Characterization of four plasma membrane aquaporins in tulip petals: A putative homolog is regulated by phosphorylation
Characterization of four plasma membrane aquaporins in tulip petals: A putative homolog is regulated by phosphorylation
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DOI：
10.1093/pcp/pcn095
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发表时间：
2008-08-01
影响因子：
4.9
通讯作者：
Shibata, Hitoshi
中科院分区：
文献类型：
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作者：
Azad, Abul Kalam;Katsuhara, Maki;Shibata, Hitoshi
我们以前建议，温度依赖性郁金香（郁金香）花瓣运动，伴随着水的运输是由一个身份不明的质膜内在蛋白（PIP）的可逆磷酸化调节。本研究从郁金香花瓣中鉴定并克隆了4个PIP的全长cDNA。两个PIP，即TgPIP 1; 1和TgPIP 1; TgPIP 2;1和TgPIP 2;2属于水通道蛋白的PIP 2亚家族，并根据植物中PIP基因的命名法命名。在这四个同源物中，只有TgPIP 2;2在使用非洲爪蟾卵母细胞的异源表达测定中显示出显著的水通道活性。水通道活性的这种功能亚型被废除汞和蛋白激酶和蛋白磷酸酶的抑制剂的影响。使用定点突变的方法，取代几个丝氨酸残基与丙氨酸，并评估水通道活性，使用甲基营养型酵母巴斯德毕赤酵母表达测定，我们表明，Ser 35，Ser 116和Ser 274是TgPIP 2的推定磷酸化位点;2。实时荧光定量RT-PCR分析表明，TgPIP 1;1和TgPIP 1;2在郁金香花瓣、茎、叶、鳞茎和根中的转录水平较TgPIP 2;1和TgPIP 2;2低。TgPIP 2;1的转录水平在根中可忽略不计，并且TgPIP 2;2在所有器官中以显著的转录水平普遍表达。从本文报道的数据，我们建议TgPIP 2;2可能通过磷酸化和去磷酸化调节水通道活性，并可能在所有郁金香器官的跨细胞水运输中发挥作用。
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 transcriptionPCR 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.