(6):3730-8
references.pmid: 14645064
references.citation: Plant Physiol. 1992 Jun;99(2):456-63
references.pmid: 16668907
references.citation: Plant J. 2000 Feb;21(3):259-67
references.pmid: 10758477
references.citation: Plant Cell. 1999 Apr;11(4):677-90
references.pmid: 10213786
references.citation: BMC Plant Biol. 2012 May 09;12:66
references.pmid: 22571503
references.citation: J Biol Chem. 2009 Sep 25;284(39):26526-32
references.pmid: 19636075
references.citation: Nature. 2006 Aug 24;442(7105):939-42
references.pmid: 16878138
references.citation: Plant Cell. 2003 Feb;15(2):347-64
references.pmid: 12566577
references.citation: Proc Natl Acad Sci U S A. 2010 Feb 16;107(7):3251-6
references.pmid: 20133698
references.citation: Anal Biochem. 1976 May 7;72:248-54
references.pmid: 942051
references.citation: Cell. 2009 Sep 18;138(6):1064-6
references.pmid: 19766561
references.citation: Cell Res. 2011 Jul;21(7):1116-30
references.pmid: 21445098
references.citation: Proc Natl Acad Sci U S A. 2006 Aug 15;103(33):12625-30
references.pmid: 16895985
references.citation: Planta. 1992 Jul;187(4):554-7
references.pmid: 24178152
references.citation: Plant Physiol. 1985 Jan;77(1):200-5
references.pmid: 16664008
references.citation: Plant Physiol. 2006 May;141(1):196-207
references.pmid: 16581873
references.citation: Cell. 2009 Sep 18;138(6):1184-94
references.pmid: 19766570
references.citation: Biochim Biophys Acta. 2000 May 1;1465(1-2):37-51
references.pmid: 10748246
references.citation: J Exp Bot. 2004 Sep;55(405):2005-14
references.pmid: 15310822
references.citation: Proc Natl Acad Sci U S A. 1985 Jun;82(11):3683-7
references.pmid: 16593570
references.citation: Plant Cell. 2007 May;19(5):1590-602
references.pmid: 17540716
references.citation: Plant Physiol. 1982 Nov;70(5):1335-40
references.pmid: 16662676
references.citation: Nucleic Acids Res. 2005 Nov 28;33(20):6494-506
references.pmid: 16314312
references.citation: Anal Biochem. 1980 Feb;102(1):110-9
references.pmid: 6986823
references.citation: Mol Plant. 2013 Nov;6(6):1984-7
references.pmid: 23645597
references.citation: FEBS Lett. 2007 May 25;581(12):2204-14
references.pmid: 17412324
references.citation: EMBO J. 2003 Mar 3;22(5):1005-13
references.pmid: 12606566
references.citation: Genome Res. 2000 Apr;10(4):516-22
references.pmid: 10779491
references.citation: Plant Physiol. 1992;99:1124-33
references.pmid: 11537883
references.citation: Physiol Plant. 2013 Feb;147(2):207-17
references.pmid: 22607526
references.citation: Cell. 1993 Mar 12;72(5):705-13
references.pmid: 8453665
references.citation: Plant Cell Physiol. 2010 Jun;51(6):960-8
references.pmid: 20430762
references.citation: J Plant Physiol. 2003 May;160(5):523-30
references.pmid: 12806781
references.citation: Plant Cell. 1999 Aug;11(8):1381-92
references.pmid: 10449574
references.citation: Proc Natl Acad Sci U S A. 1994 Aug 30;91(18):8547-51
references.pmid: 8078920
references.citation: Planta. 2012 Feb;235(2):311-23
references.pmid: 21904872
references.citation: Plant Physiol. 2007 Jan;143(1):425-33
references.pmid: 17085507
references.citation: Plant Physiol. 1986 Jun;81(2):497-502
references.pmid: 16664845
references.citation: Nat Genet. 2009 Dec;41(12):1275-81
references.pmid: 19881527
references.citation: J Exp Bot. 2003 Aug;54(389):1851-64
references.pmid: 12869520
references.citation: Plant Physiol. 1990 Aug;93(4):1426-32
references.pmid: 16667635
references.citation: Plant Physiol. 2010 Nov;154(3):1232-43
references.pmid: 20805328
references.citation: Plant J. 2007 Nov;52(3):473-84
references.pmid: 17825054
references.citation: Planta. 2004 Aug;219(4):714-21
references.pmid: 15107992
references.citation: Proc Natl Acad Sci U S A. 2006 Apr 25;103(17):6518-23
references.pmid: 16618929
references.citation: J Exp Bot. 2011 Oct;62(14):4903-16
references.pmid: 21705389
references.citation: Plant J. 1999 Mar;17(5):563-8
references.pmid: 10205909
references.citation: Plant Physiol. 1995 Dec;109(4):1277-1283
references.pmid: 12228668
references.citation: Biochim Biophys Acta. 2000 May 1;1465(1-2):219-35
references.pmid: 10748256
references.citation: Planta. 1988 Nov;176(2):235-40
references.pmid: 24220778
references.citation: Plant Physiol. 1987 Mar;83(3):490-6
references.pmid: 16665277
references.citation: Cell. 2006 Jun 30;125(7):1347-60
references.pmid: 16814720
references.citation: Annu Rev Plant Biol. 2011;62:25-51
references.pmid: 21275645
references.citation: Plant Physiol. 2000 Nov;124(3):1335-48
references.pmid: 11080309
references.citation: FEBS Lett. 2007 May 25;581(12):2290-300
references.pmid: 17481610