组成激活型CPK34对花粉管顶端生长的影响
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摘要
在花粉管的顶端生长过程中,胞内钙信号可以被钙传感器所识别,进而作用到下游多条信号通路上,以调控花粉管的生长和导向。CPKs作为一类重要的钙传感器,包含一个激酶区和一个钙调素样区,因此CPKs既是钙传感器,又是效应因子。基因芯片的数据显示存在5个花粉高量表达的CPKs,其中CPK34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。研究结果显示CPK34过量表达可以引起明显的花粉管生长障碍,导致花粉管顶端形成一个膨大的肿块。随后CPK34去除自抑制区和钙调素样区,只保留蛋白激酶区,从而构建了CPK34的组成激活(constitutively active, CA)形式(CACPK34)。相对于CPK34所造成的花粉管表型,CACPK34过量表达显示出更严重的去极性生长表型。花粉管的正常生长需要适宜的胞外钙离子浓度,过高或过低的钙浓度可以直接抑制花粉管的伸长。过量表达CPK34的花粉管表现出对低钙胁迫敏感性降低(极性生长部分恢复),而对高钙胁迫敏感性增加(去极化生长显著增强)。而对于缺少钙结合区域的CACPK34,其过量表达的花粉管在不同胁迫条件下未表现出明显的区别。实验结果对于深入了解细胞极性生长过程中的信号转导网络,将来利用分子工具进行人为干预,提高授粉效率等都具有一定的理论指导意义。
In the process of pollen tube growth, intracellular calcium signals can be recognized by calcium sensors, so as to act on multiple downstream signal pathways and to control the growth and orientation of the pollen tube. Therefore, CPKs was not only a calcium sensor, but also an effect factor. As a kind of important calcium sensor, CPKs was consist of a kinase domain and calmodulin-like domain. The data of gene chip show that there were five CPKs with high expression of pollen, wherein the CPK34 was fused with a green fluorescent protein(GFP), to made transient expression on tobacco pollen by gene gun technology. The results showed that CPK34 overexpression could cause a significant barrier to the growth of pollen tube, leading to a obvious growth disorder of pollen tube. Cpk34 removed the self-inhibition region and calmodulin-like region, and only retained the protease region, to constructed the constitutively active form(CA) of CPK34. Compared with the pollen tube phenotype caused by CPK34, CACPK34 overexpression showed more serious depolarization growth phenotype.The normal growth of pollen tube requires appropriate extracellular calcium concentration. Excessive or too low calcium can directly inhibit the elongation of pollen tube. The overexpression of CPK34 in the pollen tube showed a low sensitivity to low calcium stress(partial recovery of polar growth), but it was increased to high calcium stress(depolarization growth enhanced significantly). Due to the lack of calcium binding region, overexpressed pollen tube did not show significant difference under various stress conditions. The results have certain theoretical significance for further understanding the signal network in the process of cell polar growth, artificial intervention by molecular tools and improving pollination efficiency in the future.
In the process of pollen tube growth, intracellular calcium signals can be recognized by calcium sensors, so as to act on multiple downstream signal pathways and to control the growth and orientation of the pollen tube. Therefore, CPKs was not only a calcium sensor, but also an effect factor. As a kind of important calcium sensor, CPKs was consist of a kinase domain and calmodulin-like domain. The data of gene chip show that there were five CPKs with high expression of pollen, wherein the CPK34 was fused with a green fluorescent protein(GFP), to made transient expression on tobacco pollen by gene gun technology. The results showed that CPK34 overexpression could cause a significant barrier to the growth of pollen tube, leading to a obvious growth disorder of pollen tube. Cpk34 removed the self-inhibition region and calmodulin-like region, and only retained the protease region, to constructed the constitutively active form(CA) of CPK34. Compared with the pollen tube phenotype caused by CPK34, CACPK34 overexpression showed more serious depolarization growth phenotype.The normal growth of pollen tube requires appropriate extracellular calcium concentration. Excessive or too low calcium can directly inhibit the elongation of pollen tube. The overexpression of CPK34 in the pollen tube showed a low sensitivity to low calcium stress(partial recovery of polar growth), but it was increased to high calcium stress(depolarization growth enhanced significantly). Due to the lack of calcium binding region, overexpressed pollen tube did not show significant difference under various stress conditions. The results have certain theoretical significance for further understanding the signal network in the process of cell polar growth, artificial intervention by molecular tools and improving pollination efficiency in the future.
引文
Batistic O.,and Kudla J.,2012,Analysis of calcium signaling pathways in plants,Biochim.Biophys.Acta.,1820(8):1283-1293
Bloch D.,and Yalovsky S.,2013,Cell polarity signaling,Curr.Opin.Plant Biol.,16(6):734-742
Boudsocq M.,and Sheen J.,2013,CDPKs in immune and stress signaling,Trends Plant Sci.,18(1):30-40
Chang F.,Yan A.,Zhao L.N.,Wu W.H.,and Yang Z.,2007,Aputativ e calcium-permeable cyclic nucleotide-gated channel,CNGC18,regulates polarized pollen tube growth,J.Integr.Plant Biol.,49(8):1261-1270
Frietsch S.,Wang Y.F.,Sladek C.,Poulsen L.R.,Romanowsky S.M.,Schroeder J.I.,and Harper J.F.,2007,A cyclic nucleotide-gated channel is essential for polarized tip growth of pollen,Proc.Natl.Acad.Sci.USA,104(36):14531-14536
Fu Y.,Wu G.,and Yang Z.,2001,Rop GTPase-dependent dynamics of tip-localized F-actin controls tip growth in pollentubes,J.Cell Biol.,152(5):1019-1032
Guan Y.F.,Guo J.Z.,Li H.,and Yang Z.B.,2013,Signaling in pollen tube growth:crosstalk,feedback,and missing links,Mol.Plant,6(4):1053-1064
Hashimoto K.,and Kudla J.,2011,Calcium decoding mechanisms in plants,Biochimie,93(12):2054-2059
Hedrich R.,2012,Ion channels in plants,Physiol.Rev.,92(4):1777-1811
Hepler P.K.,Kunkel J.G.,Rounds C.M.,and Winship L.J.,2011,Calcium entry into pollen tubes,Trends Plant Sci.,17(1):32-38
Holdaway-Clarke T.L.,Feijo J.A.,Hackett G.R.,Kunkel J.G.,and Hepler P.K.,1997,Pollen tube growth and the intracellular cytosolic calcium gradient oscillate in phase while extracellular calcium influx is delayed,Plant Cell,9(11):1999-2010
Malho R.,and Trewavas A.J.,1996,Localized apical increases of cytosolic free calcium control pollen tube orientation,Plant Cell,8(11):1935-1949
Messerli M.,and Robinson K.R.,1997,Tip localized Ca2+pulses are coincident with peak pulsatile growth rates in pollen tubes of Lilium longiflorum,J.Cell Sci.,110(11):1269-1278
Michard E.,Lima P.T.,Borges F.,Silva A.C.,Portes M.T.,Carvalho J.E.,Gilliham M.,Liu L.H.,Obermeyer G.,and Feijo J.A.,2011,Glutamate receptor-like genes form Ca2+channels in pollen tubes and are regulated by pistil D-serine,Science,332(6028):434-437
Myers C.,Romanowsky S.M.,Barron Y.D.,Garg S.,Azuse C.L.,Curran A.,Davis R.M.,Hatton J.,Harmon A.C.,and Harper J.F.,2009,Calcium-dependent protein kinases regulate polarized tip growth in pollen tubes,Plant J.,59(4):528-539
Rounds C.M.,and Bezanilla M.,2013,Growth mechanisms in tip-growing plant cells,Annu.Rev.Plant Biol.,64:243-265
Schulz P.,Herde M.,and Romeis T.,2013,Calcium-dependent protein kinases:hubs in plant stress signaling and development,Plant Physiol.,163(2):523-530
Steinhorst L.,and Kudla J.,2013,Calcium-a central regulator of pollen germination and tube growth,BBA-Mol.Cell Res.,1833(7):1573-1581
Yang Z.,2008,Cell polarity signaling in Arabidopsis,Annu.Rev.Cell Dev.Biol.,24:551-575
Yoon G.M.,Dowd P.E.,Gilroy S.,and McCubbin A.G.,2006,Calcium-dependent protein kinase isoforms in Petunia have distinct functions in pollen tube growth,including regulating polarity,Plant Cell,18(4):867-878
Zhao L.N.,Shen L.K.,Zhang W.Z.,Zhang W.,Wang Y.,and Wu W.H.,2013,Ca2+-dependent protein kinase11 and 24 modulate the activity of the inward rectifying K+channels in Arabidopsis pollen tubes,Plant Cell,25(2):649-661
Zhou L.M.,Fu Y.,and Yang Z.B.,2009,A genome-wide functional characterization of Arabidopsis regulatory calcium sensors in pollen tubes,J.Integr.Plant Biol.,51(8):751-761
Zhou L.M.,Lan W.Z.,Jiang Y.Q.,Fang W.,and Luan S.,2014,A calcium-dependent protein kinase interacts with and activates a calcium channel to regulate pollen tube growth,Mol.Plant,7(2):369-376
Bloch D.,and Yalovsky S.,2013,Cell polarity signaling,Curr.Opin.Plant Biol.,16(6):734-742
Boudsocq M.,and Sheen J.,2013,CDPKs in immune and stress signaling,Trends Plant Sci.,18(1):30-40
Chang F.,Yan A.,Zhao L.N.,Wu W.H.,and Yang Z.,2007,Aputativ e calcium-permeable cyclic nucleotide-gated channel,CNGC18,regulates polarized pollen tube growth,J.Integr.Plant Biol.,49(8):1261-1270
Frietsch S.,Wang Y.F.,Sladek C.,Poulsen L.R.,Romanowsky S.M.,Schroeder J.I.,and Harper J.F.,2007,A cyclic nucleotide-gated channel is essential for polarized tip growth of pollen,Proc.Natl.Acad.Sci.USA,104(36):14531-14536
Fu Y.,Wu G.,and Yang Z.,2001,Rop GTPase-dependent dynamics of tip-localized F-actin controls tip growth in pollentubes,J.Cell Biol.,152(5):1019-1032
Guan Y.F.,Guo J.Z.,Li H.,and Yang Z.B.,2013,Signaling in pollen tube growth:crosstalk,feedback,and missing links,Mol.Plant,6(4):1053-1064
Hashimoto K.,and Kudla J.,2011,Calcium decoding mechanisms in plants,Biochimie,93(12):2054-2059
Hedrich R.,2012,Ion channels in plants,Physiol.Rev.,92(4):1777-1811
Hepler P.K.,Kunkel J.G.,Rounds C.M.,and Winship L.J.,2011,Calcium entry into pollen tubes,Trends Plant Sci.,17(1):32-38
Holdaway-Clarke T.L.,Feijo J.A.,Hackett G.R.,Kunkel J.G.,and Hepler P.K.,1997,Pollen tube growth and the intracellular cytosolic calcium gradient oscillate in phase while extracellular calcium influx is delayed,Plant Cell,9(11):1999-2010
Malho R.,and Trewavas A.J.,1996,Localized apical increases of cytosolic free calcium control pollen tube orientation,Plant Cell,8(11):1935-1949
Messerli M.,and Robinson K.R.,1997,Tip localized Ca2+pulses are coincident with peak pulsatile growth rates in pollen tubes of Lilium longiflorum,J.Cell Sci.,110(11):1269-1278
Michard E.,Lima P.T.,Borges F.,Silva A.C.,Portes M.T.,Carvalho J.E.,Gilliham M.,Liu L.H.,Obermeyer G.,and Feijo J.A.,2011,Glutamate receptor-like genes form Ca2+channels in pollen tubes and are regulated by pistil D-serine,Science,332(6028):434-437
Myers C.,Romanowsky S.M.,Barron Y.D.,Garg S.,Azuse C.L.,Curran A.,Davis R.M.,Hatton J.,Harmon A.C.,and Harper J.F.,2009,Calcium-dependent protein kinases regulate polarized tip growth in pollen tubes,Plant J.,59(4):528-539
Rounds C.M.,and Bezanilla M.,2013,Growth mechanisms in tip-growing plant cells,Annu.Rev.Plant Biol.,64:243-265
Schulz P.,Herde M.,and Romeis T.,2013,Calcium-dependent protein kinases:hubs in plant stress signaling and development,Plant Physiol.,163(2):523-530
Steinhorst L.,and Kudla J.,2013,Calcium-a central regulator of pollen germination and tube growth,BBA-Mol.Cell Res.,1833(7):1573-1581
Yang Z.,2008,Cell polarity signaling in Arabidopsis,Annu.Rev.Cell Dev.Biol.,24:551-575
Yoon G.M.,Dowd P.E.,Gilroy S.,and McCubbin A.G.,2006,Calcium-dependent protein kinase isoforms in Petunia have distinct functions in pollen tube growth,including regulating polarity,Plant Cell,18(4):867-878
Zhao L.N.,Shen L.K.,Zhang W.Z.,Zhang W.,Wang Y.,and Wu W.H.,2013,Ca2+-dependent protein kinase11 and 24 modulate the activity of the inward rectifying K+channels in Arabidopsis pollen tubes,Plant Cell,25(2):649-661
Zhou L.M.,Fu Y.,and Yang Z.B.,2009,A genome-wide functional characterization of Arabidopsis regulatory calcium sensors in pollen tubes,J.Integr.Plant Biol.,51(8):751-761
Zhou L.M.,Lan W.Z.,Jiang Y.Q.,Fang W.,and Luan S.,2014,A calcium-dependent protein kinase interacts with and activates a calcium channel to regulate pollen tube growth,Mol.Plant,7(2):369-376