植物中钙依赖蛋白激酶(CDPK)的研究进展

武志刚，武舒佳，王迎春，郑琳琳

(内蒙古大学生命科学学院，内蒙古 呼和浩特 010021)

植物中存在着复杂的信号转导网络，用于调控植物的新陈代谢，以应对不断变化的生存环境。Ca2+作为重要的第二信使，经Ca2+受体蛋白、钙调素(CaM)和钙依赖蛋白激酶(calcium-dependent protein kinase，CDPK)等协同作用[1]，将钙信号向下游传递并级联放大，促进响应蛋白的产生，从而调控植物的生长发育以及免疫应答和胁迫响应。

CDPK是目前植物中研究较多、了解较为清楚的一类丝氨酸/苏氨酸蛋白激酶，在植物发育及其对生物和非生物胁迫信号转导的应答中执行不同的生物学功能[2-4]。基因组数据分析显示，在拟南芥(Arabidopsisthaliana)、水稻(Oryzasativa)、小麦(Triticumaestivum)、玉米(Zeamays)及杨树(Populustomentosa)中分别含有34、31、20、40、20个CDPK基因[5-6]。后续研究发现CDPK同样存在于绿藻类、卵菌和纤毛虫、顶覆虫等部分原生动物中。CDPK在不同组织中的表达、亚细胞定位、作用底物以及与不同蛋白体形成复合物等方面的差异，决定了该家族成员功能的差异，因此，本研究拟从这些方面对CDPKs进行全面的概述。

1 CDPK的结构特征及调控机制

CDPK首次发现于豌豆(Pisumsativum)中，第1次从大豆(Glycinemax)中得到纯化和鉴定。与其他钙结合蛋白不同，CDPK是单肽链结构。如图1所示，CDPK具有4个典型的结构域，从N端到C端依次为N-末端的可变域、催化域(激酶域)、连接域(自抑制域)和调控域(类钙调素域/CaM-LD)。

图1 CDPK的结构特征Fig.1 Structural features of CDPK

N-末端可变域序列相似性低，保守性差[7]。多数 CDPK 在N-端可变域含有与膜定位相关的豆蔻酰化位点(第2位的甘氨酸残基)以及棕榈酰化位点(第4或第5位的半胱氨酸残基)[8]，参与CDPK的膜结合过程。此外，N-末端可变域还在调节底物特异性过程中扮演关键的角色[9-10]；催化域也称激酶域，具有典型的Ser/Thr蛋白激酶的保守序列，同源性较高[11]，该区域突变会导致催化活性丧失；连接域高度保守，Liese等[7]的研究表明，连接域功能上相当于一种假底物，与催化域结合保证CDPK处于非活跃状态，因此也叫自抑制域；C-末端调控域上具有类似钙调素的结构，因此CDPK的激活依赖于钙离子而不依赖于钙调素。调控域包含4个可与钙离子结合的EF-手性结构，是Ca2+和CDPK的结合位点[12]。根据对Ca2+的亲和性不同将其分为两个球形结构，分别为N-lobe和C-lobe[13]，N-lobe对Ca2+亲和性低，C-lobe对Ca2+亲和性高，当Ca2+浓度低时与C-lobe结合，当Ca2+浓度高时与N-lobe结合，与连接域相互作用，导致调控域发生构象变化，解除自抑制。

2 CDPK的分布与亚细胞定位

2.1 CDPK的表达特性及其在植株内的分布

CDPK在植株中分布极其广泛，器官水平上，在植株的根、茎、叶、花、果实及种子中均能检测到基因的表达；细胞水平上，CDPK基因普遍存在于分生组织细胞、木质部细胞、保卫细胞、花粉母细胞及胚胎细胞中。CDPK的家族成员的表达特点不尽相同，有些可以在植物的大多数器官或组织中表达，如烟草(Nicotianatabacum)NtCPK1在叶片中不表达，在根、茎和花中均有表达[14]；拟南芥的AtCPK12在根、茎、叶、花和成熟果荚等组织中均有表达，但在干种子中不表达[15]；油菜(Brassicacampestris)BnCDPK1 在根、茎、叶、花和种子中都有表达，而有些却只在特定的组织表达，如AtCPK16、AtCPK17、AtCPK25、AtCPK34只在花粉管中表达[16]；此外，CDPK在不同器官中表达丰度也存在差异，油菜BnCDPK1在叶片中表达量最高，其次为茎、根和种子，在花中的表达量最低[17]。

2.2 CDPK的亚细胞定位及其影响因素

通过亚细胞定位研究表明，CDPK可定位于细胞质膜、内质网膜、胚乳贮藏囊泡、细胞骨架、线粒体、染色质、细胞核、细胞质、过氧化物酶体、油体及液泡膜等部位，N-末端可变域是CDPK亚细胞定位和功能的关键[18]。利用CDPK与绿色荧光蛋白(GFP)融合表达的方法，发现拟南芥CDPK几乎全部定位于质膜，少数定位于核。大多拟南芥CDPK具有专一的膜结合位点，即N-末端可变域的酰基化位点，其中豆蔻酰化位点与靶细胞膜形成一个不可逆转的松散的结合，而棕榈酰化位点则具有可逆的稳定的膜锚定结合[19]。豆蔻酰化位点上的甘氨酸突变为丙氨酸(如AtCPK2、AtCPK3、AtCPK5、AtCPK6、AtCPK9和AtCPK13)使CDPK的膜结合能力降低[20]，棕榈酰化位点突变则使亚细胞定位由膜向核转变。另外，CDPK的亚细胞定位也受非生物胁迫的影响，如冰草(Agropyroncristatum)McCPK1响应低温胁迫而改变亚细胞定位，由定位于质膜向定位于细胞核、内质网(ER)和肌动蛋白丝转变[21]。

3 CDPK的靶蛋白

了解CDPK在植物体内作用的靶蛋白，对研究其功能具有重要意义。到目前为止，已发现多种CDPK作用的底物(表1)，它们参与多种细胞过程，如初级和次级代谢、胁迫响应、离子和水分运输、转录过程、信号转导途径等。

4 CDPK与14-3-3蛋白的相互作用

在上述CDPK的靶蛋白中，14-3-3蛋白因其与CDPK之间的交叉磷酸化作用受到学者的广泛关注。14-3-3蛋白既与磷酸化的CDPK结合也可被CDPK磷酸化，在真核生物中通过磷酸化及与蛋白质互作调节多种生物学过程。

4.1 14-3-3蛋白对CDPK的调节

14-3-3蛋白对CDPK的活性有直接调节作用。拟南芥AtCPK1是第1个被发现的受14-3-3蛋白调控的CDPK[45]，但在体外实验中，AtCPK1未能与14-3-3ω蛋白相结合，因此推测AtCPK1和14-3-3ω之间的相互作用可能发生在胞内[46]，作用位点仍有待鉴定。14-3-3蛋白除了参与CDPK活性的直接调节外，还可以控制CDPK的稳定性。拟南芥AtCPK3已被证实为14-3-3的相互作用蛋白[47-48]，尽管体外实验显示14-3-3蛋白并不影响AtCPK3的激酶活性，但近期研究发现，饥饿诱导胁迫的AtCPK3在与14-3-3蛋白质相互作用丧失后被选择性切割[49]，证明拟南芥细胞内的14-3-3与AtCPK3的互作可以稳定AtCPK3的活性。此外，在拟南芥细胞程序性死亡过程中，坏死性真菌产生的毒素FB1(Fumonisin B1)诱导拟南芥中程序性死亡因子PHS(phytosphingosine)表达，PHS水平的增加导致胞内Ca2+升高，从而激活AtCPK3活性并磷酸化14-3-3蛋白质，14-3-3的磷酸化使得14-3-3/AtCPK3复合体解离，AtCPK3被切割[50-52]，这一过程进一步证实了14-3-3蛋白对CDPK具有保护作用。

4.2 CDPK对14-3-3蛋白的调节

14-3-3蛋白不仅结合并调节磷酸化后的CDPK，它们自身也被磷酸化。动植物14-3-3s的各个位点都有可能被磷酸化，磷酸化在调节14-3-3与靶蛋白的相互作用过程中发挥了极其重要的作用[53-54]。在动物细胞中，程序性死亡因子PHS诱导两种不同的蛋白激酶PKA(protein kinase A)和PKCδ(protein kinase Cδ)识别并磷酸化14-3-3ζ二聚体界面处的丝氨酸残基(Ser58)，Ser58磷酸化破坏14-3-3s的二聚体结构，从而完成了细胞凋亡过程(图2)；在植物细胞中，与过氧化物酶体结合的AtCPK1和AtCPK3、AtCPK6、AtCPK8、AtCPK24、AtCPK28均可在多个位点磷酸化蛋白14-3-3χ和14-3-3ε[24]，其中对14-3-3χ和14-3-3ε蛋白磷酸化最快速的是AtCPK3和AtCPK28，因为二者N-末端可变域序列具有高度的同源性。

表1 已有文献报道的CDPK在植物体内作用的靶蛋白及其调控作用Table 1 The target protein and its regulatory role of CDPK

5 CDPK/14-3-3复合体的活性调控作用

拟南芥具有13个14-3-3蛋白编码基因，在植物发育的不同阶段和不同组织中差异表达。CDPK/14-3-3复合体在植物信号通路中协同作用，共同调控代谢、激素信号传导和气孔运动，以及对非生物和生物胁迫的响应等重要的生理生化过程[55-56]。

5.1 CDPK/14-3-3复合体在初级代谢过程中的活性调控作用

当光合作用不活跃时，拟南芥硝酸还原酶NR(nitrate reductase)在丝氨酸残基(Ser534)处被AtCPK17或AtCPK28磷酸化，14-3-3蛋白通过结合叶片中磷酸化的NR，阻止亚硝酸盐的产生，使其不能进一步还原成铵，最终导致NR失活[24,42]。菠菜(Spinaciaoleracea)硝酸还原酶SoNR则在丝氨酸残基(Ser543)处被相关蛋白激酶SnRK(SNF1-related protein kinase)磷酸化后与抑制性蛋白14-3-3结合，由于拟南芥AtCPK3与菠菜SnRK具有非常相似的氨基酸序列，因此拟南芥AtCPK3能够使菠菜NR磷酸化[41]。14-3-3蛋白也可通过结合被AtCPK3磷酸化的拟南芥6-磷酸果糖-2-激酶F2KP(fructose-2,6-bisphosphatase)参与碳循环过程[25,46]。

图2 PHS诱导的细胞程序性死亡过程[52]Fig.2 PHS-induced programmed cell death process[52] PKA: 蛋白激酶A Protein kinase A; PKC: 蛋白激酶C Protein kinase C; P: 磷酸化Phosphorylation.

5.2 CDPK/14-3-3复合体在植物激素合成过程中的活性调控作用

CDPK与赤霉素(GA)信号传导存在紧密的联系。1995年，在水稻中首次发现由GA诱导的未知的膜定位CDPK[43]。10年后，Abbasi等[56]研究显示，OsCDPK13在转录水平响应GA而被活化；拟南芥AtCPK28为GA和茉莉酸(JA)的调节剂，AtCPK28突变体改变了维持GA稳态的关键酶的表达，表现出枝条和叶柄生长缓慢的特性[57-58]；烟草中参与GA生物合成的反馈调节因子芽生长抑制因子RSG(repression of shoot growth)被鉴定为NtCDPK1的直接靶标，RSG通过控制GA生物合成基因的转录来维持GA稳态。RSG在细胞质基质和细胞核之间来回移动，当GA浓度降低时，RSG转入细胞核中激活GA合成基因的转录，合成的GA诱导细胞质基质中游离Ca2+增加，从而导致NtCDPK1构象发生改变，促进RSG在S114片段磷酸化并与14-3-3蛋白结合。NtCDPK1与RSG和14-3-3蛋白质形成异源三聚体并作为支架和媒介促进磷酸化后的RSG与14-3-3形成二聚体，RSG/14-3-3复合体从NtCDPK1脱落后，细胞质基质中的RSG失活，核内靶基因的活化被抑制[43,58-59](图3)。

图3 CDPK和14-3-3蛋白对GA稳态的调控[52]Fig.3 The regulation of CDPK and 14-3-3 protein on GA homeostasis[52] N: 细胞核Nucleus; C: 细胞质基质Cytoplasmic matrix; P: 磷酸化Phosphorylation.下同The same below.

在植物乙烯的生物合成途径中，限速酶1-氨基环丙烷-1-羧酸合酶ACS(1-aminocyclopropane-1-carboxylic acid ACC synthase)家族起到了催化调控的作用。ACS因其C-末端存在不同的序列被分为3种类型，14-3-3s与所有类型的ACS均可以相互作用[39]。拟南芥AtACS7是一种3型ACS，可以被AtCPK16在3个位点(Ser216、Thr296和Ser299)处磷酸化，然后在细胞质基质中与14-3-3ω直接相互作用形成蛋白复合体[60-61]，AtCPK16和14-3-3ω的协同作用增加了AtACS7的稳定性。

5.3 CDPK/14-3-3复合体在开花过程中的活性调控作用

凝胶内激酶测定结果显示，磷脂酰乙醇胺结合蛋白开花位点D(AtFD)T282片段可能是AtCPK6和AtCPK33的底物[62]，磷酸化后的AtFD与14-3-3蛋白形成复合体，与开花位点T(AtFT)相互作用激活花分生组织基因如拟南芥花分生组织决定基因AP1(Apetala 1)的转录，从而促进开花过程[63]。而AtCPK33突变体则表现出轻度延迟开花的现象(图4)。

6 CDPK的生物学功能

6.1 CDPKs在植物发育中的作用

图4 CDPK调控开花过程[4]Fig.4 CDPK control flowering process[4]

图5 CDPK对钙信号传导的调控[4]Fig.5 Regulation of calcium signaling of CDPK[4]

CDPK是植物所有主要发育过程包括传粉、生长、开花和衰老的调控者。许多拟南芥CDPK(AtCPK2、AtCPK14、AtCPK16、AtCPK17、AtCPK20、AtCPK24、AtCPK26和AtCPK34)主要在花粉中表达，表明它们参与花粉发育或花粉管伸长。最近的研究表明，拟南芥中的两种CDPK，依赖Ca2+的AtCPK11和不依赖于Ca2+的AtCPK24可以形成级联信号，AtCPK11磷酸化AtCPK24 N-末端结构域，在花粉管特异性钾离子内流通道SPIK(shaker pollen inward K+)定位的质膜处相互作用并抑制其活性，从而对花粉管伸长进行负调节作用。此外，其他两种花粉特异性CDPK，AtCPK17和AtCPK34也定位于细胞质膜，推测可能与花粉伸长过程相关，研究发现AtCPK17/AtCPK34双突变体在花粉管的顶端极化生长表达上存在缺陷，而单个的突变则正常表达[64-65]。

6.2 CDPK对钙信号传导的调控

在产生钙信号过程中，拟南芥内质网膜和细胞质膜上分别具有自我抑制型Ca2+泵ACA2(autoinhibted calcium ATPase 2)和ACA8(autoinhibted calcium ATPase 8)，这两种Ca2+泵调控Ca2+泵入内质网腔或泵出细胞，维持细胞质基质中较低的Ca2+浓度，二者均可以直接被CDPK磷酸化。Giacometti等[66]研究表明，AtCPK16定位于质膜，因此在质膜内磷酸化AtACA8。AtCPK1可以与过氧化物酶体和微粒体结合，推测与过氧化物酶体相结合的AtCPK1/P复合体可作用于AtACA2和AtACA8；而ER上可衍生出微粒体，因此推测与微粒体相结合的AtCPK1/LB复合体可能与AtACA2连接并调控其作用(图5)。

6.3 CDPK参与植物非生物胁迫应答

研究表明，CDPK参与植物非生物胁迫反应，低温[67]、光[68]、干旱[69]、盐害[70]、低渗透[71]、营养饥饿等多种环境因子以及赤霉素GA、生长素、细胞分裂素等激素的诱导都能引起CDPK基因的特异性表达，如表2所列。

表2 不同植物中响应非生物胁迫的CDPKTable 2 CDPK responsed to abiotic stresses in different plants

6.4 CDPK与气孔运动

ABA依赖性的气孔闭合是防止干旱胁迫期间水分流失的关键机制[80]，许多CDPK参与到气孔运动的调控过程中[81]。在拟南芥中，离子通道AtKAT1和AtKAT2以及其他K+转运蛋白具有调节气孔开放的功能[54,80]。AtCPK1则激活液泡膜上的Cl-通道，导致Cl-内流；AtCPK1介导的Cl-内流、AtKAT1/AtKAT2介导的K+内流、阴离子通道SLAC1(slowtype anion-associated 1)与SLAH3(SLAC1 homologue 3)介导的阴离子(Cl-、NO3-)外流三者协同作用，提高了保卫细胞的渗透势，导致保卫细胞膨胀，气孔打开(图6左)；AtCPK3、AtCPK13是保卫细胞中主要表达的Ca2+敏感性CDPK，磷酸化AtKAT1和AtKAT2[82]，从而抑制K+内流过程。Sato等[83]的研究表明，AtSnRK2和拟南芥AtCPK4、AtCPK13可以在细胞内磷酸化AtKAT1的C-末端片段T306和T308，表明拟南芥CDPK和SnRK2之间可能存在一致的靶位点；T306的突变导致AtKAT1和AtKAT2通道活性受损。ABA信号传导产生激活的AtCPK3、AtCPK6、AtCPK21和AtCPK23以及AtOST1的活性氧(ROS)和Ca2+信号，这些激酶在保卫细胞离子通道的调节中起主要作用，它们激活AtSLAC1和AtSLAH3阴离子通道，促进阴离子的流出[84-86]。此外，AtCPK3、AtCPK4、AtCPK5、AtCPK11和AtCPK29在液泡外磷酸化双孔钾离子通道TPK1(tandem-pore K+channel 1)，磷酸化的AtTPK1与14-3-3蛋白质形成蛋白复合体介导液泡中的K+流出[87]。质膜质子泵的抑制依赖于Ca2+的阴离子通道的活化导致质膜去极化，激活保卫细胞外向整流型K+通道GORK(guard cell outward-rectifying K+channel)，K+外流。最终，保卫细胞内溶质的流出导致其膨胀程度降低，气孔闭合(图6右)。

图6 CDPK对气孔运动过程的调控[4]Fig.6 Regulation of stomatal movement of CDPK[4]

7 结语

到目前为止，CDPK的结构、亚细胞定位以及调控机制等研究已经比较成熟，越来越多CDPK家族成员的新功能已经被发现，但其作用底物和抑制剂还有待于进一步的研究，如弓形虫寄生病的治疗。弓形虫是一种常见的原生动物，几乎可以感染所有温血动物和人类，目前尚缺乏有效的治疗药物，但由于弓形虫中有CDPK，而人体并无CDPK，因此CDPK专一性抑制剂可能成为弓形虫治疗的新型药物。此外，CDPK和14-3-3蛋白质之间存在复杂的调控网络。如本研究所述，CDPK不仅磷酸化14-3-3蛋白,其本身也被14-3-3磷酸化，尽管近年的多项研究都显示出CDPK/14-3-3复合体在植物活性调控过程中的重要作用，但CPDKs与14-3-3s通过磷酸化的交叉调节过程仍不清晰，有待于国内外学者的进一步探索。

References：

[1] Wang J J, Han S F, Li X J,etal. Calcium-dependent protein kinase (CPDKs) mediates the molecular basis of plant signal transduction. Acta Prataculturae Sinica, 2009, 18(3): 241-250.

王娇娇, 韩胜芳, 李小娟, 等. 钙依赖蛋白激酶(CPDKs)介导植物信号转导的分子基础. 草业学报, 2009, 18(3): 241-250.

[2] Hamel L P, Sheen J, Séguin A. Ancient signals: comparative genomics of green plant CDPKs. Trends in Plant Science, 2014, 19(2): 79-89.

[3] Romeis T, Herde M. From local to global: CDPKs in systemic defense signaling upon microbial and herbivore attack. Current Opinion in Plant Biology, 2014, 20: 1-10.

[4] Simeunovic A, Mair A, Wurzinger B,etal. Know where your clients are: subcellular localization and targets of calcium-dependent protein kinases. Journal of Experimental Botany, 2016, 67(13): 3855.

[5] Boudsocq M, Sheen J. CDPKs in immune and stress signaling. Trends in Plant Science, 2013, 18(1): 30-40.

[6] Cai H, Cheng J, Yan Y,etal. Genome-wide identification and expression analysis of calcium-dependent protein kinase and its closely related kinase genes inCapsicumannuum. Frontiers in Plant Science, 2015, 6: 737.

[7] Liese A, Romeis T. Biochemical regulation ofinvivofunction of plant calcium-dependent protein kinases (CDPK). Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 2013, 1833(7): 1582-1589.

[8] Rutschmann F, Stalder U, Piotrowski M,etal. LeCPK1, a calcium-dependent protein kinase from tomato. Plasma membrane targeting and biochemical characterization. Plant Physiology, 2002, 129(1): 156-168.

[9] Asai S, Ichikawa T, Nomura H,etal. The variable domain of a plant calcium-dependent protein kinase (CDPK) confers subcellular localization and substrate recognition for NADPH oxidase. Journal of Biological Chemistry, 2013, 288(20): 14332-14340.

[10] Ito T, Nakata M, Fukazawa J,etal. Alteration of substrate specificity: the variable N-terminal domain of tobacco Ca2+-dependent protein kinase is important for substrate recognition. The Plant Cell, 2010, 22(5): 1592-1604.

[11] Hrabak E M, Chan C W M, Gribskov M,etal. TheArabidopsisCDPK-SnRK superfamily of protein kinases. Plant Physiology, 2003, 132(2): 666-680.

[12] Dubrovina A S, Kiselev K V, Veselova M V,etal. Enhanced resveratrol accumulation in rolB transgenic cultures ofVitisamurensiscorrelates with unusual changes in CDPK gene expression. Journal of Plant Physiology, 2009, 166(11): 1194-1206.

[13] Christodoulou J, Malmendal A, Harper J F,etal. Evidence for differing roles for each lobe of the calmodulin-like domain in a calcium-dependent protein kinase. Journal of Biological Chemistry, 2004, 279(28): 29092-29100.

[14] Yoon G M, Cho H S, Ha H J,etal. Characterization of NtCDPK1, a calcium-dependent protein kinase gene inNicotianatabacum, and the activity of its encoded protein. Plant Molecular Biology, 1999, 39(5): 991-1001.

[15] Zhao R, Sun H L, Mei C,etal. TheArabidopsisCa2+-dependent protein kinase CPK12 negatively regulates abscisic acid signaling in seed germination and post-germination growth. New Phytologist, 2011, 192(1): 61-73.

[16] Myers C, Romanowsky S M, Barron Y D,etal. Calcium-dependent protein kinases regulate polarized tip growth in pollen tubes. The Plant Journal, 2009, 59(4): 528-539.

[17] Zhang Q P, Wen L, Wang F,etal. Cloning and expression analysis of calcium-dependent protein kinase BnCDPK1 inBrassica. Journal of Plant Genetic Resources, 2014, 15(6): 1320-1326.

张秋平, 文李, 王峰, 等. 油菜钙依赖蛋白激酶 BnCDPK1 的克隆和表达分析. 植物遗传资源学报, 2014, 15(6): 1320-1326.

[18] Dammann C, Ichida A, Hong B,etal. Subcellular targeting of nine calcium-dependent protein kinase isoforms fromArabidopsis. Plant Physiology, 2003, 132(4): 1840-1848.

[19] Resh M D. Trafficking and signaling by fatty-acylated and prenylated proteins. Nature Chemical Biology, 2006, 2(11): 584-590.

[20] Mehlmer N, Wurzinger B, Stael S,etal. The Ca2+-dependent protein kinase CPK3 is required for MAPK-independent salt-stress acclimation inArabidopsis. The Plant Journal, 2010, 63(3): 484-498.

[21] Chehab E W, Patharkar O R, Hegeman A D,etal. Autophosphorylation and subcellular localization dynamics of a salt-and water deficit-induced calcium-dependent protein kinase from ice plant. Plant Physiology, 2004, 135(3): 1430-1446.

[22] Klimecka M, Muszyńska G. Structure and functions of plant calcium-dependent protein kinases. Acta Biochimica Polonica, 2007, 54(2): 219-233.

[23] Curran A, Chang I F, Chang C L,etal. Calcium-dependent protein kinases fromArabidopsisshow substrate specificity differences in an analysis of 103 substrates. Frontiers in Plant Science, 2011, 2(12): 1085-1091.

[24] Swatek K N, Wilson R S, Ahsan N,etal. Multisite phosphorylation of 14-3-3 proteins by calcium-dependent protein kinases. Biochemical Journal, 2014, 459(1): 15-25.

[25] Kulma A, Villadsen D, Campbell D G,etal. Phosphorylation and 14-3-3 binding ofArabidopsis6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase. The Plant Journal, 2004, 37(5): 654-667.

[26] Veerabagu M, Kirchler T, Elgass K,etal. The interaction of theArabidopsisresponse regulator ARR18 with bZIP63 mediates the regulation of proline dehydrogenase expression. Molecular Plant, 2014, 7(10): 1560-1577.

[27] Mair A, Pedrotti L, Wurzinger B,etal. SnRK1-triggered switch of bZIP63 dimerization mediates the low-energy response in plants. Elife Sciences, 2015, 4: e05828.

[28] Kanchiswamy C N, Takahashi H, Quadro S,etal. Regulation ofArabidopsisdefense responses againstSpodopteralittoralisby CPK-mediated calcium signaling. BMC Plant Biology, 2010, 10(1): 97-106.

[29] Liu Y, Zhang S. Phosphorylation of 1-aminocyclopropane-1-carboxylic acid synthase by MPK6, a stress-responsive mitogen-activated protein kinase, induces ethylene biosynthesis inArabidopsis. The Plant Cell, 2004, 16(12): 3386-3399.

[30] Joo S, Liu Y, Lueth A,etal. MAPK phosphorylation-induced stabilization of ACS6 protein is mediated by the non-catalytic C-terminal domain, which also contains the cis-determinant for rapid degradation by the 26S proteasome pathway. The Plant Journal, 2008, 54(1): 129-140.

[31] Han L, Li G J, Yang K Y,etal. Mitogen-activated protein kinase 3 and 6 regulateBotrytiscinerea-induced ethylene production inArabidopsis. The Plant Journal, 2010, 64(1): 114-127.

[32] Luo X, Chen Z, Gao J,etal. Abscisic acid inhibits root growth inArabidopsisthrough ethylene biosynthesis. The Plant Journal, 2014, 79(1): 44-55.

[33] Choi H, Park H J, Park J H,etal.Arabidopsiscalcium-dependent protein kinase AtCPK32 interacts with ABF4, a transcriptional regulator of abscisic acid-responsive gene expression, and modulates its activity. Plant Physiology, 2005, 139(4): 1750-1761.

[34] Milla R, Miguel A, Uno Y,etal. A novel yeast two-hybrid approach to identify CDPK substrates: Characterization of the interaction between AtCPK11 and AtDi19, a nuclear zinc finger protein 1. FEBS Letters, 2006, 580(3): 904-911.

[35] Uno Y, Milla M A R, Maher E,etal. Identification of proteins that interact with catalytically active calcium-dependent protein kinases fromArabidopsis. Molecular Genetics and Genomics, 2009, 281(4): 375-390.

[36] Winter H, Huber S C. Regulation of sucrose metabolism in higher plants: localization and regulation of activity of key enzymes. Critical Reviews in Plant Sciences, 2000, 19(1): 31-67.

[37] Hardin S C, Winter H, Huber S C. Phosphorylation of the amino terminus of maize sucrose synthase in relation to membrane association and enzyme activity. Plant Physiology, 2004, 134(4): 1427-1438.

[38] Fedosejevs E T, Ying S, Park J,etal. Biochemical and molecular characterization of RcSUS1, a cytosolic sucrose synthase phosphorylatedinvivoat serine 11 in developing castor oil seeds. Journal of Biological Chemistry, 2014, 289(48): 33412-33424.

[39] Yoon G M. New insights into the protein turnover regulation in ethylene biosynthesis. Molecular Cells, 2015, 38(7): 597-603.

[40] Kobayashi M, Yoshioka M, Asai S,etal. StCDPK5 confers resistance to late blight pathogen but increases susceptibility to early blight pathogen in potato via reactive oxygen species burst. New Phytologist, 2012, 196(1): 223-237.

[41] Sugden C, Donaghy P G, Halford N G,etal. Two SNF1-related protein kinases from spinach leaf phosphorylate and inactivate 3-hydroxy-3-methylglutaryl-coenzyme A reductase, nitrate reductase, and sucrose phosphate synthaseinvitro. Plant Physiology, 1999, 120(1): 257-274.

[42] Lambeck I, Chi J C, Krizowski S,etal. Kinetic analysis of 14-3-3-inhibitedArabidopsisthaliananitrate reductase. Biochemistry, 2010, 49(37): 8177-8186.

[43] Ishida S, Yuasa T, Nakata M,etal. A tobacco calcium-dependent protein kinase, CDPK1, regulates the transcription factor repression of shoot growth in response to gibberellins. The Plant Cell, 2008, 20(12): 3273-3288.

[44] Cheng S H, Sheen J, Gerrish C,etal. Molecular identification of phenylalanine ammonialyase as a substrate of a specific constitutively activeArabidopsisCDPK expressed in maize protoplasts. FEBS Letters, 2001, 503(23): 185-188.

[45] Chang I F, Curran A, Woolsey R,etal. Proteomic profiling of tandem affinity purified 14-3-3 protein complexes inArabidopsisthaliana. Proteomics, 2009, 9(11): 2967-2985.

[46] Pallucca R, Visconti S, Camoni L,etal. Specificity of ε and non-ε isoforms ofArabidopsis14-3-3 proteins towards the H+-ATPase and other targets. Plos One, 2014, 9(6): e90764.

[47] Cotelle V, Meek S E, Provan F,etal. 14-3-3s regulate global cleavage of their diverse binding partners in sugar-starvedArabidopsiscells. EMBO Journal, 2000, 19(12): 2869-2876.

[48] Lachaud C, Prigent E, Thuleau P,etal. 14-3-3-regulated Ca2+-dependent protein kinase CPK3 is required for sphingolipid-induced cell death inArabidopsis. Cell Death & Differentiation, 2013, 20(2): 209-217.

[49] Denison F C, Gökirmak T, Ferl R J. Phosphorylation-related modification at the dimer interface of 14-3-3ω dramatically alters monomer interaction dynamics. Archives of Biochemistry and Biophysics, 2014, 541: 1-12.

[50] Gökirmak T, Denison F C, Laughner B J,etal. Phosphomimetic mutation of a conserved serine residue inArabidopsisthaliana14-3-3ω suggests a regulatory role of phosphorylation in dimerization and target interactions. Plant Physiology and Biochemistry, 2015, 97: 296-303.

[51] Boer A H D, Kleeff P J M V, Jing G. Plant 14-3-3 proteins as spiders in a web of phosphorylation. Protoplasma, 2013, 250(2): 425.

[52] Wilson R S, Swatek K N, Thelen J J. Regulation of the regulators: post-translational modifications, subcellular, and spatiotemporal distribution of plant 14-3-3 proteins. Frontiers in Plant Science, 2016, 7: 611.

[53] Ormancey M, Thuleau P, Mazars C,etal. CDPKs and 14-3-3 proteins: Emerging Duo in signaling. Trends in Plant Science, 2017, 22(3): 263-272.

[54] Lozano-Durán R, Robatzek S. 14-3-3 proteins in plant-pathogen interactions. Molecular Plant-Microbe Interactions, 2015, 28(5): 511-518.

[55] Cotelle V, Leonhardt N. 14-3-3 proteins in guard cell signaling. Frontiers in Plant Science, 2016, 6(e90734): 1210.

[56] Abbasi F, Onodera H, Toki S,etal. OsCDPK13, a calcium-dependent protein kinase gene from rice, is induced by cold and gibberellin in rice leaf sheath. Plant Molecular Biology, 2004, 55(4): 541-552.

[57] Matschi S, Werner S, Schulze W X,etal. Function of calcium-dependent protein kinase CPK28 ofArabidopsisthalianain plant stem elongation and vascular development. The Plant Journal, 2013, 73(6): 883-896.

[58] Ito T, Nakata M, Fukazawa J,etal. Scaffold function of Ca2+-dependent protein kinase: NtCDPK1 transfers 14-3-3 to the substrate RSG after phosphorylation. Plant Physiology, 2014, 165(4): 1737-1750.

[59] Ito T, Nakata M, Fukazawa J,etal. Scaffold function of Ca2+-dependent protein kinase: tobacco Ca2+-dependent protein kinase1 transfers 14-3-3 to the substrate repression of shoot growth after phosphorylation. Plant Physiology, 2014, 165(4): 1737-1750.

[60] Huang S J, Chang C L, Wang P H,etal. A type III ACC synthase, ACS7, is involved in root gravitropism inArabidopsisthaliana. Journal of Experimental Botany, 2013, 64(14): 4343-4360.

[61] Lyzenga W J, Booth J K, Stone S L. TheArabidopsisRING-type E3 ligase XBAT32 mediates the proteasomal degradation of the ethylene biosynthetic enzyme, 1-aminocyclopropane-1-carboxylate synthase 7. Plant Journal for Cell & Molecular Biology, 2012, 71(1): 23-34.

[62] Kawamoto N, Sasabe M, Endo M,etal. Calcium-dependent protein kinases responsible for the phosphorylation of a bZIP transcription factor FD crucial for the florigen complex formation. Scientific Reports, 2015, 5: 8341.

[63] Taoka K, Ohki I, Tsuji H,etal. Structure and function of florigen and the receptor complex. Trends in Plant Science, 2013, 18(5): 287-294.

[64] Zhao L N, Shen L K, Zhang W Z,etal. Ca2+-dependent protein kinase 11 and 24 modulate the activity of the inward rectifying K+channels inArabidopsispollen tubes. The Plant Cell, 2013, 25(2): 649-661.

[65] Liu H, Che Z, Zeng X,etal. Genome-wide analysis of calcium-dependent protein kinases and their expression patterns in response to herbivore and wounding stresses in soybean. Functional & Integrative Genomics, 2016, 16(5): 481-493.

[66] Giacometti S, Marrano C A, Bonza M C,etal. Phosphorylation of serine residues in the N-terminus modulates the activity of ACA8, a plasma membrane Ca2+-ATPase ofArabidopsisthaliana. Journal of Experimental Botany, 2012, 63(3): 1215-1224.

[67] Li W G, Komatsu S. Cold stress-induced calcium-dependent protein kinase (s) in rice (OryzasativaL.) seedling stem tissues. Theoretical and Applied Genetics, 2000, 101(3): 355-363.

[68] Giammaria V, Grandellis C, Bachmann S,etal. StCDPK2 expression and activity reveal a highly responsive potato calcium-dependent protein kinase involved in light signalling. Planta, 2011, 233(3): 593.

[69] Zou J J, Wei F J, Wang C,etal.Arabidopsiscalcium-dependent protein kinase CPK10 functions in abscisic acid-and Ca2+-mediated stomatal regulation in response to drought stress. Plant Physiology, 2010, 154(3): 1232-1243.

[70] Xu J, Tian Y S, Peng R H,etal. AtCPK6, a functionally redundant and positive regulator involved in salt/drought stress tolerance inArabidopsis. Planta, 2010, 231(6): 1251-1260.

[71] Romeis T, Ludwig A A, Martin R,etal. Calcium-dependent protein kinases play an essential role in a plant defence response. EMBO Journal, 2001, 20(20): 5556-5567.

[72] Zhang M, Liang S, Lu Y T. Cloning and functional characterization of NtCPK4, a new tobacco calcium-dependent protein kinase. Biochimica et Biophysica Acta (BBA)-Gene Structure and Expression, 2005, 1729(3): 174-185.

[73] Ma S Y, Wu W H. AtCPK23 functions inArabidopsisresponses to drought and salt stresses. Plant Molecular Biology, 2007, 65(4): 511-518.

[74] Liu G, Chen J, Wang X. VfCPK1, a gene encoding calcium-dependent protein kinase fromViciafaba, is induced by drought and abscisic acid. Plant, Cell & Environment, 2006, 29(11): 2091-2099.

[75] Yu X C, Zhu S Y, Gao G F,etal. Expression of a grape calcium-dependent protein kinase ACPK1 inArabidopsisthalianapromotes plant growth and confers abscisic acid-hypersensitivity in germination, postgermination growth, and stomatal movement. Plant Molecular Biology, 2007, 64(5): 531-538.

[76] Berberich T, Kusano T. Cycloheximide induces a subset of low temperature-inducible genes in maize. Molecular and General Genetics MGG, 1997, 254(3): 275-283.

[77] Wan B, Lin Y, Mou T. Expression of rice Ca2+-dependent protein kinases (CDPKs) genes under different environmental stresses. FEBS Letters, 2007, 581(6): 1179-1189.

[78] Ye S, Wang L, Xie W,etal. Expression profile of calcium-dependent protein kinase (CDPKs) genes during the whole lifespan and under phytohormone treatment conditions in rice (OryzasativaL. ssp. indica). Plant Molecular Biology, 2009, 70(3): 311-325.

[79] Lanteri M L, Pagnussat G C, Lamattina L. Calcium and calcium-dependent protein kinases are involved in nitric oxide-and auxin-induced adventitious root formation in cucumber. Journal of Experimental Botany, 2006, 57(6): 1341-1351.

[80] Munemasa S, Hauser F, Park J,etal. Mechanisms of abscisic acid-mediated control of stomatal aperture. Current Opinion in Plant Biology, 2015, 28: 154-162.

[81] Zhang T, Chen S, Harmon A C. Protein phosphorylation in stomatal movement. Plant Signaling & Behavior, 2014, 9(11): e972845.

[82] Ronzier E, Corratgé-Faillie C, Sanchez F,etal. CPK13, a noncanonical Ca2+-dependent protein kinase, specifically inhibits KAT2 and KAT1 shaker K+channels and reduces stomatal opening. Plant Physiology, 2014, 166(1): 314-326.

[83] Sato A, Sato Y, Fukao Y,etal. Threonine at position 306 of the KAT1 potassium channel is essential for channel activity and is a target site for ABA-activated SnRK2/OST1/SnRK2. 6 protein kinase. Biochemical Journal, 2009, 424(3): 439-448.

[84] Geiger D, Scherzer S, Mumm P,etal. Guard cell anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca2+affinities. Proceedings of the National Academy of Sciences, 2010, 107(17): 8023-8028.

[85] Mori I C, Murata Y, Yang Y,etal. CDPKs, CPK6 and CPK3 function in ABA regulation of guard cell S-type anion-and Ca2+-permeable channels and stomatal closure. PLoS Biology, 2006, 4(10): e327.

[86] Brandt B, Brodsky D E, Xue S,etal. Reconstitution of abscisic acid activation of SLAC1 anion channel by CPK6 and OST1 kinases and branched ABI1 PP2C phosphatase action. Proceedings of the National Academy of Sciences, 2012, 109(26): 10593-10598.

[87] Latz A, Mehlmer N, Zapf S,etal. Salt stress triggers phosphorylation of theArabidopsisvacuolar K+channel TPK1 by calcium-dependent protein kinases (CDPKs). Molecular Plant, 2013, 6(4): 1274-1289.