拟南芥CBL9的质膜定位对花粉管顶端生长的影响
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摘要
旨在揭示拟南芥CBL9(Calcineurin B-like protein 9)在花粉管顶端生长中的作用。基于花粉管瞬时表达体系,通过基因枪技术将带有黄色荧光蛋白(YFP)标签的CBL9在烟草花粉中进行瞬时表达,观察其亚细胞定位及过表达表型。针对CBL9的N端酰基化的保守位点,通过点突变技术构建CBL9G2A突变体,对比研究其表型及定位的改变情况。结果显示CBL9-YFP定位于花粉管顶端质膜及颗粒状细胞器上,CBL9过量表达可以引起显著的花粉管去极化生长。而CBL9G2A-YFP则表现出非特异性的胞内弥散定位,同时CBL9G2A过量表达没有显著影响花粉管的顶端生长。CBL9的N端酰基化位点是CBL9质膜定位的重要因素,而正确定位的CBL9才能发挥调控花粉管生长的相关功能。
This work is aimed to understand the role of Arabidopsis thaliana CBL9(calcineurin B-like protein 9)in the growth of pollen tube tip. Based on the transient expression system,the CBL9 with YFP-tag were transiently expressed in tobacco pollen grains by gene gun,and the overexpression phenotypes and subcellular localization patterns of CBL9-YFP were observed. At the conservative sites for N-terminal acylation of CBL9,a mutant version of CBL9,CBL9 G2 A was constructed via overlap-PCR-mediated site mutagenesis,and the changes in their phenotypes and localization were compared and studied. The results demonstrated that CBL9-YFP was localized to plasma membrane(PM)of growing pollen tubes and granular organelles. CBL9 overexpression induced a severe loss of growth polarity,leading to a short tube with the swelling tip. CBL9 G2 A was evenly distributed in cytosol of pollen tubes,and its overexpression did not cause the depolarization growth of pollen tubes. These results suggested that N-terminal acylation site of CBL9 is an important factor in correct targeting to PM,which is required for its function in polarized growth of pollen tubes.
This work is aimed to understand the role of Arabidopsis thaliana CBL9(calcineurin B-like protein 9)in the growth of pollen tube tip. Based on the transient expression system,the CBL9 with YFP-tag were transiently expressed in tobacco pollen grains by gene gun,and the overexpression phenotypes and subcellular localization patterns of CBL9-YFP were observed. At the conservative sites for N-terminal acylation of CBL9,a mutant version of CBL9,CBL9 G2 A was constructed via overlap-PCR-mediated site mutagenesis,and the changes in their phenotypes and localization were compared and studied. The results demonstrated that CBL9-YFP was localized to plasma membrane(PM)of growing pollen tubes and granular organelles. CBL9 overexpression induced a severe loss of growth polarity,leading to a short tube with the swelling tip. CBL9 G2 A was evenly distributed in cytosol of pollen tubes,and its overexpression did not cause the depolarization growth of pollen tubes. These results suggested that N-terminal acylation site of CBL9 is an important factor in correct targeting to PM,which is required for its function in polarized growth of pollen tubes.
引文
[1]Rounds CM, Bezanilla M. Growth mechanisms in tip-growing plant cells[J]. Annu Rev Plant Biol, 2013, 64(2):43-65.
[2]Qin Y, Yang Z. Rapid tip growth:insights from pollen tubes[J].Semin Cell Dev Biol, 2011, 22(8):816-824.
[3]Steinhorst L, Kudla J. Calcium-a central regulator of pollen germination and tube growth[J]. BBA-Mol Cell Res, 2013, 1833(7):1573-1581.
[4]Konrad KR, Wudick MM, Feijo JA. Calcium regulation of tip growth:new genes for old mechanisms[J]. Curr Opin Plant Biol,2011, 14(6):721-730.
[5]Hepler PK, Kunkel JG, Rounds CM, et al. Calcium entry into pollen tubes[J]. Trends Plant Sci, 2011, 17(1):32-38.
[6]Kroeger J, Geitmann A. The pollen tube paradigm revisited[J].Curr Opin Plant Biol, 2012, 15(6):618-624.
[7]Pierson E, Miller D, Callaham D, et al. Pollen tube growth is coupled to the extracellular calcium ion flux and the intracellular calcium gradient:effect of BAPTA-type buffers and hypertonic media[J].The Plant Cell, 1994, 6(12):1815-1828.
[8]Rathore K, Cork R, Robinson K. A cytoplasmic gradient of Ca2+is correlated with the growth of lily pollen tubes[J]. Dev Biol, 1991,148(2):612-619.
[9]Michard E, Lima PT, Borges F, et al. Glutamate receptor-like genes form Ca2+channelsinpollentubesandareregulatedbypistil D-serine[J]. Science, 2011, 332(6028):434-437.
[10]Frietsch S, Wang YF, Sladek C, et al. A cyclic nucleotide-gated channel is essential for polarized tip growth of pollen[J]. PNAS,2007, 104(36):14531-14536.
[11]Schiott M, Romanowsky SM, Baekgaard L, et al. A plant plasma membrane Ca2+pump is required for normal pollen tube growth and fertilization[J]. PNAS, 2004, 101(25):9502-9507.
[12]Gu Y, Fu Y, Dowd P, et al. A Rho family GTPase controls actin dynamics and tip growth via two counteracting downstream pathways in pollen tubes[J]. J Cell Biol, 2005, 169(1):127-138.
[13]Luan S. The CBL-CIPK network in plant calcium signaling[J].Trends Plant Sci, 2009, 14(1):37-42.
[14]HashimotoK,KudlaJ.Calciumdecodingmechanismsin plants[J]. Biochimie, 2011, 93(12):2054-2059.
[15]Kudla J, Batistic O, Hashimoto K. Calcium signals:The lead currency of plant information processing[J]. The Plant Cell,2010, 22(3):541-563.
[16]Xu J, Li HD, Chen LQ, et al. A protein kinase, interacting with two calcineurin B-like proteins, regulates K+transporterAKT1in Arabidopsis[J]. Cell, 2006, 125(7):1347-1360.
[17]Liu J, Zhu JK. A calcium sensor homolog required for plant salt tolerance[J]. Science, 1998, 280(5371):1943-1945.
[18]Mahs A, Steinhorst L, Han JP, et al. The calcineurin B-like Ca2+sensors CBL1 and CBL9 function in pollen germination and pollen tube growth in Arabidopsis[J]. Mol Plant, 2013, 6(4):1149-1162.
[19]Bloch D, Yalovsky S. Cell polarity signaling[J]. Curr Opin Plant Biol, 2013, 16(6):734-742.
[20]Chang F, Yan A, Zhao LN, et al. A putative calcium-permeable cyclic nucleotide-gated channel, CNGC18, regulates polarized pollen tube growth[J]. J Integr Plant Biol, 2007, 49(8):1261-1270.
[21]Batistic O. Plant calcineurin B-like proteins and their interacting protein kinases[J]. BBA-Mol Cell Res, 2009, 1793(6):985-992.
[22]Defalco TA, Bender KW, Snedden WA. Breaking the code:Ca2+sensors in plant signalling[J]. Biochem J, 2010, 425(1):27-40.
[2]Qin Y, Yang Z. Rapid tip growth:insights from pollen tubes[J].Semin Cell Dev Biol, 2011, 22(8):816-824.
[3]Steinhorst L, Kudla J. Calcium-a central regulator of pollen germination and tube growth[J]. BBA-Mol Cell Res, 2013, 1833(7):1573-1581.
[4]Konrad KR, Wudick MM, Feijo JA. Calcium regulation of tip growth:new genes for old mechanisms[J]. Curr Opin Plant Biol,2011, 14(6):721-730.
[5]Hepler PK, Kunkel JG, Rounds CM, et al. Calcium entry into pollen tubes[J]. Trends Plant Sci, 2011, 17(1):32-38.
[6]Kroeger J, Geitmann A. The pollen tube paradigm revisited[J].Curr Opin Plant Biol, 2012, 15(6):618-624.
[7]Pierson E, Miller D, Callaham D, et al. Pollen tube growth is coupled to the extracellular calcium ion flux and the intracellular calcium gradient:effect of BAPTA-type buffers and hypertonic media[J].The Plant Cell, 1994, 6(12):1815-1828.
[8]Rathore K, Cork R, Robinson K. A cytoplasmic gradient of Ca2+is correlated with the growth of lily pollen tubes[J]. Dev Biol, 1991,148(2):612-619.
[9]Michard E, Lima PT, Borges F, et al. Glutamate receptor-like genes form Ca2+channelsinpollentubesandareregulatedbypistil D-serine[J]. Science, 2011, 332(6028):434-437.
[10]Frietsch S, Wang YF, Sladek C, et al. A cyclic nucleotide-gated channel is essential for polarized tip growth of pollen[J]. PNAS,2007, 104(36):14531-14536.
[11]Schiott M, Romanowsky SM, Baekgaard L, et al. A plant plasma membrane Ca2+pump is required for normal pollen tube growth and fertilization[J]. PNAS, 2004, 101(25):9502-9507.
[12]Gu Y, Fu Y, Dowd P, et al. A Rho family GTPase controls actin dynamics and tip growth via two counteracting downstream pathways in pollen tubes[J]. J Cell Biol, 2005, 169(1):127-138.
[13]Luan S. The CBL-CIPK network in plant calcium signaling[J].Trends Plant Sci, 2009, 14(1):37-42.
[14]HashimotoK,KudlaJ.Calciumdecodingmechanismsin plants[J]. Biochimie, 2011, 93(12):2054-2059.
[15]Kudla J, Batistic O, Hashimoto K. Calcium signals:The lead currency of plant information processing[J]. The Plant Cell,2010, 22(3):541-563.
[16]Xu J, Li HD, Chen LQ, et al. A protein kinase, interacting with two calcineurin B-like proteins, regulates K+transporterAKT1in Arabidopsis[J]. Cell, 2006, 125(7):1347-1360.
[17]Liu J, Zhu JK. A calcium sensor homolog required for plant salt tolerance[J]. Science, 1998, 280(5371):1943-1945.
[18]Mahs A, Steinhorst L, Han JP, et al. The calcineurin B-like Ca2+sensors CBL1 and CBL9 function in pollen germination and pollen tube growth in Arabidopsis[J]. Mol Plant, 2013, 6(4):1149-1162.
[19]Bloch D, Yalovsky S. Cell polarity signaling[J]. Curr Opin Plant Biol, 2013, 16(6):734-742.
[20]Chang F, Yan A, Zhao LN, et al. A putative calcium-permeable cyclic nucleotide-gated channel, CNGC18, regulates polarized pollen tube growth[J]. J Integr Plant Biol, 2007, 49(8):1261-1270.
[21]Batistic O. Plant calcineurin B-like proteins and their interacting protein kinases[J]. BBA-Mol Cell Res, 2009, 1793(6):985-992.
[22]Defalco TA, Bender KW, Snedden WA. Breaking the code:Ca2+sensors in plant signalling[J]. Biochem J, 2010, 425(1):27-40.