Plant and Cell Physiology Advance Access originally published online on June 20, 2008
Plant and Cell Physiology 2008 49(8):1196-1208; doi:10.1093/pcp/pcn095
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Characterization of Four Plasma Membrane Aquaporins in Tulip Petals: A Putative Homolog is Regulated by Phosphorylation
1Department of Life Science and Biotechnology, Shimane University, Shimane, 690-8504 Japan
2Department of Biotechnology, Shahjalal University of Science and Technology, Sylhet 3114, Bangladesh
3Research Institute of Bioresources, Okayama University, Japan
*Corresponding author: E-mail, shibata{at}life.shimane-u.ac.jp; Fax, +81-0852-32-6092.
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Abstract |
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We suggested previously that temperature-dependent tulip (Tulipa gesneriana) petal movement that is concomitant with water transport is regulated by reversible phosphorylation of an unidentified plasma membrane intrinsic protein (PIP). In this study, four full-length cDNAs of PIPs from tulip petals were identified and cloned. Two PIPs, namely TgPIP1;1 and TgPIP1;2, are members of the PIP1 subfamily, and the remaining two PIPs, namely TgPIP2;1 and TgPIP2;2, belong to the PIP2 subfamily of aquaporins and were named according to the nomenclature of PIP genes in plants. Of these four homologs, only TgPIP2;2 displayed significant water channel activity in the heterologous expression assay using Xenopus laevis oocytes. The water channel activity of this functional isoform was abolished by mercury and was affected by inhibitors of protein kinase and protein phosphatase. Using a site-directed mutagenesis approach to substitute several serine residues with alanine, and assessing water channel activity using the methylotrophic yeast Pichia pastoris expression assay, we showed that Ser35, Ser116 and Ser274 are the putative phosphorylation sites of TgPIP2;2. Real-time reverse transcription–PCR analysis revealed that the transcript levels of TgPIP1;1 and TgPIP1;2 in tulip petals, stems, leaves, bulbs and roots are very low when compared with those of TgPIP2;1 and TgPIP2;2. The transcript level of TgPIP2;1 is negligible in roots, and TgPIP2;2 is ubiquitously expressed in all organs with significant transcript levels. From the data reported herein, we suggest that TgPIP2;2 might be modulated by phosphorylation and dephosphorylation for regulating water channel activity, and may play a role in transcellular water transport in all tulip organs.
Keywords: Aquaporin - Phosphorylation - PIP - Tulip - Water channel activity
Abbreviations: cRNA, complementary RNA; DMSO, dimethylsulfoxide; MBS, modified Barth's solution; OA, okadaic acid; ORF, open reading frame; Pf, osmotic water permeability coefficient; PIP, plasma membrane intrinsic protein; RACE, rapid amplification of cDNA ends; RMS, root-mean-square; RT–PCR, reverse transcription–PCR; TgPIP, Tulipa gesneriana PIP; TM, tramsmembrane; UTR, untranslated region
(Received May 4, 2008; Accepted June 17, 2008)
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