持续激活型CIPK9在花粉管生长过程中的生物学功能及亚细胞定位分析
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摘要
植物的花粉管生长是一个多因素参与的生理学过程,需要多种信号传导系统来引导植物细胞完成。钙离子作为第二信使,可以通过钙传感器CBLs激活下游的蛋白激酶CIPKs参与调控细胞的极性发育过程。该研究中CIPK9被确定为候选基因,其C端与绿色荧光蛋白(GFP)相融合,通过基因枪技术在烟草花粉中进行瞬时表达,观察对应的亚细胞定位及花粉管中诱导的表型。结果表明:(1) GFP标记的CIPK9定位于花粉管中高速运动的颗粒状细胞器,并可随胞质环流进行规律的运动,为进一步探究CIPK9的生物学功能,还构建了持续激活型CIPK9(CACIPK9)。(2)与全长CIPK9相比较,CACIPK9缺少C末端的调控区域,并在激酶区域的激活环中进行了点突变,从而表现出不受调控的持续高活性。(3)缺少C端调控区的CACIPK9表现出非特异性的亚细胞定位,即与GFP对照相同的胞内弥散定位,说明CIPK9的C末端调控区对于其在花粉管中的正确定位发挥重要的调控作用。另外,CACIPK9过表达可以引起花粉管的去极化生长表型。这表明CIPK9作为钙信号下游家族的一员参与了花粉管极性生长的相关过程,并对花粉管的生长具有一定的调控作用。
Pollen tube growth is a physiological process in which multi-factors are involved. The entire growth process requires multiple signaling systems to regulate plant cell growth and orientation. Calcium,as the second messenger,is achieved by calcium sensors CBLs,and then activates downstream target CIPKs to participate in the regulation of cell polarity development. In this study,CIPK9 was identified as a candidate gene,and subsequently the PCR-amplified coding region of CIPK9 was cloned into p LAT52-GFP vector for transient expression in pollen tubes. The C-terminus of CIPK9 was fused to green fluorescent protein( GFP),and the resulting constructs were transiently expressed in tobacco pollen grains by gene-gun transform technology. CIPK9-GFP subcellular localization and biological function in pollen tube were studied by transient expression system( particle bombardment). The results were as follows:( 1) GFP-labeled CIPK9 was localized in granular organelles with high-speed movement in pollen tubes,and shifted regularly with the cytoplasmic circulation. In order to further study the biological function of CIPK9,a constitutively active CIPK9( CACIPK9) was constructed.( 2) Compared with full-length CIPK9,CACIPK9 lacked the regulatory region at the Cterminal and had a Thr-178-to-Asp point mutation in the activation loop containing conserved Asp-Phe-Gly and Ala-ProGlu motifs. CACIPK9 thus had sustained high kinase activity without regulation.( 3) While CACIPK9 lacking Cterminal regulatory region evenly distributed in the pollen tube cytoplasm,similar to that of GFP control,suggesting that the C-terminal of CIPK9 plays an important role in the correct subcellular localization of CIPK9 in pollen tube. In addition,overexpression of CACIPK9 could induce the depolarization of pollen tube growth. In conclusion,CIPK9,as a member of the downstream family of calcium signaling,participates in the process of pollen tube polarity growth and plays a certain role in pollen tube growth.
Pollen tube growth is a physiological process in which multi-factors are involved. The entire growth process requires multiple signaling systems to regulate plant cell growth and orientation. Calcium,as the second messenger,is achieved by calcium sensors CBLs,and then activates downstream target CIPKs to participate in the regulation of cell polarity development. In this study,CIPK9 was identified as a candidate gene,and subsequently the PCR-amplified coding region of CIPK9 was cloned into p LAT52-GFP vector for transient expression in pollen tubes. The C-terminus of CIPK9 was fused to green fluorescent protein( GFP),and the resulting constructs were transiently expressed in tobacco pollen grains by gene-gun transform technology. CIPK9-GFP subcellular localization and biological function in pollen tube were studied by transient expression system( particle bombardment). The results were as follows:( 1) GFP-labeled CIPK9 was localized in granular organelles with high-speed movement in pollen tubes,and shifted regularly with the cytoplasmic circulation. In order to further study the biological function of CIPK9,a constitutively active CIPK9( CACIPK9) was constructed.( 2) Compared with full-length CIPK9,CACIPK9 lacked the regulatory region at the Cterminal and had a Thr-178-to-Asp point mutation in the activation loop containing conserved Asp-Phe-Gly and Ala-ProGlu motifs. CACIPK9 thus had sustained high kinase activity without regulation.( 3) While CACIPK9 lacking Cterminal regulatory region evenly distributed in the pollen tube cytoplasm,similar to that of GFP control,suggesting that the C-terminal of CIPK9 plays an important role in the correct subcellular localization of CIPK9 in pollen tube. In addition,overexpression of CACIPK9 could induce the depolarization of pollen tube growth. In conclusion,CIPK9,as a member of the downstream family of calcium signaling,participates in the process of pollen tube polarity growth and plays a certain role in pollen tube growth.
引文
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LIU J,ISHITANI M,HALFTER U,et al.,2000.The Arabidopsis thaliana SOS2 gene encodes a protein kinase that is required for salt tolerance[J].PNAS,97(7):3730-3734.
LUAN S,KUDLA J,RODRIGUEZ-CONCEPCION M,et al.,2002.Calmodulins and calcineurin B-like proteins:Calcium sensors for specific signal response coupling in plants[J].Plant Cell,14(Suppl.):389-400.
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MCCORMACK E,TSAI YC,BRAAM J,2005.Handling calcium signaling:Arabidopsis Ca Ms and CMLs[J].Trends Plant Sci,10(8):383-389.
OHTA M,GUO Y,HALFTER U,et al.,2003.A novel domain in the protein kinase SOS2 mediates interaction with the protein phosphatase 2C ABI2[J].PNAS,100(20):11771-11776.
QUAN R,LIN H,MENDOZA I,et al.,2007.SCABP8/CBL10,a putative calcium sensor,interacts with the protein kinase SOS2 to protect Arabidopsis shoots from salt stress[J].Plant Cell,19(4):1415-1431.
ROUNDS CM,BEZANILLA M,2013.Growth mechanisms in tipgrowing plant cells[J].Ann Rev Plant Biol,64:243-265.
SHI J,KIM KN,RITZ O,et al.,1999.Novel protein kinases associated with calcineurin B-like calcium sensors in Arabidopsis[J].Plant Cell,11(12):2393-2405.
WU G,GU Y,LI S,et al.,2001.A genome-wide analysis of Arabidopsis Rop-interactive CRIB motif-containing proteins that act as Rop GTPase targets[J].Plant Cell,13(12):2841-2856.
XU J,LI HD,CHEN LQ,et al.,2006.A protein kinase,interacting with two calcineurin B-like proteins,regulates K+transporter AKT1 in Arabidopsis[J].Cell,125(7):1347-1360.
YANG Z,2002.Small GTPases versatile signaling switches in plants[J].Plant Cell,14(Suppl.1):375-388.
YANG Z,2008.Cell polarity signaling in Arabidopsis[J].Ann Rev Cell Dev Biol,24:551-575.
CHEONG YH,PANDEY GK,GRANT JJ,et al.,2007.Two calcineurin B-like calcium sensors,interacting with protein kinase CIPK23,regulate leaf transpiration and root potassium uptake in Arabidopsis[J].Plant J,52(2):223-239.
FU Y,WU G,YANG Z,2001.Rop GTPase-dependent dynamics of tip-localized F-actin controls tip growth in pollen tubes[J].J Cell Biol,152(5):1019-1032.
GUO Y,HALFTER U,ISHITANI M,et al.,2001.Molecular characterization of functional domains in the protein kinase SOS2 that is required for plant salt tolerance[J].Plant Cell,13(6):1383-1400.
HEPLER PK,KUNKEL JG,ROUNDS CM,et al.,2011.Calcium entry into pollen tubes[J].Trends Plant Sci,17(1):32-38.
KIM BG,WAADT R,CHEONG YH,et al.,2007.The calcium sensor CBL10 mediates salt tolerance by regulating ion homeostasis in Arabidopsis[J].Plant J,52(3):473-484.
KIM KN,CHEONG YH,GUPTA R,et al.,2000.Interaction specificity of Arabidopsis calcineurin B-like calcium sensors and their target kinases[J].Plant Physiol,124(4):1844-1853.
KOLUKISAOGLU U,WEINL S,BLAZEVIC D,et al.,2004.Calcium sensors and their interacting protein kinases:Genomics of the Arabidopsis and rice CBL-CIPK signaling networks[J].Plant Physiol,134(1):43-58.
KROEGER J,GEITMANN A,2012.The pollen tube paradigm revisited[J].Curr Opin Plant Biol,15(6):618-624.
LIU J,ISHITANI M,HALFTER U,et al.,2000.The Arabidopsis thaliana SOS2 gene encodes a protein kinase that is required for salt tolerance[J].PNAS,97(7):3730-3734.
LUAN S,KUDLA J,RODRIGUEZ-CONCEPCION M,et al.,2002.Calmodulins and calcineurin B-like proteins:Calcium sensors for specific signal response coupling in plants[J].Plant Cell,14(Suppl.):389-400.
LUAN S,2009.The CBL-CIPK network in plant calcium signaling[J].Trends Plant Sci,14(1):37-42.
MCCORMACK E,TSAI YC,BRAAM J,2005.Handling calcium signaling:Arabidopsis Ca Ms and CMLs[J].Trends Plant Sci,10(8):383-389.
OHTA M,GUO Y,HALFTER U,et al.,2003.A novel domain in the protein kinase SOS2 mediates interaction with the protein phosphatase 2C ABI2[J].PNAS,100(20):11771-11776.
QUAN R,LIN H,MENDOZA I,et al.,2007.SCABP8/CBL10,a putative calcium sensor,interacts with the protein kinase SOS2 to protect Arabidopsis shoots from salt stress[J].Plant Cell,19(4):1415-1431.
ROUNDS CM,BEZANILLA M,2013.Growth mechanisms in tipgrowing plant cells[J].Ann Rev Plant Biol,64:243-265.
SHI J,KIM KN,RITZ O,et al.,1999.Novel protein kinases associated with calcineurin B-like calcium sensors in Arabidopsis[J].Plant Cell,11(12):2393-2405.
WU G,GU Y,LI S,et al.,2001.A genome-wide analysis of Arabidopsis Rop-interactive CRIB motif-containing proteins that act as Rop GTPase targets[J].Plant Cell,13(12):2841-2856.
XU J,LI HD,CHEN LQ,et al.,2006.A protein kinase,interacting with two calcineurin B-like proteins,regulates K+transporter AKT1 in Arabidopsis[J].Cell,125(7):1347-1360.
YANG Z,2002.Small GTPases versatile signaling switches in plants[J].Plant Cell,14(Suppl.1):375-388.
YANG Z,2008.Cell polarity signaling in Arabidopsis[J].Ann Rev Cell Dev Biol,24:551-575.