,, 赵雷霖, 翟红红, 王远, 孟志刚, 梁成真, 张锐, 郭三堆, 孙国清,中国农业科学院生物技术研究所,北京 100081Functional Characterization of AtNEK6 Overexpression in Cotton Under Drought and Salt Stress
FAN Xin,, ZHAO LeiLin, ZHAI HongHong, WANG Yuan, MENG ZhiGang, LIANG ChengZhen, ZHANG Rui, GUO SanDui, SUN GuoQing,Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081通讯作者:
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收稿日期:2018-06-8接受日期:2018-07-23网络出版日期:2018-11-16
| 基金资助: |
Received:2018-06-8Accepted:2018-07-23Online:2018-11-16
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范鑫, 赵雷霖, 翟红红, 王远, 孟志刚, 梁成真, 张锐, 郭三堆, 孙国清. AtNEK6在棉花旱盐胁迫响应中的功能分析[J]. 中国农业科学, 2018, 51(22): 4230-4240 doi:10.3864/j.issn.0578-1752.2018.22.002
FAN Xin, ZHAO LeiLin, ZHAI HongHong, WANG Yuan, MENG ZhiGang, LIANG ChengZhen, ZHANG Rui, GUO SanDui, SUN GuoQing.
0 引言
【研究意义】棉花是世界上重要的经济作物,在中国及世界经济发展中占有重要地位。干旱、盐碱是影响棉花产量的主要非生物胁迫因素。AtNEK6是在拟南芥中发现的一种NIMA相关激酶,在拟南芥中过表达AtNEK6能够促进植物的生长发育,提高植物的耐盐耐旱性。【前人研究进展】NIMA（never-in-mitosis- A-related kinase,NEKs）相关激酶被称为有丝分裂酶第三家族,属于丝氨酸/苏氨酸激酶家族,位于纺锤体极体,主要调控细胞周期G2到M期过渡,有调节细胞有丝分裂和减数分裂功能[1]。OAKLEY等[2]最早在细胞分裂周期突变体的遗传筛选中发现了构巢曲霉NIMA相关蛋白。研究表明,NIMA存在于大部分生物中,如,原生生物衣藻、疟原虫和四膜虫等,真核生物果蝇、非洲爪蟾、小鼠和人类等[3];当NIMA缺失后,致使G2期阻滞,NIMA过表达后,导致细胞较早进入有丝分裂[4,5]。目前,人类基因组已发现11个NIMA基因,命名为NEK1—NEK11[3],更重要的是,从真菌到人类,许多NEKs都在各自的微管组织中心找到[6,7,8,9]。进一步研究发现,NEK1在DDR（DNA damage response）和纤毛功能中起重要作用[10];NEK2、NEK6、NEK7和NEK9主要参与细胞从间期转变为有丝分裂过程中中心体分离和有丝分裂纺锤体装配以及染色质的浓缩,核孔复合体（nuclear pore complex,NPC）的分解和核膜破裂等结构变化[11,12];NEK3不仅参与催乳素介导的信号途径,而且可以调节神经元中微管的乙酰化状态[13,14];NEK11通过磷酸化CDC25A蛋白促进其在DNA损伤诱导的细胞周期检查点中的降解,且该基因突变诱导癌症发生[15];NEK4主要参与DNA损伤反应、纤毛维持、微管稳定、细胞凋亡信号、RNA剪接及应激反应[16]。目前,在植物中已经发现了很多NEK基因。ZHANG等[17]在金鱼草中发现了第一个NIMA类基因。随后,拟南芥、番茄、水稻等植物中也发现了NIMA基因家族。在番茄中的NIMA蛋白与SP蛋白相互作用,调控茎的发育和开花[18]。在拟南芥中过表达杨树PNEK1导致植株的花和果荚异常发育,表明可能参与了花器官发育的调控[19]。FUJII等[20]发现在水稻中,OsNEK3被线粒体磷酸酶2C DCW11调控,控制花粉的萌发。目前在植物中研究的比较系统的是拟南芥中的AtNEK6,张博[21]等报道过表达AtNEK6促进莲座叶生长、侧根形成、结种数以及胁迫耐受能力。突变体nek6-1的叶子和果荚都很短,侧根数也减少,同时对盐胁迫和渗透胁迫过敏感。此外,过表达AtNEK6上调周期蛋白基因CYCB1;1和CYCA3;1的表达,而突变体nek6-1下调细胞周期蛋白的表达,但突变体nek6-1表现出乙烯相关基因表达上调和乙烯含量的积累。结果表明,AtNEK6通过细胞周期蛋白基因的激活和乙烯的抑制来调节植物的生长和胁迫应答过程。大豆中,GmNEK1与AtNEK6亲缘关系较近,将其转入拟南芥后,提高了植株的耐盐耐寒性[22]。【本研究切入点】过表达AtNEK6促进了拟南芥的生长发育并提高了其耐逆性。然而,将AtNEK6转入棉花等经济作物后是否同样可以促进棉花植株的生长发育并提高其耐逆性并未见报道。【拟解决的关键问题】本研究鉴于AtNEK6在拟南芥模式植物中过表达赋予受体植株较优良的抗逆性状,通过将AtNEK6转化棉花,并分析其在棉花中的功能,以期获得优良抗逆种质,为培育抗逆广适的棉花新品种提供支持。1 材料与方法
1.1 试验材料
AtNEK6由中国科学院遗传与发育研究所陈受宜研究员惠赠。陆地棉（Gossypium hirsutum L.）品种R18、农杆菌（Agrobacterium tumefaciens）菌株GV3101为生物技术研究所作物分子育种实验室保存,植物表达载体pBI121-AtNEK6由生物技术研究所作物分子育种实验室构建并保存。植物DNA提取试剂盒购于天根生化科技公司,植物总RNA提取试剂盒购于原平皓生物技术公司,反转录试剂盒购于全式金生物技术公司,实时荧光定量PCR（qRT-PCR）试剂盒购于东洋纺生物科技公司,检测植物生理指标的试剂盒购于北京索莱宝科技公司,其余常规试剂购于拜尔迪生物技术公司,引物（电子附表1）由上海生工生物工程公司合成。1.2 转化棉花及阳性植株的鉴定
通过电击法将pBI121-AtNEK6质粒转化GV3101农杆菌感受态,通过农杆菌介导法转化棉花,使用诱导筛选培养基培养2—3个月后,转到诱导分化培养基上,4个月后分化出胚状体,然后转移到胚萌发培养基上,2个月后获得转基因棉花。将种子种植于花盆中,待棉花长出真叶,提取棉花基因组DNA,PCR鉴定阳性转基因植株[23]。通过卡那霉素和PCR鉴定筛选阳性植株,温室培养收获T1代种子,将T1代植株在花盆中于温室培养,通过筛选鉴定阳性植株,获得T2代种子。
1.3 实时荧光定量PCR（qPCR）分析
用RNA提取试剂盒提取棉花样品总RNA,反转录获得cDNA,按照KOD SYBR qPCR MIX（QKD-201）的说明书配置荧光定量PCR反应体系,使用仪器ABI公司的FAST-7500进行反应和分析,其中以棉花GhActin作为内参基因,利用Primer3设计内参基因和目的基因的引物（电子附表1）,按照2-ΔΔCt法计算目的基因的相对表达量。1.4 转基因棉花表型分析
对1月龄棉花进行株高及叶面积（第三叶至第十叶）测量,参考TAO等[24]方法进行植株表皮细胞测量,取自下而上第五叶片进行扫描电子显微镜（scanning electron microscope,SEM）分析,每个材料进行4次重复。1.5 转基因棉花的耐盐耐旱性分析
将消毒后的转基因棉花种子和野生型棉花种子种于含250 mmol·L-1甘露醇和200 mmol·L-1NaCl的1/2MS培养基上,待种子萌发培养1周后,每个株系选取6株进行根长、侧根数、鲜重和干重的测定。在温室中,将不同株系的种子种植于含1:1（v/v）蛭石和珍珠盐的小盆（直径12 cm）中,用Hogland营养液培养1个月,每隔3 d浇灌100 mL/次,待棉花长至1个月,进行胁迫处理,盐处理组每隔3 d浇灌100 mL含250 mmol·L-1 NaCl营养液;干旱处理组每隔3 d浇灌100 mL含300 mmol·L-1甘露醇营养液;对照组每隔3 d浇100 mL营养液。处理24 h时取棉花植株叶片材料,检测相关胁迫因子的表达量,并测定SOD、CAT活性和MDA含量,每个株系取4个重复。胁迫处理2周后,观察表型并拍照。
2 结果
2.1 转基因棉花阳性植株鉴定
通过农杆菌介导法将植物表达载体pBI121- AtNEK6转化棉花,共获得10个不同的转基因株系。根据qPCR的检测结果,选择表达量较高的3个不同的转基因株系L7、L17、L25进行下一步试验。（图1-A—图1-D）在转基因植株中AtNEK6均有表达,野生型植株未检测到表达（图1-E）。图1
新窗口打开|下载原图ZIP|生成PPT图1转基因棉花的获得和鉴定
A：pBI121-AtNEK6载体图谱;B：愈伤组织;C：转基因植株幼苗;D：阳性转基因植株鉴定,WT：野生型对照;1—15：转基因株系;E：转基因株系中AtNEK6的表达量检测。**表示在P<0.01水平上差异显著。下同
Fig. 1Acquisition and identification of transgenic cotton
A: The schematic structure of pBI121-AtNEK6 vector; B: Callus of tobacco; C: Seeding of transgenic plants; D: Identification of positive transgenic cotton plants; WT: CK; 1-15: AtNEK6 transgenic line; E: Expression level of AtNEK6. ** indicate significant difference at P<0.01 level. The same as below
2.2 在棉花中过表达AtNEK6促进植物生长
待棉花长至1个月,正常生长条件下进行表型观测,转基因株系株高、叶片大小和叶面积均高于野生型（图2-A—图2-D）,通过扫描电镜观察转基因株系和野生型叶片表皮细胞的变化,结果表明,转基因株系的叶片表皮细胞和野生型植株的叶片表皮细胞大小相似（图2-E和图2-F）。图2
新窗口打开|下载原图ZIP|生成PPT图2AtNEK6促进了转基因棉花的生长发育
A：野生型植株和转基因植株的表型,Bar=10 cm;B：野生型植株和转基因植株的第三叶至第十叶,Bar=5 cm;C：野生型植株和转基因植株的株高;D：野生型植株和转基因植株的叶面积,1—8：第一片真叶到第八片真叶;E：扫面电镜观察野生型植株和转基因植株的叶表皮细胞,Bar=50 μm;F：野生型植株和转基因植株的表皮细胞面积;G：细胞周期和生长发育相关基因的表达量检测。*表示在P<0.05水平差异显著。下同
Fig. 2AtNEK6 promotes the growth of transgenic cotton
A: Growth phenotype of plant from WT and transgenic lines, Bar=10 cm; B: Morphology of leaves from WT and transgenic lines, Bar=5 cm; C: Height of plant from WT and transgenic lines; D: The difference of leaf area between WT and transgenic lines, 1-8: The first true leaf to the eighth true leaf; E: Leaf epidermal cells of WT and transgenic lines under SEM, Bar=50 μm; F: Leaf epidermal cell area of WT and transgenic lines; G: Transcript levels of cell cycle-related/ growth-related genes in WT and transgenic lines revealed by qRT-PCR. * indicate significant difference at P<0.05 level, respectively. The same as below
为了解AtNEK6促进棉花生长发育的分子机制,利用qPCR技术分别检测野生型植株和转基因株系棉花中细胞周期蛋白基因CYCB1;1、CYCA3;1和生长发育相关基因GhGRF5[24]、GhEOD[25]、GhAN3[26]、GhEBP1[27]在幼苗期的表达情况。结果表明,与野生型相比,转基因植株中CYCB1;1、CYCA3;1、GhGRF5、GhEOD、GhAN3和GhEBP1均上调表达（图2-G）。
2.3 转基因棉花的耐盐耐旱性分析
为了研究转AtNEK6棉花的耐旱性和耐盐性,分别将转基因棉花和野生型棉花种子种植于含甘露醇和NaCl的1/2MS培养基中进行胁迫处理（图3-A）。结果表明,正常条件下,转基因株系的根长、鲜重和干重与野生型相比均无明显差异（图3-A—图3-B和图3-D—图3-E）,但转基因株系侧根数均高于野生型（图3-C）,平均侧根数分别提高了11.49%、22.41%和22.41%;甘露醇胁迫处理7 d后,与野生型植株相比,转基因植株根系更加发达,且平均根长分别提高了19.89%、22.04%和29.56%,平均侧根数分别提高了86.84%、71.05%和90.78%,平均鲜重分别增加了1.23倍、1.07倍和1倍,平均干重分别增加了1倍、0.72倍和1倍;NaCl胁迫处理7 d后,转基因株系和野生型植株根长没有明显差异,但转基因株系平均侧根数分别提高了1.31倍、1.10倍和1.23倍,平均鲜重增加了0.75倍、0.90倍和0.62倍,平均干重增加了0.73倍、1.04倍和0.82倍。图3
新窗口打开|下载原图ZIP|生成PPT图3干旱和盐碱胁迫下转基因植株性状分析
A：在含250 mmol·L-1甘露醇和200 mmol·L-1 NaCl的1/2MS培养基上生长7 d的野生型植株和转基因植株;B：根长;C：侧根数;D：鲜重;E：干重
Fig. 3Characteristics of transgenic plants under drought and salt stress
A: Performances of WT and transgenic cotton after treatment of 250 mmol·L-1 Mannitol and 200 mmol·L-1 NaCl for 7 days; B: Root length; C: Lateral roots number; D: Fresh weight; E: Dry weight
室温条件下,对正常培养1月龄的转基因植株和野生型植株进行干旱和盐胁迫处理,胁迫处理前,转基因株系和野生型植株均正常生长,但转基因株系株高均高于野生型植株（图4-A）;甘露醇处理后,野生型植株萎蔫程度较转基因株系严重,盐胁迫处理后,野生型植株和转基因株系均未出现萎蔫（图4-A）。
图4
新窗口打开|下载原图ZIP|生成PPT图4转AtNEK6棉花在温室中耐盐耐旱性鉴定和酶活测定
A：胁迫处理后野生型植株和转基因植株的表型;B：CAT活性;C：SOD活性;D：MDA含量
Fig. 4Identification of drought and salt tolerance of AtNEK6-overexpressing cotton in greenhouse and enzyme activity detection
A: Performances of WT and transgenic cotton under drought and salt stress; B: CAT activity; C: SOD activity; D: MDA content
SOD和CAT的活性以及MDA含量是植物在抗逆性方面的重要生理指标。正常条件下,野生型植株与转基因植株的CAT活性、SOD活性和MDA含量并无明显差异。甘露醇处理组中,野生型棉花的SOD酶活为89.71 U·g-1,转基因植株L7、L17和L25的SOD酶活分别为148.12、127.39和219.91 U·g-1,比野生型提高了0.65倍、0.42倍和1.45倍（图4-B）;野生型棉花的CAT酶活为311.88 U·g-1,转基因植株L7、L17和L25的CAT酶活分别为517.54、513.02和442.96 U·g-1,比野生型提高了0.65倍、0.64倍和0.42倍（图4-C）;野生型棉花的MDA含量为39.35 nmol·g-1,转基因植株L7、L17和L25的MDA含量分别为19.11、23.05和30.50 nmol·g-1,比野生型棉花降低了0.51倍、0.41倍和0.22倍（图4-D）。盐处理组中,野生型棉花的SOD酶活为141.30 U·g-1,转基因植株L7、L17和L25的SOD酶活分别为197.12、206.88和205.72 U·g-1,比野生型提高了0.40倍、0.46倍和0.46倍;野生型棉花的CAT酶活为216.96 U·g-1,转基因植株L7、L17和L25的CAT酶活分别为345.78、334.48和409.06 U·g-1,比野生型提高了0.59倍、0.54倍和0.89倍;野生型棉花的MDA含量为38.61 nmol·g-1,转基因植株L7、L17和L25的MDA含量分别为31.07、32.12和33.02 nmol·g-1,比野生型棉花降低了0.2倍、0.17倍和0.14倍。
2.4 逆境胁迫相关基因在转基因棉花中的表达分析
通过实时荧光定量（qRT-PCR）分析表明,在甘露醇和NaCl的处理下,逆境胁迫相关基因GhAREB（ABA-responsive element binding）、GhDREB（dehydration-responsive element binding）、GhNCED（9-cis-epoxycarotenoid dioxygenase）、GhLEA5（late embryogenesis-abundant protein）均上调表达（图6）。在正常条件下,GhAREB在野生型和转基因植株中的表达量没有明显差异,而在盐和甘露醇胁迫后,转基因植株中GhAREB的表达量相比野生型约提高1倍。正常条件下,GhDREB、GhNCED和GhLEA5在转基因植株中的表达量显著高于野生型,进行胁迫处理后,这些基因在野生型中的表达量无明显变化,而胁迫处理后的转基因植株中这三个基因的表达量均显著提高。图5
新窗口打开|下载原图ZIP|生成PPT图5胁迫处理下转AtNEK6棉花中逆境胁迫相关基因的表达分析
Fig. 5Expression analyses of stress response genes in WT and AtNEK6-overexpressing cotton under drought and salt conditions
3 讨论
3.1 在棉花中过表达AtNEK6对植物生长发育的影响
植物在不良环境中自身会产生一系列的生理代谢反应,如代谢和生长的可逆性抑制,严重时甚至引起不可逆的伤害,导致整个植株死亡。本研究在棉花中过表达AtNEK6,在提高植物耐盐耐旱性的同时,也促进了植物的生长发育。通过观察发现,转基因株系株高、叶片大小和叶面积均高于野生型,这与在拟南芥的研究结果一致[21],进一步通过扫描电镜观察棉花叶片表皮细胞,发现野生型和转基因株系的表皮细胞面积并无明显差异,说明AtNEK6是通过促进细胞增殖影响了植株的生长发育。在过表达AtNEK6拟南芥中,转基因株系的细胞周期相关基因CYCB1;1和CYCA3;1的表达量均上调,本研究对转AtNEK6棉花中的细胞周期基因以及些重要的生长发育相关基因GhGRF5、GhEOD、GhAN3和GhEBP1的棉花同源基因进行表达量分析,亦得到相似结果,因此推测在棉花中AtNEK6可能是通过上调这些基因的表达促进细胞增殖,进而促进了叶片的生长。此外有研究发现,一些细胞周期调控基因是应激诱导型的,可以提高植物的耐受能力[28,29],因此,AtNEK6很有可能是通过调节细胞周期依赖性蛋白,促进了植物的生长发育进而提高了其耐受能力。3.2 AtNEK6对转基因棉花幼苗的影响
通过在无菌的1/2MS培养基上播种棉花种子,分析在甘露醇和盐的胁迫下,转基因棉花种子的萌发情况以及根的发育情况。发现转基因植株的根系不论在盐胁迫下还是甘露醇胁迫下,相对于野生型都要更加发达,根长更长、侧根数更多;进一步称量并统计了鲜重和干重,转基因植株的鲜重和干重相较于野生型均显著提高,说明在盐和甘露醇的胁迫下,AtNEK6通过维持植株的根系发育,提高了转基因植株在萌发期的耐盐耐旱能力。3.3 AtNEK6对棉花植株的抗氧化能力的影响
在逆境胁迫下,植物体内会产生大量的超氧化物阴离子和过氧化氢,SOD可以清除过量的阴离子,CAT可以清除过量的过氧化氢,并且它们的活性与非生物胁迫下的氧化损伤相关[30]。本研究转基因植株中的SOD和CAT的活性显著高于野生型,表明AtNEK6通过促进植株的活性氧清除能力,使棉花避免氧化损伤。MDA是细胞膜脂过氧化的产物之一,反应膜脂过氧化程度,MDA含量越低代表植物受损伤程度越低,转基因植株中的MDA含量显著低于野生型,因此,MDA含量的降低可能是转基因植株耐盐耐旱能力增强的一个重要因素。3.4 AtNEK6对棉花相关耐逆基因的表达影响
耐逆基因在一定程度上反应了植物的耐逆能力,GmNEK1通过直接上调RH3的表达提高了过表达GmNEK1拟南芥的耐盐耐寒能力[22]。本研究检测了逆境胁迫类的重要基因GhAREB（ABA反应元件）、GhDREB（脱水反应元件）、GhNCED（9-顺式-环氧加氧酶）、GhLEA5（水通道蛋白）,这些基因表达的升高也表明AtNEK6可能是通过影响多条途径来调控植株的胁迫反应过程,进而提高了植物的耐旱性和耐盐性。众所周知,各种胁迫导致微管重组和解聚,这对于抗逆性至关重要[31]。在膜蛋白和膜相关蛋白和脂质的调节下,微管在响应植物激素和胁迫中起重要作用[32,33];另外,微管对于植物细胞壁的延伸性至关重要,与细胞生长相关[34]。先前研究表明,拟南芥中NEK6、NEK4和NEK5彼此相互作用以调节表皮细胞扩增期间的定向细胞的生长和微管组织[35],所以植物中的NEK很可能通过控制细胞形态发生和扩张来调节细胞生长和胁迫应答,而这个过程主要由β-微管蛋白磷酸化和微管去稳定化介导[36]。基于这些结果,AtNEK6除了通过调节逆境胁迫相关基因的表达来实现功能,猜测AtNEK6也可能通过自身的激酶活性与微管相关作用,参与微管的重组,从而使植物在不利环境下适应性生长。
总之,作为丝氨酸/苏氨酸激酶家族成员基因,在棉花过表达AtNEK6可能通过多种途径促进了植株的生长发育,并提高了其耐盐耐旱性。该转基因棉花种质资源的获得,为今后培育品质优良、抗逆性高的棉花材料提供了试验依据。
4 结论
转AtNEK6棉花的耐盐耐旱能力得到提高,表现为萌发期根系发育更好、清除活性氧能力增强和不同逆境胁迫相关基因的表达提高。AtNEK6通过影响生长发育的相关基因促进棉花植株的生长发育,通过多条胁迫响应途径调控棉花植株的耐盐耐旱性。（责任编辑 李莉）
参考文献 原文顺序
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被引期刊影响因子
URLPMID:3294854 [本文引用: 1]
The temperature-sensitive cell cycle mutation nimA5 causes nuclei of Aspergillus nidulans to be blocked in late G2 at restrictive temperature. Under these conditions the spindle pole body divides but does not separate and the mitotic index drops to zero. If nimA5 is blocked for more than one doubling time and then shifted from restrictive to permissive temperature, nuclei immediately enter mitosis, the mitotic spindle forms, and the chromosomes condense (Oakley, B. R., and N. R. Morris, 1983, J. Cell Biol., 96:1155-8). We have cloned the wild-type nimA gene by DNA-mediated complementation of the nimA5 mutant phenotype and have characterized nimA mRNA expression by Northern blot analysis. The transcript is 3.6 kb in length and is under tight nuclear cycle regulation. In synchronously dividing cells, the levels of nimA mRNA become elevated as cells enter mitosis and drop sharply as cells progress through mitosis. Cells blocked in S-phase with hydroxyurea have very low levels of nimA mRNA. Cells blocked in mitosis, either by the antimitotic agent benomyl or by the cell cycle mutation bimE7, maintain elevated levels of the nimA transcript. These data demonstrate not only that nimA is required for entry into mitosis, but because the transcript is normally expressed cyclically and is under tight cell cycle control, they suggest that nimA may play a regulatory role in the initiation of mitosis.
[本文引用: 1]
DOI:10.1242/jcs.111195URLPMID:23132929 [本文引用: 2]
Abstract Genetic screens for cell division cycle mutants in the filamentous fungus Aspergillus nidulans led to the discovery of never-in-mitosis A (NIMA), a serine/threonine kinase that is required for mitotic entry. Since that discovery, NIMA-related kinases, or NEKs, have been identified in most eukaryotes, including humans where eleven genetically distinct proteins named NEK1 to NEK11 are expressed. Although there is no evidence that human NEKs are essential for mitotic entry, it is clear that several NEK family members have important roles in cell cycle control. In particular, NEK2, NEK6, NEK7 and NEK9 contribute to the establishment of the microtubule-based mitotic spindle, whereas NEK1, NEK10 and NEK11 have been implicated in the DNA damage response. Roles for NEKs in other aspects of mitotic progression, such as chromatin condensation, nuclear envelope breakdown, spindle assembly checkpoint signalling and cytokinesis have also been proposed. Interestingly, NEK1 and NEK8 also function within cilia, the microtubule-based structures that are nucleated from basal bodies. This has led to the current hypothesis that NEKs have evolved to coordinate microtubule-dependent processes in both dividing and non-dividing cells. Here, we review the functions of the human NEKs, with particular emphasis on those family members that are involved in cell cycle control, and consider their potential as therapeutic targets in cancer.
DOI:10.1016/0092-8674(91)90180-7URLPMID:1913824 [本文引用: 1]
We show that in Aspergillus nidulans, p34tyrosine dephosphorylation accompanies activation of p34as an H1 kinase at mitosis. However, the mutation arrests cells in G2 with p34tyrosine dephosphorylated and fully active as an H1 kinase. Activation of NIMA is therefore not required for p34activation. Furthermore, mutation of , which encodes a protein with 50% similarity to fission yeast cdc25, causes a G2 arrest and prevents tyrosine dephosphorylation of p34but does not prevent full activation of the NIMA protein kinase. Mitotic activation of p340by tyrosine dephosphorylation is therefore not required for activation of NIMA. These data suggest that activation of either the p34protein kinase or the NIMA protein kinase alone is not sufficient to initiate mitosis. Parallel activation of both cell cycle-regulated protein kinases is required to trigger mitosis.
DOI:10.1016/0092-8674(88)90385-6URLPMID:3359487 [本文引用: 1]
There may be a causal relationship between expression of the G2-specific gene nimA and mitotic regulation in Aspergillus. To test this relationship we have introduced extra inducible copies of nimA into Aspergillus and determined the effect of nimA overproduction on mitotic regulation. The results show that nimA overexpression causes mitotic induction in less than a cell cycle and maintains chromatin in a condensed state. These effects occur even if cells are first blocked in S phase. Sequence analysis shows that the nimA gene encodes a potential protein kinase. These data indicate that there is indeed a causal relationship between expression of nimA and the regulation of mitosis and further implicate protein phosphorylation in mitotic control.
DOI:10.1093/emboj/21.7.1713URL [本文引用: 1]
NIMA kinases appear to be the least functionally conserved mitotic regulators, being implicated in chromosome condensation in fungi and in spindle function in metazoans. We demonstrate here that the fission yeast NIMA homologue, Fin1p, can induce profound chromosome condensation in the absence of the condensin and topoisomerase II, indicating that Fin1p-induced condensation differs from mitotic condensation. Fin1p expression is transcriptionally and post-translationally cell cycle-regulated, with Fin1p kinase activity maximal from the metaphase090009anaphase transition to G1. Fin1p is localized to the spindle pole body and fin10200 cells are hypersensitive to anti-microtubule drugs, synthetically lethal with a number of spindle mutants and require the spindle checkpoint for viability. Moreover, fin10200 cells show unusual and extensive elaborations of the nuclear envelope. These data support a role for Fin1p in spindle function and nuclear envelope transactions at or after the metaphase090009anaphase transition that may be generally applicable to other NIMA-family members.
[本文引用: 1]
DOI:10.1016/j.yexcr.2004.04.052URLPMID:15572022 [本文引用: 1]
The Nima-related kinase 2 (Nek2) has been implicated in the regulation of centrosome integrity and separation in several species and is a candidate for cell transformation. We now show that reduction of levels of the Drosophila Nek2 by RNAi in cultured cells leads to both dispersal of centrosomal antigens and formation of ectopic bodies of centrosomal antigens. Overexpression of the active DmNek2 kinase resulted in an increase in the number of mitotic cells with fragmented centrosomes. The DmNek2 protein kinase is associated with punctuate bodies within the centrosome consistent with its presence on centrioles. In addition, it is present at lower levels on the midbody during cytokinesis. Midbody association was enhanced following overexpression, whereupon the DmNek2 protein kinase also localised to the cell cortex becoming concentrated in the region of the cleavage furrow in late telophase. Many of such cells showed abnormalities in the organisation of anillin and actin in the cleavage furrow that was associated with formation of ectopic membrane protrusions between each daughter cell. We discuss potential roles for DmNek2 in maintaining centrosome integrity in mitosis, during cytokinesis, and consequently for the fidelity of chromosome segregation.
DOI:10.1091/mbc.E05-05-0450URLPMID:1474788 [本文引用: 1]
ABSTRACT The genome of Tetrahymena thermophila contains 39 loci encoding NIMA-related kinases (NRKs), an extraordinarily large number for a unicellular organism. Evolutionary analyses grouped these sequences into several subfamilies, some of which have orthologues in animals, whereas others are protist specific. When overproduced, NRKs of three subfamilies caused rapid shortening of cilia. Ultrastructural studies revealed that each NRK triggered ciliary resorption by a distinct mechanism that involved preferential depolymerization of a subset of axonemal microtubules, at either the distal or proximal end. Overexpression of a kinase-inactive variant caused lengthening of cilia, indicating that constitutive NRK-mediated resorption regulates the length of cilia. Each NRK preferentially resorbed a distinct subset of cilia, depending on the location along the anteroposterior axis. We also show that normal Tetrahymena cells maintain unequal length cilia. We propose that ciliates used a large number of NRK paralogues to differentially regulate the length of specific subsets of cilia in the same cell.
DOI:10.1016/j.ejcb.2012.03.009URLPMID:22613497 [本文引用: 1]
The NIMA-related kinase Nek2 promotes centrosome separation at the G2/M transition and, consistent with this role, is known to be concentrated at the proximal ends of centrioles. Here, we show by immunofluorescence microscopy that Nek2 also localises to the distal portion of the mother centriole. Its accumulation at this site is cell cycle-dependent and appears to peak in late G2. These findings are consistent with previous data implicating Nek2 in promoting reorganisation of centrosome-anchored microtubules at the G2/M transition, given that microtubules are anchored at the subdistal appendages of the mother centriole in interphase. In addition, we report that siRNA-mediated depletion of Nek2 compromises the ability of cells to resorb primary cilia before the onset of mitosis, while overexpression of catalytically active Nek2A reduces ciliation and cilium length in serum-starved cells. Based on these findings, we propose that Nek2 has a role in promoting cilium disassembly at the onset of mitosis. We also present evidence that recruitment of Nek2 to the proximal ends of centrioles is dependent on one of its substrates, the centrosome cohesion protein C-Nap1.
DOI:10.1186/1747-1028-2-25URL [本文引用: 1]
DOI:10.4161/cc.10.22.18226URL [本文引用: 1]
DOI:10.1242/jcs.048975URL [本文引用: 1]
NIMA-related kinases (Neks) belong to a large family of Ser/Thr kinases that have critical roles in coordinating microtubule dynamics during ciliogenesis and mitotic progression. The Nek kinases are also expressed in neurons, whose axonal projections are, similarly to cilia, microtubule-abundant structures that extend from the cell body. We therefore investigated whether Nek kinases have additional, non-mitotic roles in neurons. We found that Nek3 influences neuronal morphogenesis and polarity through effects on microtubules. Nek3 is expressed in the cytoplasm and axons of neurons and is phosphorylated at Thr475 located in the C-terminal PEST domain, which regulates its catalytic activity. Although exogenous expression of wild-type or phosphomimic (T475D) Nek3 in cultured neurons has no discernible impact, expression of a phospho-defective mutant (T475A) or PEST-truncated Nek3 leads to distorted neuronal morphology with disturbed polarity and deacetylation of microtubules via HDAC6 in its kinase-dependent manner. Thus, the phosphorylation at Thr475 serves as a regulatory switch that alters Nek3 function. The deacetylation of microtubules in neurons by unphosphorylated Nek3 raises the possibility that it could have a role in disorders where axonal degeneration is an important component.
DOI:10.1038/sj.onc.1210264URL [本文引用: 1]
DOI:10.1038/ncb1969URLPMID:19734889 [本文引用: 1]
Abstract DNA damage-induced cell-cycle checkpoints have a critical role in maintaining genomic stability. A key target of the checkpoints is the CDC25A (cell division cycle 25 homologue A) phosphatase, which is essential for the activation of cyclin-dependent kinases and cell-cycle progression. To identify new genes involved in the G2/M checkpoint we performed a large-scale short hairpin RNA (shRNA) library screen. We show that NIMA (never in mitosis gene A)-related kinase 11 (NEK11) is required for DNA damage-induced G2/M arrest. Depletion of NEK11 prevents proteasome-dependent degradation of CDC25A, both in unperturbed and DNA-damaged cells. We show that NEK11 directly phosphorylates CDC25A on residues whose phosphorylation is required for beta-TrCP (beta-transducin repeat-containing protein)-mediated polyubiquitylation and degradation of CDC25A. Furthermore, we demonstrate that CHK1 (checkpoint kinase 1) directly activates NEK11 by phosphorylating it on Ser 273, indicating that CHK1 and NEK11 operate in a single pathway that controls proteolysis of CDC25A. Taken together, these results demonstrate that NEK11 is an important component of the pathway enforcing the G2/M checkpoint, suggesting that genetic mutations in NEK11 may contribute to the development of human cancer.
DOI:10.1186/s12953-014-0057-yURLPMID:25628518 [本文引用: 1]
Background Chemoresistance remains a significant challenge in chronic myelogenous leukemia (CML) management, which is one of the most critical prognostic factors. Elucidation the molecular mechanisms underlying the resistance to chemoresistance may lead to better clinical outcomes. Results In order to identify potential protein targets involved in the drug-resistant phenotype of leukemia, especially the chronic myelogenous leukemia (CML), we used a high-resolution ltra-zoom 2DE-based proteomics approach to characterize global protein expression patterns in doxorubicin-resistant myelogenous leukemia cells compared with parental control cells. Ultra-high resolution of 2DE was achieved by using a series of slightly overlapping narrow-range IPG strips during isoelectric focusing (IEF) separation. A total number of 44 proteins with altered abundances were detected and identified by MALDI -TOF or LC-MS /MS. Among these proteins, enolase, aldolase, HSP70 and sorcin were up-regulated in doxorubicin-resistant myelogenous leukemia cell line, whereas HSP27 was down-regulated. Some of the results have been validated by Western blotting. Both enolase and aldolase were first reported to be involved in chemoresistance, suggesting that process of glycolysis in doxorubicin-resistant myelogenous leukemia cells was accelerated to some extent to provide more energy to survive chemical stress. Possible roles of most of the identified proteins in development of chemoresistance in myelogenous leukemia cells were fully discussed. The results presented here could provide clues to further study for elucidating the mechanisms underlying drug resistance in leukemia. Conclusions As a whole, under the chemical stress, the doxorubicin-resistant myelogenous leukemia cells may employ various protective strategies to survive. These include: (i) pumping the cytotoxic drug out of the cells by P-glycoprotein, (ii) increased storage of fermentable fuel, (iii) sophisticated cellular protection by molecular chaperones, (iv) improved handling of intracellular calcium, (v) increased glucose utilization via increased rates of glycolysis. In the present study, proteomic analysis of leukemia cells and their drug resistant variants revealed multiple alterations in protein expression. Our results indicate that the development of drug resistance in doxorubicin-resistant myelogenous leukemia cells is a complex phenomenon undergoing several mechanisms.
DOI:10.1046/j.1365-2818.1996.110390.xURLPMID:8919984 [本文引用: 1]
In plants, cell proliferation occurs mostly within meristems but a significant amount also occurs at other well-defined sites during specific stages of development. We have developed molecular markers to follow the location and progress of cell division within multicellular plant organs and thereby gain some insight into how cell division might be regulated during morphogenesis. As in other eukaryotes, cell division in plants is regulated by a highly conserved set of protein kinases and phosphatases. The molecular information available on these molecules from other eukaryotes has allowed the design of strategies by which plant homologues can be isolated. In this report we describe the identification of a nimA -like gene from Antirrhinum majus and describe the pattern in which its transcript is expressed. Comparison of the pattern of AmnimA gene expression with that of genes which are expressed in a cell cycle-dependent manner suggests that this gene is expressed in actively dividing tissues but expression is not specific to any particular phase of the cell cycle nor specific to any particular tissue type.
DOI:10.2307/3871528URL [本文引用: 1]
Divergent architecture of shoot models in flowering plants reflects the pattern of production of vegetative and reproductive organs from the apical meristem. The SELF-PRUNING (SP) gene of tomato is a member of a novel CETS family of regulatory genes (CEN, TFL1, and FT) that controls this process. We have identified and describe here several proteins that interact with SP (SIPs) and with its homologs from other species: a NIMA-like kinase (SPAK), a bZIP factor, a novel 10-kD protein, and 14-3-3 isoforms. SPAK, by analogy with Raf1, has two potential binding sites for 14-3-3 proteins, one of which is shared with SP. Surprisingly, overexpression of 14-3-3 proteins partially ameliorates the effect of the sp mutation. Analysis of the binding potential of chosen mutant SP variants, in relation to conformational features known to be conserved in this new family of regulatory proteins, suggests that associations with other proteins are required for the biological function of SP and that ligand binding and protein-protein association domains of SP may be separated. We suggest that CETS genes encode a family of modulator proteins with the potential to interact with a variety of signaling proteins in a manner analogous to that of 14-3-3 proteins.
DOI:10.1016/j.febslet.2005.07.036URLPMID:16098516 [本文引用: 1]
Meristems are sites of undifferentiated cell division, which carry on developing into functional organs. Using the two-hybrid system with a poplar 14-3-3, we uncovered poplar NIMA-related kinase 1 (PNek1) as an interacting protein. PNek1 shows high homology to the mammalian NIMA-related kinases, which are thought to be involved in cell cycle progression. Using a synchronized poplar cell suspension, we observed an accumulation of PNek1 mRNA at the G 1/S transition and throughout the G 2-to-M progression. Moreover, PNek1-GFP fusion protein localized in the cytoplasm and in both the nuclear and nucleolar regions. Overexpression of PNek1-GFP in Arabidopsis caused morphological abnormalities in flower and siliques. Overall, these results suggest that PNek1 is involved in plant development.
DOI:10.1093/pcp/pcp026URLPMID:19224952 [本文引用: 1]
Abstract OsNek3 (Oryza sativa L. NIMA-related kinase) and DCW11 encoding a mitochondrial putative protein phosphatase 2C were found in our previous microarray study as down-regulated genes in the rice CW-CMS line, which lacked pollen germination ability. Further analysis of DCW11 revealed that DCW11 is strongly correlated with CW-CMS occurrence. Here we show the relationship between OsNek3 and DCW11. OsNek3 was preferentially expressed in mature pollen. A knockout mutant with Tos17 inserted into OsNek3 did not show any pollen-defective phenotype. On the other hand, plants overexpressing OsNek3 occasionally produced a peculiar pollen structure in which the outer cell wall of four pollen grains fused together even at the mature pollen stages, which resembled that of quartet mutants in Arabidopsis. OsNek3 was shown to interact with a LIM domain-containing protein, OsPLIM2b, whose expression was strongly specific in mature pollen, suggesting that OsNek3 might play a role in pollen germination. OsNek3 was shown to be down-regulated in DCW11-knockdown lines, whereas osnek3 mutation did not result in DCW11 down-regulation. These results suggest that OsNek3 is downstream of DCW11 in retrograde signaling from the mitochondria to the nucleus and is involved in CW-CMS.
DOI:10.1111/j.1365-313X.2011.04733.xURLPMID:21801253 [本文引用: 2]
The NIMA-related kinases (NEKs) are a family of serine/threonine kinases involved largely in cell cycle control in fungi, mammals and other eukaryotes. In Arabidopsis, NEK6 is involved in the regulation of epidermal cell morphogenesis. However, other roles of NEK6 in plants are less well understood. Here we report functions of NEK6 in plant growth, development and stress responses in Arabidopsis. NEK6 transcripts and proteins are induced by ethylene precursor ACC and salt stress. Expression of other NEK genes except NEK5 is also responsive to the two treatments. Overexpression and mutant analysis disclose that the NEK6 gene increases rosette growth, seed yield and lateral root formation. However, NEK6 appears to play a negative role in the control of seed size. The gene also promotes plant tolerance to salt stress and osmotic stress in its overexpressing plants. The NEK6 gene may achieve its function through suppression of ethylene biosynthesis and activation of CYCB1;1 and CYCA3;1 expression. Our present study reveals new functions of the NEK6 gene in plant growth and stress tolerance, and manipulation of NEK6 may improve important agronomic traits in crop plants.
DOI:10.1093/pcp/pcx060URLPMID:28444301 [本文引用: 2]
NEK (NIMA-related kinase) is known as a family of serine/threonine kinases which mainly participate in microtubule-related mitotic events in fungi, mammals and other eukaryotes. Our previous studies found that Arabidopsis NEK6 plays an important role in plant response to abiotic stress. We further investigated roles of the NEK family in soybean and found that at least eight members can respond to abiotic stresses. Among them, only GmNEK1, a novel NEK member which is distantly related to Arabidopsis NEK6, enhanced plant growth and promoted salt and cold tolerance in transgenic Arabidopsis plants. The growth of soybean plants harboring GmNEK1-overexpressing hairy roots under saline condition was also improved. A series of stress-related genes including RH3, CORI3 and ALDH10A8 were found to be up-regulated in GmNEK1-overexpressing Arabidopsis plants and soybean hairy roots. Moreover, soybean plants with GmRH3-overexpressing hairy roots exhibited increased salt tolerance, while soybean plants with GmRH3-RNAi (RNA interference) roots were more sensitive to salt stress than the wild-type plants. Our study uncovers a novel role for GmNEK1 in promoting plant adaptive growth under adverse conditions at least partially through up-regulation of GmRH3. Manipulation of these genes in soybean or other crops may improve growth and production under stress conditions.
DOI:10.3969/j.issn.1002-7807.2005.01.001URL [本文引用: 1]
以pmi基因作为筛选标记基因,以甘露糖作为筛选剂,通过农杆菌介导法将GFP基因导入棉花细胞并得到再生植株,经过PCR检测、Southern杂交证实外源基因已经整合到棉花基因组中,Westbloting与荧光显微镜检测证明GFP基因得到表达。本文研究讨论了甘露糖作为棉花转化细胞的筛选剂在农杆菌介导的转化中的应用浓度及方法,即:甘露糖的筛选浓度在30～50g·L 1之间,在愈伤组织诱导初期适当低一点,随着愈伤组织的生长而加大筛选浓度。由于甘露糖不利于再生胚的分化,当愈伤组织转入分化培养基时,要以葡萄糖代替甘露糖。
DOI:10.3969/j.issn.1002-7807.2005.01.001URL [本文引用: 1]
以pmi基因作为筛选标记基因,以甘露糖作为筛选剂,通过农杆菌介导法将GFP基因导入棉花细胞并得到再生植株,经过PCR检测、Southern杂交证实外源基因已经整合到棉花基因组中,Westbloting与荧光显微镜检测证明GFP基因得到表达。本文研究讨论了甘露糖作为棉花转化细胞的筛选剂在农杆菌介导的转化中的应用浓度及方法,即:甘露糖的筛选浓度在30～50g·L 1之间,在愈伤组织诱导初期适当低一点,随着愈伤组织的生长而加大筛选浓度。由于甘露糖不利于再生胚的分化,当愈伤组织转入分化培养基时,要以葡萄糖代替甘露糖。
DOI:10.1104/pp.15.00355URLPMID:25941315 [本文引用: 2]
Abstract Ethylene is an important phytohormone in the regulation of plant growth, development, and stress response throughout the lifecycle. Previously, we discovered that a subfamily II ethylene receptor tobacco (Nicotiana tabacum) Histidine Kinase1 (NTHK1) promotes seedling growth. Here, we identified an NTHK1-interacting protein translationally controlled tumor protein (NtTCTP) by the yeast (Saccharomyces cerevisiae) two-hybrid assay and further characterized its roles in plant growth. The interaction was further confirmed by in vitro glutathione S-transferase pull down and in vivo coimmunoprecipitation and bimolecular fluorescence complementation assays, and the kinase domain of NTHK1 mediates the interaction with NtTCTP. The NtTCTP protein is induced by ethylene treatment and colocalizes with NTHK1 at the endoplasmic reticulum. Overexpression of NtTCTP or NTHK1 reduces plant response to ethylene and promotes seedling growth, mainly through acceleration of cell proliferation. Genetic analysis suggests that NtTCTP is required for the function of NTHK1. Furthermore, association of NtTCTP prevents NTHK1 from proteasome-mediated protein degradation. Our data suggest that plant growth inhibition triggered by ethylene is regulated by a unique feedback mechanism, in which ethylene-induced NtTCTP associates with and stabilizes ethylene receptor NTHK1 to reduce plant response to ethylene and promote plant growth through acceleration of cell proliferation. 2015 American Society of Plant Biologists. All Rights Reserved.
DOI:10.1111/j.1365-313X.2005.02429.xURLPMID:15960617 [本文引用: 1]
The development of the flat morphology of leaf blades is dependent on the control of cell proliferation as well as cell expansion. Each process has a polarity with respect to the longitudinal and transverse axes of the leaf blade. However, only a few regulatory components of these processes have been identified to date. We have characterized two genes from Arabidopsis thaliana : ANGUSTIFOLIA3 ( AN3 ), which encodes a homolog of the human transcription coactivator SYT, and GROWTH-REGULATING FACTOR5 ( AtGRF5 ), which encodes a putative transcription factor. AN3 is identical to GRF-INTERACTING FACTOR1 ( AtGIF1 ). The an3 and atgrf5 mutants exhibit narrow-leaf phenotypes due to decreases in cell number. Conversely, cell proliferation in leaf primordia is enhanced and leaves grow larger than normal when AN3 or AtGRF5 is overexpressed. Both genes are expressed in leaf primordia, and in the yeast two-hybrid assay, the gene products were found to interact with each other through their N-terminal domains. These results suggest that AN3 and AtGRF5 act together and are required for the development of appropriate leaf size and shape through the promotion and/or maintenance of cell proliferation activity in leaf primordia.
[本文引用: 1]
DOI:10.1038/sj.emboj.7601362URLPMID:17024182 [本文引用: 1]
Plant organ size shows remarkable uniformity within species indicating strong endogenous control. We have identified a plant growth regulatory gene, functionally and structurally homologous to human EBP1. Plant EBP1 levels are tightly regulated; gene expression is highest in developing organs and correlates with genes involved in ribosome biogenesis and function. EBP1 protein is stabilised by auxin. Elevating or decreasing EBP1 levels in transgenic plants results in a dose-dependent increase or reduction in organ growth, respectively. During early stages of organ development, EBP1 promotes cell proliferation, influences cell-size threshold for division and shortens the period of meristematic activity. In postmitotic cells, it enhances cell expansion. EBP1 is required for expression of cell cycle genes; CyclinD3;1, ribonucleotide reductase 2 and the cyclin-dependent kinase B1;1. The regulation of these genes by EBP1 is dose and auxin dependent and might rely on the effect of EBP1 to reduce RBR1 protein level. We argue that EBP1 is a conserved, dose-dependent regulator of cell growth that is connected to meristematic competence and cell proliferation via regulation of RBR1 level.
[本文引用: 1]
[本文引用: 1]
DOI:10.1104/pp.111.184531URLPMID:22106097 [本文引用: 1]
Nitric oxide (NO) is a key redox-active, small molecule involved in various aspects of plant growth and development. Here, we report the identification of an NO accumulation mutant, nitric oxide excessl (noel), in rice (Oryza sativa), the isolation of the corresponding gene, and the analysis of its role in NO-mediated leaf cell death. Map-based cloning revealed that NOE1 encoded a rice catalase, OsCATC. Furthermore, noe1 resulted in an increase of hydrogen peroxide (H60O60) in the leaves, which consequently promoted NO production via the activation of nitrate reduclase. The removal of excess NO reduced cell death in both leaves and suspension cultures derived from noe1 plants, implicating NO as an important endogenous mediator of H60O60-induced leaf cell death. Reduction of intracellular S-nitrosothiol (SNO) levels, generated by overexpression of rice S-nitrosoglutathione reducíase gene (GSNOR1), which regulates global levels of protein S-nitrosylation, alleviated leaf cell death in noe1 plants. Thus, S-nitrosylation was also involved in light-dependent leaf cell death in noe1. Utilizing the biotin-switch assay, nanoliquid chromatography, and tandem mass spectrometry, S-nitrosylated proteins were identified in both wild-type and noe1 plants. NO targets identified only in noe1 plants included glyceraldehyde 3-phosphate dehydrogenase and thioredoxin, which have been reported to be involved in S-nitrosylation-regulated cell death in animals. Collectively, our data suggest that both NO and SNOs are important mediators in the process of H60O60-induced leaf cell death in rice.
DOI:10.3389/fpls.2015.00947URLPMID:4628123 [本文引用: 1]
N-MYC DOWNREGULATED-LIKE proteins (NDL), members of the alpha/beta hydrolase superfamily were recently rediscovered as interactors of G-protein signaling inArabidopsis thaliana. Although the precise molecular function of NDL proteins is still elusive, in animals these proteins play protective role in hypoxia and expression is induced by hypoxia and nickel, indicating role in stress. Homology of NDL1 with animal counterpart N-MYC DOWNREGULATED GENE (NDRG) suggests similar functions in animals and plants. It is well established that stress responses leads to the microtubule depolymerization and reorganization which is crucial for stress tolerance. NDRG is a microtubule-associated protein which mediates the microtubule organization in animals by causing acetylation and increases the stability of -tubulin. As NDL1 is highly homologous to NDRG, involvement of NDL1 in the microtubule organization during plant stress can also be expected. Discovery of interaction of NDL with protein kinesin light chain- related 1, enodomembrane family protein 70, syntaxin-23, tubulin alpha-2 chain, as a part of G protein interactome initiative encourages us to postulate microtubule stabilizing functions for NDL family in plants. Our search for NDL interactors in G protein interactome also predicts the role of NDL proteins in abiotic stress tolerance management. Based on published report in animals and predicted interacting partners for NDL in G protein interactome lead us to hypothesize involvement of NDL in the microtubule organization during abiotic stress management in plants.
DOI:10.1016/j.bbrc.2011.01.081URLPMID:21277286 [本文引用: 1]
Past decades have brought great advances in understanding the relationship between turgor pressure and plant cell growth. New studies have provided evidence that turgor pressure acts as a stimulus for cell growth, and is also a developmental cue for post-embryonic organogenesis. However, the subcellular mechanisms underlying plant cell turgor pressure sensing remain unclear. Here, using the relatively simple undifferentiated cells from suspension cultures, we report real-time in vivo observations of the reorganization of microtubules and actin microfilaments induced by turgor pressure changes. We found that these two cytoskeletal elements differed in their reorganization patterns. Our results will be useful in the understanding of the relationship between the cytoskeleton, turgor pressure, and stress in plant cell morphogenesis.
DOI:10.1007/s13238-015-0233-6URLPMID:4742386 [本文引用: 1]
房间分割和扩大要求微导管的订的安排，它由发展、环境的因素服从于空间、时间的修正。理解信号怎么在外皮的微导管组织翻译到变化具有基本重要性。外皮的微导管数组的一个定义特征是它有血浆膜的协会；血浆膜的模块被认为在微导管组织的调停起重要作用。在这评论，我们响应植物激素和压力由联系膜、拴住膜的蛋白质和类脂化合物在关于外皮的微导管组织的规定的研究加亮进展。transmembrane kinase 受体象 Rho 一样 guanosine triphosphatase， phospholipase D， phosphatidic 酸，和 phosphoinositides 在微导管组织与他们的角色的一个焦点被讨论。
DOI:10.1093/jxb/erv511URLPMID:26608646 [本文引用: 1]
The advent of user-friendly instruments for measuring force/deflection curves of plant surfaces at high spatial resolution has resulted in a recent outpouring of reports of the 'Young's modulus' of plant cell walls. The stimulus for these mechanical measurements comes from biomechanical models of morphogenesis of meristems and other tissues, as well as single cells, in which cell wall stress feeds back to regulate microtubule organization, auxin transport, cellulose deposition, and future growth directionality. In this article I review the differences between elastic modulus and wall extensibility in the context of cell growth. Some of the inherent complexities, assumptions, and potential pitfalls in the interpretation of indentation force/deflection curves are discussed. Reported values of elastic moduli from surface indentation measurements appear to be 10- to >1000-fold smaller than realistic tensile elastic moduli in the plane of plant cell walls. Potential reasons for this disparity are discussed, but further work is needed to make sense of the huge range in reported values. The significance of wall stress relaxation for growth is reviewed and connected to recent advances and remaining enigmas in our concepts of how cellulose, hemicellulose, and pectins are assembled to make an extensible cell wall. A comparison of the loosening action of -expansin and Cel12A endoglucanase is used to illustrate two different ways in which cell walls may be made more extensible and the divergent effects on wall mechanics.
DOI:10.1111/j.1365-313X.2011.04652.xURLPMID:21605211 [本文引用: 1]
NimA-related kinase 6 (NEK6) has been implicated in microtubule regulation to suppress the ectopic outgrowth of epidermal cells; however, its molecular functions remain to be elucidated. Here, we analyze the function of NEK6 and other members of the NEK family with regard to epidermal cell expansion and cortical microtubule organization. The functional NEK6 reen fluorescent protein fusion localizes to cortical microtubules, predominantly in particles that exhibit dynamic movement along microtubules. The kinase-dead mutant of NEK6 (ibo1-1) exhibits a disturbance of the cortical microtubule array at the site of ectopic protrusions in epidermal cells. Pharmacological studies with microtubule inhibitors and quantitative analysis of microtubule dynamics indicate excessive stabilization of cortical microtubules in ibo1/nek6 mutants. In addition, NEK6 directly binds to microtubules in vitro and phosphorylates -tubulin. NEK6 interacts and co-localizes with NEK4 and NEK5 in a transient expression assay. The ibo1-3 mutation markedly reduces the interaction between NEK6 and NEK4 and increases the interaction between NEK6 and NEK5. NEK4 and NEK5 are required for the ibo1/nek6 ectopic outgrowth phenotype in epidermal cells. These results demonstrate that NEK6 homodimerizes and forms heterodimers with NEK4 and NEK5 to regulate cortical microtubule organization possibly through the phosphorylation of -tubulins.
DOI:10.1007/s10265-015-0751-6URLPMID:26354760 [本文引用: 1]
Microtubules are highly dynamic structures that control the spatiotemporal pattern of cell growth and division. Microtubule dynamics are regulated by reversible protein phosphorylation involving both...
