谷晓勇, 刘扬, 刘利静 ^,山东大学生命科学学院,青岛 266237

Progress on the biosynthesis and signal transduction of phytohormone salicylic acid

Xiaoyong Gu, Yang Liu, Lijing Liu ^,School of Life Sciences, Shandong University, Qingdao 266237, China

通讯作者: 刘利静，博士，教授，研究方向：植物免疫。E-mail: ljliu@sdu.edu.cn

责任编辑: 储成才
收稿日期:2020-06-12修回日期:2020-07-14网络出版日期:2020-09-20

基金资助:

山东大学齐鲁青年学科建设经费项目资助编号. 11200087963080

Received:2020-06-12Revised:2020-07-14Online:2020-09-20

Fund supported:

Supported by the Qilu Scholarship from Shandong University No.11200087963080

作者简介 About authors
谷晓勇,硕士,助理实验师,研究方向：植物免疫。E-mail:guxy18@sdu.edu.cn。





摘要
植物激素水杨酸(salicylic acid,SA)是广泛存在于植物体中的小分子酚类物质,参与植物多种生理过程,特别是在植物免疫中发挥重要功能。植物免疫过程中体内SA大量合成,SA信号通路被激活从而诱导抗病相关基因表达。近年来,随着研究的不断深入,SA生物合成和信号转导都取得一系列重要进展：进一步完善了SA生物合成的异分支酸合酶(isochorismate synthase, ICS)和苯丙氨酸解氨酶(phenylalanine ammonia-lyase, PAL)途径;明确了NPR1 (nonexpresser of PR genes 1)和其同源蛋白NPR3、NPR4是植物接收SA的受体;发现II类TGA (TGACG-binding factor)转录因子通过与不同SA受体互作激活或抑制下游基因表达等。本文系统介绍了SA生物合成和信号转导领域的相关进展,以期为深入研究SA调控植物生长发育和环境胁迫响应提供理论参考。
关键词： 水杨酸;水杨酸生物合成;水杨酸受体;水杨酸信号转导

Abstract
The phenolic phytohormone salicylic acid (SA) is widely produced in plants, and is a key player in many processes of plant physiology, especially in plant immunity. During pathogen infection, SA is accumulated and the SA signaling pathway is activated to induce the expression of defense-related genes. Recently, a series of SA-related studies have been published. These researches filled gaps in the two SA biosynthesis pathways: the isochorismate synthase (ICS) pathway and the phenylalanine ammonia-lyase (PAL) pathway. The NPR1 (nonexpresser of PR genes 1) and its paralogs, NPR3 and NPR4, were identified as SA receptors. The effect of type II TGAs (TGACG-binding factor) on SA downstream genes was shown to depend on the SA receptor they interacted with. This review will systematically introduce the progress on SA biosynthesis and signal transduction, aiming to provide a theoretical reference for in-depth study of SA regulation on plant development and defense responses.
Keywords：salicylic acid;salicylic acid biosynthesis;salicylic acid receptors;salicylic acid signal transduction


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本文引用格式
谷晓勇, 刘扬, 刘利静. 植物激素水杨酸生物合成和信号转导研究进展. 遗传[J], 2020, 42(9): 858-869 doi:10.16288/j.yczz.20-173
Xiaoyong Gu. Progress on the biosynthesis and signal transduction of phytohormone salicylic acid. Hereditas(Beijing)[J], 2020, 42(9): 858-869 doi:10.16288/j.yczz.20-173


水杨酸(salicylic acid, SA)是一种广泛存在于细菌和植物体中的小分子酚类物质。早在公元前4世纪,古苏美尔人和古埃及人就已经开始使用柳树(Salix L.)和杨树(Populus L.)的树皮和树叶来缓解眼疾、风湿、分娩和发烧引起的疼痛^{[ 1]}。直到1828年,德国科学家Johann Buchner提取出柳树皮中有效成分,并以白柳的拉丁文学名Salix alba将其命名为水杨苷(SA glycoside, SAG)。1838年,Raffaele Piria进一步分解水杨苷得到SA。1898年拜耳公司将乙酰水杨酸以阿司匹林商标上市,并迅速成为世界最畅销药物之一^{[ 2]}。直到20世纪90年代初,人们才逐渐认识到SA在植物中的重要作用,并将其定义为植物激素^{[ 3]}。

植物激素SA参与植物生长发育的多个过程,并在植物对环境胁迫响应中发挥重要功能。SA调控植物种子萌发、出芽、开花、坐果和果实成熟等过程^{[ 4, 5]}。最新研究表明,SA通过抑制乙烯信号途径影响植物出芽过程中顶端弯钩的形成^{[ 6]}。SA介导植物对非生物胁迫的抗性,如SA能改变植物对重金属、热、冷、干旱、高盐等胁迫环境的适应性^{[ 4]}。SA是重要的免疫激素,目前对SA的研究主要集中在植物免疫领域^{[ 7, 8]}。植物先天免疫系统包括病原相关分子模式触发的免疫反应(pathogen associated molecular pattern-triggered immunity, PTI)和效应子触发的免疫反应(effector-triggered immunity, ETI),SA在这两种免疫反应中都发挥重要作用,如在SA合成突变体中,PTI和ETI对病原菌的生长抑制效果都被严重削弱^{[ 9, 10]}。SA对植物系统获得性抗性(systemic acquired resistance, SAR)至关重要,SA积累或信号转导缺失突变体不能正常产生SAR^{[ 11, 12]}。

SA对植物生长发育和环境胁迫响应的调控是通过改变植物体内SA浓度和SA下游基因表达强度来实现。在病原菌侵染时,植物体内SA生物合成和信号转导被增强,SA诱导抗病相关基因的表达从而提高植物抗病能力。因此,对SA生物合成和信号转导过程的认知是探究植物自身发育和其与环境互作的重要前提^{[ 13]}。异分支酸合酶(isochorismate synthase, ICS)途径和苯丙氨酸解氨酶(phenylalanine ammonia-lyase, PAL)途径是植物合成SA的主要方式,但参与这两条途径的部分酶类还未被解析^{[ 8]}。NPR1 (nonexpresser of PR genes 1)是SA信号转导的关键调控因子,但NPR1是否参与SA信号接收还存在争论^{[ 13]}。近几年,科研人员在SA研究领域发表了多篇重量级论文,进一步完善了SA生物合成途径,也使争论多年的SA受体问题尘埃落定。鉴于此,本文系统介绍了SA生物合成和信号转导研究的相关进展,以期为SA领域的相关研究提供借鉴和参考。

1 水杨酸的生物合成

植物通过两条通路合成水杨酸：ICS途径和PAL途径。它们都起始于叶绿体,以分支酸(chorismate)为前体,并涉及多个酶促反应( 图1)^{[ 8]}。两条途径对SA合成的贡献存在差异,在拟南芥(Arabidopsis thaliana)中,与免疫相关SA主要由ICS途径产生^{[ 14]}。

图1

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图1水杨酸合成示意图

植物通过两条途径合成SA。一是ICS途径：ICS催化分支酸产生异分支酸,异分支酸经EDS5转运至细胞质后PBS3催化其与谷氨酸结合生成IC-9-Glu,IC-9-Glu自主分解或经EPS1催化加速分解最终产生水杨酸;二是PAL途径：分支酸经催化产生苯丙氨酸,苯丙氨酸进入细胞质后由PALs催化产生反式肉桂酸,反式肉桂酸进入过氧化物酶体后经β-氧化产生苯甲酸,苯甲酸转运至细胞质后可能由BA2H羟化产生水杨酸。绿色箭头所示为发生在叶绿体中的反应;棕色箭头所示为发生在过氧化物酶体中的反应;蓝色箭头所示为细胞质中的反应过程。ICS：isochorismate synthase;EDS5：enhanced disease susceptibility 5;PBS3：avrPphB susceptible 3;EPS1：enhanced pseudomonas susceptibility 1;PAL：phenylalanine ammonia-lyase;AIM：abnormal inflorescence meristem 1;BA2H：benzoic acid 2-hydroxylase。
Fig. 1The schematic diagram of salicylic acid biosynthesis

1.1 ICS途径

ICS途径起始于分支酸,经异分支酸(isochorismate)、异分支酸-9-谷氨酸(isochorismate-9-glutamate, IC-9-Glu)最终合成水杨酸^{[ 8]}。在拟南芥中该途径有3种酶参与,分别是ICS、PBS3 (avrPphB susceptible 3)和EPS1 (enhanced pseudomonas susceptibility 1)^{[ 8]}。

1.1.1 ICS

ICS1是第一个被报道参与ICS途径的酶类,它以分支酸为底物合成异分支酸。通过正向遗传学的方法筛选病原菌处理后植物体内SA积累减少的突变体,发现了sid2 (SA induction deficient 2),其突变的基因位点就是ICS1^{[ 15]}。拟南芥基因组编码2个ICS——ICS1和ICS2,都定位于叶绿体。病原菌侵染或紫外线处理植物能诱导SA生物合成,而在ics1突变体中SA积累大幅度降低,ics1 ics2双突变体完全丧失诱导合成SA的能力^{[ 16]}。这些结果说明异分支酸的合成发生在叶绿体中,由ICS1和ICS2共同介导。

1.1.2 PBS3

植物中异分支酸如何进一步催化生成SA一直是科学界的未解之谜。虽然该过程在铜绿假单胞菌(Pseudomonas aeruginosa)中已被解析：异分支酸由异分支酸丙酮酸裂解酶(isochorismate pyruvate lyase, IPL)直接裂解为SA,但植物中没有IPL的同源蛋白^{[ 17]}。为了寻找这一谜题的答案,科研人员进一步探究低SA含量的拟南芥突变体,如pbs3和eps1等^{[ 18, 19]} SA生物合成减少的机理。Rekhter等^{[ 20]}在snc2-1D (suppressor of npr1-1, constitutive 2) npr1双突变体背景下突变PBS3。该双突变体本身SA含量偏高,植物生长受阻。虽然突变PBS3并未恢复snc2-1D npr1的生长表型,但是对突变体中SA及其前体和代谢物含量进行测定显示,SA和SAG的含量在snc2-1D npr1 pbs3中显著低于snc2-1D npr1,而异分支酸的含量在二者之间没有显著区别^{[ 20]}。这一结果说明PBS3作用于异分支酸下游介导SA的生物合成。而Torrens-Spence等^{[ 21]}发现PBS3突变可以互补另一高SA含量双突变体s3h (SA 3-hydroxylase) dmr6 (downy mildew resistant 6)的植株矮小表型。PBS3编码氨基转移酶(aminotransferase),体外生物化学实验表明PBS3可以促进底物谷氨酸化^{[ 22]}。Rekhter等^{[ 20]}和Torrens-Spence等^{[ 21]}都证明在ICS途径中PBS3负责将谷氨酸加在异分支酸上合成IC-9-Glu,IC-9-Glu可以自我衰变为SA。PBS3定位在细胞质中,因此异分支酸合成后需从叶绿体转运到细胞质中才能参与后续SA生物合成过程^{[ 20]}。Rekhter等^{[ 20]}和Torrens-Spence等^{[ 21]}的研究进一步完善了植物SA生物合成的ICS途径。

PBS3的发现表明植物已经进化出一种独特的、不同于细菌的ICS途径。PBS3存在于多种开花植物中,暗示该蛋白广泛参与植物SA生物合成过程^{[ 23]}。

1.1.3 EDS5

EDS5 (enhanced disease susceptibility 5)属于多种药物和毒素排出(multidrug and toxin extrusion, MATE)转运蛋白家族,定位于叶绿体膜。eds5突变体中SA含量降低,推测其介导SA或SA前体的转运^{[ 24]}。Serrano等^{[ 25]}将原生质体孵育在¹⁴C标记的SA溶液中,然后分离叶绿体,检测叶绿体中SA含量,结果显示EDS5可以介导SA在细胞质和叶绿体之间的转运。而在eds5突变体中,人为改变PBS3细胞质定位特性使之定位到叶绿体,植物能够正常合成SA^{[ 20]}。因此推测EDS5在PBS3上游发挥作用,负责异分支酸从叶绿体到细胞质的运输。

1.1.4 EPS1

EPS1是BAHD (BEAT、AHCT、HCBT和DAT)乙酰转移酶家族蛋白,eps1突变体在丁香假单胞菌(Pseudomonas syringae)侵染后植物体内SA积累少于野生型,对病原菌的抗性降低^{[ 19]}。EPS1突变可以互补s3h dmr6双突变体植株矮小表型,暗示其参与SA生物合成过程^{[ 21]}。EPS1具有异羟甲基-谷氨酸A丙酮酰谷氨酸裂解酶(isochorismoyl-glutamate A pyruvoylglutamate lyase)活性,催化IC-9-Glu裂解产生SA。在体外反应中加入EPS1可以加快IC-9-Glu裂解产生SA的速度^{[ 21]}。EPS1仅存在于十字花科植物中,表明其他科植物SA的合成可能依赖于IC-9-Glu自发衰变,或进化出了其他酶类来辅助该过程^{[ 21]}。

1.2 PAL途径

通过同位素标记实验发现在烟草(Nicotiana tabacum L.)中苯丙氨酸(phenylalanine, Phe)可以经反式肉桂酸(trans-cinnamic acid, t-CA)、苯甲酸(benzoic acid)进而合成SA^{[ 26]}。已知PAL和AIM (abnormal inflorescence meristem 1)分别是催化合成反式肉桂酸和苯甲酸的关键酶类,而苯甲酸可能被BA2H (benzoic acid 2-hydroxylase)羟基化产生SA,但在植物体内编码BA2H的基因尚未被解析^{[ 27]}。

1.2.1 PAL

大麦(Hordeum vulgare)的PAL最早被分离,并被证实具有苯丙氨酸脱氨酶活性^{[ 28]}。拟南芥基因组中含有4个PAL基因(PAL1~PAL4)。相比于野生型,pal1 pal2 pal3 pal4四突变体中基础PAL活性降低90%,正常生长状况下SA积累减少75%,病原菌侵染后突变体SA积累减少50%,说明PAL途径影响植物正常生长和病原菌侵染时SA的生物合成^{[ 29]}。这也解释了为什么在ics1 ics2双突变体中仍可以检测到SA^{[ 14]}。

水稻(Oryza sativa L.)PAL蛋白家族包括9个成员(OsPAL1~OsPAL9),大多数OsPAL在水稻中的表达受病原菌和昆虫诱导。OsPAL高表达增强水稻对病原菌和昆虫的抗性,说明PAL途径介导的SA生物合成对水稻免疫至关重要^{[ 30, 31, 32, 33]}。在此之前,对PAL的研究主要集中在植物抗菌领域,He等^{[ 33]}研究发现PAL的抗虫功能,增强了对PAL和SA功能的认知,具有重要的科学意义。

1.2.2 AIM1

反式肉桂酸可以通过β氧化途径在过氧化物酶体中合成苯甲酸。已知有3类酶参与该过程,分别是肉桂酸:辅酶A连接酶(cinnamate: CoA ligase)、羟酰辅酶A水解酶(hydroxyacyl-CoA hydrolyase)和3-酮酰基辅酶A硫醇酶(3-ketoacyl CoA thiolase, KAT1)^{[ 34, 35, 36]}。AIM1编码羟酰辅酶A水解酶,是拟南芥种子中合成苯甲酸代谢物的重要酶类^{[ 37]}。在水稻aim1突变体中,肉桂酸含量升高,苯甲酸和SA含量大幅度降低,说明AIM1参与反式肉桂酸到苯甲酸的β氧化过程^{[ 38]}。

综上所述,植物SA生物合成的ICS途径已基本被解析,PAL途径也进一步被完善,但PAL途径的部分反应过程,如苯甲酸如何羟基化形成SA等还有待进一步探究。虽然SA生物合成过程的大部分酶类是在拟南芥和水稻中发现的,但在烟草、番茄(Lycopersicon esculentum Mill.)、杨树、红花(Carthamus tinctorius L.)和黄瓜(Cucumis sativus L.)中同样发现SA经由ICS或PAL途径合成,表明这两种SA生物合成途径在进化上具有保守性^{[ 39, 40, 41, 42]}。拟南芥中病原菌侵染时SA生物合成主要依赖于ICS途径,但在烟草感染病毒后,体内苯甲酸和SA大量合成,暗示烟草中病原体诱导的SA主要通过PAL途径产生^{[ 43]}。因此这两种途径在不同植物中对SA生物合成的贡献具有物种特异性。

1.3 水杨酸的合成调控

在病原菌侵染时SA生物合成途径中的基因,如ICS1被诱导表达,促进SA积累进而增强植物抗病性^{[ 15]}。迄今为止,已有多个正调控ICS1表达的转录因子被报道,包括TCP (teosinte branched1/cycloidea/pcf)、WRKY (WRKY DNA binding protein)和CBP60 (CaM-binding protein 60)类蛋白等^{[ 44, 45, 46]}。其中,对CBP60类蛋白SARD1(SAR-deficient 1)和CBP60g的研究较为深入^{[ 47]}。病原菌通过TGA1 (TGACG-binding factor 1)和TGA4诱导SARD1和CBP60g表达^{[ 48]}。ChIP-Seq (chromatin immunoprecipitation-sequencing)分析显示SARD1和CBP60g能够结合ICS1、EDS5和PBS3等SA合成相关基因的启动子序列^{[ 49]}。Wang等^{[ 50]}研究显示,病原菌诱导的ICS1高表达和SA积累在sard1 cbp60g双突变体中被阻断,而相对于野生型,SRAD1过表达植株中积累更多SA。这些结果表明SARD1和CBP60g是诱导SA合成的关键因子。

丁香假单胞菌通过冠菌素(coronatine)抑制植物体内SA的合成^{[ 51]}。冠菌素是一种茉莉酸类似物,被茉莉酸受体COI1 (coronatine insensitive 1)接收,并通过茉莉酸信号通路发挥功能^{[ 51]}。Zheng等^{[ 51]}研究发现冠菌素通过MYC2激活ANAC (abscisic acid-responsive NAC)类转录因子ANAC019、ANAC055和ANAC072进而抑制ICS1的表达,减少SA的合成从而降低植物的抗病能力。其他转录因子如WRKY54、WRKY70、EIN3 (ethylene insensitive 3)和CBP60a等也是ICS1表达的抑制子^{[ 52, 53, 54]}。

2 水杨酸信号接收

2.1 NPR1

植物识别病原菌后内源SA被诱导合成从而增强抗病相关基因表达。为了解析SA信号转导过程,科研人员通过多个正向遗传学筛选寻找SA不敏感突变体,发现npr1、nim1 (non-inducible immunity 1)和sai1 (salicylic acid insensitive 1)这3个突变体突变同一个基因NPR1^{[ 11, 55~ 57]}。NPR1突变使植物丧失SA诱导下游基因高表达和抗病性^{[ 55]}。Wang等^{[ 52]}研究表明,SA调控2280个基因的表达,其中2248个基因表达改变依赖于NPR1。这些数据表明NPR1是SA信号通路的关键调控因子。SA通过多种蛋白修饰影响NPR1的转录激活活性从而调控下游基因表达^{[ 58, 59]}。当植物体内SA含量较少时,NPR1形成多聚体并定位于细胞质中。NPR1第55和59位丝氨酸被磷酸化,抑制NPR1的转录激活活性。当SA积累时NPR1从多聚体还原为单体并转移到细胞核中。在细胞核中NPR1被相素化,进而促进11和15位丝氨酸磷酸化,增强NPR1转录激活活性,促进SA下游基因表达。对NPR1的泛素化修饰导致其被26S蛋白酶体降解,一方面降低NPR1含量,另一方面使新的NPR1蛋白被募集到转录位点,增强下游基因表达^{[ 59]}。

2012年Wu等^{[ 60]}通过平衡透析配体结合实验发现NPR1结合SA,Ding等^{[ 61]}通过常规的受体-配体结合实验进一步证实了该结论。Wu等^{[ 60]}研究显示NPR1通过其羧基端第521和529位半胱氨酸结合金属铜和SA;通过螯合作用去除金属将解除NPR1和SA的结合;SA结合导致NPR1羧基端反式激活结构域构象发生改变,从而使其从NPR1氨基端具有抑制功能的BTB/POZ (broad-complex, tramtrack and bric a brac/poxvirus and zinc finger)结构域中释放出来,诱导下游基因转录。而Ding等^{[ 61]}研究显示NPR1的第432位精氨酸在结合SA过程中发挥重要作用,将其突变为谷氨酰胺将大幅度降低NPR1结合SA的能力。虽然这两篇文章关注的氨基酸位点不同,但都证明NPR1羧基端在SA接收中的重要性。

2.2 NPR3和NPR4

NPR3和NPR4 (NPR3/4)是NPR1的同源蛋白,二者功能冗余,共同抑制植物对丁香假单胞菌的抗性^{[ 62]}。2012年Fu等^{[ 63]}研究指出NPR3/4是SA受体。通过常规受体-配体结合实验,他们发现NPR3/4结合SA,NPR4具有较高的SA结合能力,NPR3结合能力弱于NPR4;SA的结合促进NPR3与NPR1相互作用,抑制NPR4与NPR1互作;NPR3/4含有BTB结构域,直接与CUL3 (culin3)互作形成E3复合体促进NPR1降解;遗传学证据表明NPR3/4对植物免疫反应ETI的调控依赖于NPR1。因此他们提出假说,NPR3和NPR4分别感受植物体内不同浓度SA,通过促进NPR1降解介导植物对SA的响应。当植物体内SA浓度很低时,NPR4介导NPR1降解(正常生长状态下)。植物体内SA浓度很高时(ETI),NPR3介导NPR1降解。只有植物体内SA浓度处于中等水平时(SAR),SA足以干扰NPR4和NPR1互作,但不足以促进NPR3和NPR1互作,NPR1在植物体内积累,促进下游基因表达^{[ 64]}。

2018年Ding等^{[ 61]}进一步证实NPR3/4是SA受体。与Fu等^{[ 63]}研究结果一致,该研究同样发现NPR4与SA的结合能力高于NPR3。但该研究提出不同的NPR3/4作用模型。通过筛选snc2-1D npr1的抑制子,Ding等^{[ 61]}发现一个NPR4功能获得性突变形式npr4-4D;npr4-4D的突变位点是NPR4蛋白第419位精氨酸,该位点是NPR4结合SA的关键位点;SA结合NPR4解除其转录抑制活性,而npr4-4D不能与SA结合,持续抑制SA下游基因表达,使植物对丁香假单胞菌抗性减弱。由于npr1-1和npr4-4D突变体对SA下游基因表达和植物抗病性具有叠加效应,因此Ding等^{[ 61]}认为SA下游有两条平行的信号通路：一方面,当植物体内SA浓度很低时,NPR3和NPR4抑制SA下游基因表达,当病原菌侵染导致SA浓度升高后,NPR3/4活性被抑制,其对SA下游基因转录抑制作用被解除;另一方面,植物体内SA积累激活NPR1转录激活活性,进一步诱导SA下游抗病相关基因表达^{[ 65]}。两种不同模型的存在可能是由于NPR3/4有多个底物,而NPR1也受多种蛋白调控所致^{[ 58, 66, 67]}。

2.3 SABPs

虽然现有研究证明NPR1和NPR3/4是SA主要受体,但事实上植物中有多个SA结合蛋白(SA-binding protein, SABP)^{[ 68]}。如NPR1同源蛋白NPR2能够结合SA并互补NPR1的功能,另外两个NPR1同源蛋白BOP1 (block of cell proliferation 1)和BOP2也与SA有弱的结合能力^{[ 68, 69]}。长期以来,科研人员一直试图通过生物化学方法寻找SA受体,并陆续鉴定出多个SABPs,如过氧化氢酶(SABP1)、水杨酸甲酯酯化酶(SABP2)和叶绿体碳酸酐酶(SABP3)等^{[ 70]}。虽然这些SABPs缺乏作为SA受体的遗传学证据,但它们确实参与特定SA代谢或信号转导过程,例如SABP2以水杨酸甲酯为底物并将其转化为SA^{[ 71]}。在这些SABPs中,对SABP1同源蛋白过氧化氢酶2 (Catalase 2, CAT2)的功能解析近年获得了新进展^{[ 72]}。

烟草SABP1是最早报道的SA结合蛋白,其编码过氧化氢酶催化过氧化氢分解成水和氧气^{[ 73, 74]}。SA与SABP1结合抑制它的酶活性,导致植物体内过氧化氢积累和SA下游基因诱导表达。CAT2是拟南芥中SABP1的同源蛋白,与SABP1具有78%序列同源性。SA抑制CAT2酶活性,导致病原菌侵染后植物体内过氧化氢增加^{[ 72]}。过氧化氢增加促进色氨酸合成酶亚基1 (tryptophan synthetase subunit 1, TSB1)第308位半胱氨酸磺基化(sulfenylation),导致其活性受到抑制,减少生长素合成从而解除其对SA介导免疫反应的抑制作用。同时,SA解除CAT2对茉莉酸生物合成过程中乙酰辅酶A氧化酶(acyl-CoA oxidases)活性的促进作用,抑制茉莉酸合成,解除茉莉酸对SA介导免疫反应的负调控效应^{[ 72]}。这些结果表明CAT2特异性地调控SA信号转导的特定过程。由于NPR类SA受体与该过程的关系还未被探究,所以CAT2在SA信号转导中是否不依赖于NPRs独立发挥功能还有待新的实验证据。

3 水杨酸信号通路的转录因子

NPR类蛋白和CAT2都不具备直接结合DNA的能力,因此需要通过转录因子(transcription factor, TF)来调控SA下游基因表达。参与SA信号通路的TF包括TGA、WRKY和NIMIN(NIM1 interacting)等^{[ 75, 76]}。

3.1 TGA

由于NPR1在SA信号转导中的重要作用,科研人员通过筛选其互作蛋白以期解析SA信号通路。酵母双杂交实验结果显示NPR1与bZIP (basic leucine zipper protein)类转录因子TGA家族蛋白互作^{[ 77, 78, 79]}。TGA结合的顺式作用元件AS-1-like存在于多个SA调控基因启动子序列中^{[ 78]}。拟南芥TGA家族由10个蛋白(TGA1~TGA10)组成,其中TGA1~ TGA7与NPR1互作。这7个TGA分为3个亚家族,分别是TGA1和TGA4(I),TGA2、TGA5和TGA6(II)以及TGA3和TGA7(III)。其中II类TGA负调控SA下游基因基础表达和介导病原菌侵染时SA对下游基因的诱导表达,在植物响应SA信号中发挥关键作用^{[ 80]}。II类TGA不仅与NPR1互作,也与另外两个SA受体NPR3/4互作^{[ 61]}。正常生长状态下tga2 tga5 tga6三突变体中SA下游基因PR1 (pathogenesis-related 1)的表达量高于野生型,但该三突变体完全丧失SA诱导PR1表达的能力,植物不能产生SAR^{[ 80]}。Ding等^{[ 61]}研究发现II类TGA转录因子对SA信号响应由NPR1和NPR3/4共同调控。在正常生长状态下NPR3/4和II类TGA互作抑制SA下游基因转录,而在SA积累时这种抑制作用解除。同时NPR1与组蛋白乙酰转移酶HACs (histone acetyltransferases)形成的协同激活因子复合物与II类TGA结合,通过组蛋白乙酰化介导的表观遗传重编程激活下游基因转录^{[ 61, 81]}。由此可见,II类TGA对SA信号的调控取决于和其互作的SA受体蛋白。

3.2 NIMIN

通过酵母双杂交还发现另一类受SA诱导表达的NPR1互作蛋白——NIMIN蛋白家族^{[ 82]}。拟南芥中该家族由3个成员(NIMIN1~NIMIN3)组成,其中NIMIN1和NIMIN2与NPR1的羧基端互作,而NIMIN3与NPR1的羟基端互作^{[ 82]}。NIMIN1过表达植株对病原菌敏感性增强,降低SA诱导的免疫反应和下游PR基因表达,而nimin1敲除突变体在SA诱导后PR表达水平高于野生型^{[ 83]}。酵母三杂交显示NIMIN与NPR1和II类TGA蛋白形成复合体抑制SA下游基因表达。NIMIN蛋白含有EAR (ERF- associated amphiphilic repression)结构域,可能通过结合TPL(topless)介导对基因表达的抑制作用^{[ 83]}。在正常生长的植物中,NIMIN3发挥主要作用。病原菌侵染或SA处理后,NIMIN1和NIMIN2被快速诱导,NIMIN1防止植物免疫反应被过早激活,而NIMIN2不参与抑制基因表达,在早期SA反应中作用未知^{[ 84]}。

3.3 WRKY

Maleck等^{[ 85]}通过转录组分析发现并非所有SA调控基因启动子中都含有TGA结合位点。相反,特异性结合WRKY转录因子的W-box顺式元件在这些基因启动子中更为常见,表明WRKY家族转录因子可能在SA信号通路中具有重要作用。拟南芥WRKY家族有74个成员,其中43个参与病原菌胁迫反应或响应SA信号^{[ 86, 87, 88]}。Wang等^{[ 52]}通过转录组数据分析得到8个NPR1直接转录调控的WRKY蛋白,其中WRKY18的突变使植物对病原菌敏感性增加并减弱SA诱导的获得性抗性,而wrky58突变增强对病原菌的抗性和对SA诱导免疫的响应。WKRY18与NPR1和CDK8互作使RNA聚合酶II结合到NPR1和SA下游基因的启动子区,调控NPR1和约20% SA响应基因的表达^{[ 52, 89]}。WRKY不仅识别启动子区的W-box,WRKY50结合在PR1启动子位于TGA转录因子结合位点附近的非W-box位点,通过与II类TGA相互作用协同促进PR1表达^{[ 90]}。

综上所述,植物SA信号转导是一个复杂网路,通过多个转录因子促进或抑制SA下游基因表达,将植物对SA的响应控制在合理范围内,防止其过度激活导致植物生长受阻^{[ 91]}。

4 结语与展望

目前,人们对SA生物合成和信号转导的认知已经有了长足进步。SA生物合成涉及两个代谢途径：ICS途径和PAL途径。迄今为止ICS途径已基本被解析,PAL途径也被进一步完善。SA信号转导过程中植物主要通过NPR1和其同源蛋白NPR3/4接收SA信号,进而调控TGA、NIMIN和WRKY等多种转录因子改变下游基因的表达模式。但在SA生物合成和信号转导领域仍有一些问题有待进行深入探究：(1)在不同物种中两条SA生物合成途径对合成SA的贡献尚不明确;(2)虽然已发现到一些SA合成调控因子,但从生长发育或/和环境信号到SA生物合成还存在许多未知过程;(3) NPR1和NPR3/4之间是否以及何时存在上下游关系仍需进一步验证;(4)虽然已鉴定出一些SA下游作用分子和转录因子,但已有知识还无法形成一个完善的体系,对SA信号转导的理解还有待加强。进一步探索SA生物合成、信号转导及其功能研究将加深人们对植物免疫系统分子机制的理解,并开辟新的研究领域,如SA对生长发育的调控机理以及对生长免疫平衡的影响等。更重要的是,这些研究成果还将为作物标记辅助选择和分子设计育种提供新目标,从而促进现代农业的可持续性发展。

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