宋松泉^1,4, 刘军², 徐恒恒², 刘旭^,3, 黄荟⁴1 中国科学院植物研究所,北京100093
2 广东省农业科学院农业生物基因研究中心,广州 510640
3 中国农业科学院作物科学研究所,北京100081
4 怀化学院民族药用植物资源研究与利用湖南省重点实验室/生物与食品工程学院,湖南怀化418008

ABA Metabolism and Signaling and Their Molecular Mechanism Regulating Seed Dormancy and Germination

SONG SongQuan^1,4, LIU Jun², XU HengHeng², LIU Xu^,3, HUANG Hui⁴1 Institute of Botany, Chinese Academy of Sciences, Beijing 100093
2 Agro-Biological Gene Research Center, Guangdong Academy of Agricultural Sciences, Guangzhou 510640
3 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081
4 Key Laboratory of Research and Utilization of Ethnomedicinal Plant Resources of Hunan Province, Huaihua University/College of Biological and Food Engineering, Huaihua 418008, Hunan

通讯作者: 刘旭,E-mail：liuxu01@caas.cn

责任编辑: 李莉
收稿日期:2019-05-21接受日期:2019-09-30网络出版日期:2020-03-01

基金资助:

国家科技支撑计划.2012BAC01B05 国家自然科学基金.31871716 国家自然科学基金.31871716

Received:2019-05-21Accepted:2019-09-30Online:2020-03-01
作者简介 About authors
宋松泉,E-mail：sqsong@ibcas.ac.cn。







摘要
种子休眠是许多植物在长期系统发育进程中获得的一种适应环境变化的特性,是调控种子萌发和幼苗形成的最适时空分布的一种有效方式,也是物种成功繁衍与传播的一种选择性策略。种子休眠与萌发的激素调控可能是一种高度保守的机制,其中脱落酸（ABA）在种子休眠解除与萌发中起关键作用,赤霉素（GA）在休眠被解除后促进种子萌发。ABA在种子休眠与萌发中的作用主要受ABA代谢（生物合成和分解代谢）和信号传递途径的调控。为此,本文在综述ABA代谢和信号传递研究进展的基础上,阐述了ABA在种子发育、休眠与萌发中的作用,以及种子休眠特异性基因DOG1（萌发延迟1）与ABA信号组分的关系。研究表明,C40环氧类胡萝卜素是ABA生物合成的前体,玉米黄质环氧化酶和9-顺式-环氧类胡萝卜素二加氧酶是ABA生物合成的主要调节酶;ABA的分解代谢包括羟基化作用和与葡萄糖结合,CYP707A家族催化ABA C-8'位置上的羟基化作用,这是ABA分解代谢的重要步骤。在核心ABA信号传递途径中,ABA与PYR/PYL/RCAR受体结合并触发受体发生构象变化,从而允许受体-ABA复合物与2C类蛋白磷酸酶（PP2C）结合并抑制其活性,导致激酶如蔗糖非发酵-1相关的蛋白激酶2（SnRK2）的去抑制和活化。然后,这些激酶磷酸化和活化转录因子（transcription factors,TF）,TF与靶启动子结合和诱导下游的ABA反应基因表达。ABA在种子成熟中后期积累,合子组织中合成的ABA诱导初生休眠和促进种子成熟;在发育中积累和在干种子中存留的ABA含量在种子吸胀初期下降。ABA是种子休眠诱导和维持的正调控因子,是萌发的负调控因子。DOG1在种子成熟过程中表达和发挥作用,其表达受可变剪接和可变多腺苷酸化调控。反义DOG1是种子休眠的一种抑制因子,通过干扰转录和转录延伸负调控DOG1的表达和种子休眠。种子的休眠与萌发除了被核心ABA信号途径调控外,也被DOG1-AHG1（ABA过敏感萌发1）/AHG3途径调控。DOG1能与AHG1/AHG3结合,通过结合ABA信号传递的负调控因子和增加对ABA的敏感性而引起种子休眠。最后,提出了该领域需要进一步研究的科学问题,包括ABA代谢中ABA 8'-羟化酶、ABA葡糖基转移酶和β-葡糖苷酶及其基因怎样响应发育和环境的变化以维持正常的ABA水平。ABA的重要调控因子例如Ca ²⁺或者活性氧对核心ABA信号传递途径的影响,核心ABA信号传递途径与DOG1-AHG1/AHG3途径的下游重叠组分PP2C在整合生理条件或者环境信号时优先响应哪一条途径、这两条途径怎样被协调、以及PP2C有哪些新的靶组分。本文将为深入研究ABA调控种子休眠与萌发的分子机理提供参考。
关键词： 脱落酸;休眠;休眠基因DOG1;萌发;代谢;信号传递

Abstract
Seed dormancy is an adaptive characteristic to environmental changes acquired by many plants during long-term phylogenetic development, and is an effective way regulating the optimal spatiotemporal distribution of seed germination and seedling formation, and is also a selective strategy for the successful reproduction and propagation in species. Phytohormonal regulation of seed dormancy and germination may be a highly conserved mechanism, of which abscisic acid (ABA) plays a master role in dormancy release and germination, and gibberellin (GA) functions as stimulating seed germination after dormancy is released. The role of ABA in seed dormancy and germination is mainly regulated by its metabolism (biosynthesis and catabolism) and signaling pathways. Therefore, in this paper, we mainly summarize the research progresses of ABA metabolism and signaling, the effects of ABA on seed development, dormancy and germination as well as the relationships between DOG1 (DELAY OF GERMINATION1, a specific gene involved in seed dormancy) and ABA signaling components. The researches showed that C40 epoxycarotenoid is a precursor, and zeaxanthin epoxidase and 9-cis-epoxycarotenoid dioxygenase are the principal regulatory enzymes in ABA biosynthesis. The ABA catabolism includes hydroxylation and conjugation with glucose. The hydroxylation of ABA at C-8' position is catalyzed by the CYP707A, which is an important step for ABA catabolism. In the core ABA signaling pathway, ABA binds to PYR/PYL/RCAR receptors and triggers a conformational change that allows receptor-ABA complex to bind to and inhibit type 2C protein phosphatase (PP2C) activity, which results in de-repression and activation of kinases such as sucrose non-fermenting1-related protein kinase 2 (SnRK2). These kinases then phosphorylate and activate transcription factors (TF), which bind to the target promoters and induce the expression of ABA response gene downstream. ABA accumulates in seeds during mid- and late-maturation stages, and ABA synthesized in zygotic tissues induces primary dormancy and promotes seed maturation. ABA content accumulated during development and preserved in dry seeds declines at the early stage of seed imbibition. ABA is a positive regulator of seed dormancy induction and maintenance, and is a negative regulator of seed germination. DOG1 expresses and functions during seed maturation, and its expression is regulated by alternative splicing and alternative polyadenylation. Antisense DOG1 is a repressor of seed dormancy, which negatively regulates DOG1 expression and seed dormancy by causing transcriptional interference and affecting transcription extension. Seed dormancy and germination are regulated not only by core ABA signaling pathway, but also by DOG1-AHG1 (ABA HYPERSENSITIVE GERMINATION1)/AHG3 pathway. DOG1 can bind to AHG1/AHG3 and cause seed dormancy by sequestrating those negative regulators of ABA signaling and increasing ABA sensitivity in seeds. Finally, we propose some scientific issues required for investigation further in the future. How do ABA 8'-hydroxylase, ABA glucosyltransferase and β-glucosidase and their genes respond to developmental and environmental changes to maintain the normal ABA levels in ABA catabolism? How do the important regulators in ABA physiology such as Ca ²⁺ or reactive oxygen species influence the core ABA signaling pathway? Which pathway is preferentially responded by PP2C, a downstream overlapping component of core ABA signaling pathway and DOG1-AHG1/AHG3 pathway, when it integrates physiological conditions or environmental signals, and how are these two pathways coordinated, and what new target components does PP2C have? This paper will provide a basis to further investigate the molecular mechanism regulating seed dormancy and germination by ABA.
Keywords：abscisic acid;dormancy;dormancy gene DOG1;germination;metabolism;signaling


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本文引用格式
宋松泉, 刘军, 徐恒恒, 刘旭, 黄荟. 脱落酸代谢与信号传递及其调控种子休眠与萌发的分子机制[J]. 中国农业科学, 2020, 53(5): 857-873 doi:10.3864/j.issn.0578-1752.2020.05.001
SONG SongQuan, LIU Jun, XU HengHeng, LIU Xu, HUANG Hui. ABA Metabolism and Signaling and Their Molecular Mechanism Regulating Seed Dormancy and Germination[J]. Scientia Acricultura Sinica, 2020, 53(5): 857-873 doi:10.3864/j.issn.0578-1752.2020.05.001


植物通过长期不断地进化来调控种子的休眠与萌发,以确保其在不同的自然环境条件下萌发和形成幼苗。休眠（dormancy）是指在合适的条件下种子暂时不能完成萌发^[1]。在许多植物中,特别是一年生的种子植物,种子休眠对植物的生存与传播起关键作用,因为它只能使种子在环境条件合适时萌发^[2,3,4]。深休眠阻止种子迅速整齐地萌发,给引种驯化带来困难,严重影响植物的生长发育与产量;相反,在许多禾谷类作物中,例如水稻（Oryza sativa）、小麦（Triticum aestivum）和玉米（Zea mays）,由于长期的育种选择,种子的休眠特性被选择性地丧失,从而导致收获前成熟种子在适温和潮湿的条件下在母体植株上迅速发芽,造成农业生产中产量和质量的巨大损失^[5,6]。GUBLER等^[6]提出,目前,种子收获前发芽（pre-harvest sprouting）是禾谷类作物种子和粮食生产中的最大危害之一。

NONOGAKI^[7]提出,种子休眠与萌发的激素调控可能是种子植物中的一种高度保守的机制。在许多物种中,脱落酸（abscisic acid,ABA）是种子休眠诱导和维持的正调控因子,种子萌发的负调控因子;赤霉素（gibberellin,GA）和乙烯具有促进种子萌发和拮抗ABA的作用^[3,8-9]。高水平的ABA和低水平的GA引起种子深休眠和出苗率降低,而低水平的ABA和高水平的GA则诱导种子提早萌发,也称为胎萌（vivipary）^[4,10-11];然而,这些激素在种子休眠与萌发中的拮抗作用机制仍不够清楚。

ABA是调控植物许多发育过程包括种子休眠、萌发和幼苗生长,以及控制许多非生物胁迫反应的关键因子^[12,13,14]。研究结果证明,ABA在许多物种的种子休眠中起重要作用：外源ABA可以延迟或抑制种子和胚的萌发;在未成熟种子中,内源ABA维持种胚处于发育而不是萌芽过程;收获前的萌发也与种子中ABA含量较低有关;由ABA生物合成突变、转基因修饰或者化学抑制所引起的ABA缺乏的种子是非休眠的;ABA生物合成的化学抑制也引起一些休眠种子的萌发;ABA生物合成基因的过表达也会抑制和延迟种子萌发;在吸胀的最初几个小时,非休眠种子中的ABA含量比休眠种子下降更多^[15,16,17]。ABA在种子休眠与萌发中的作用主要受ABA代谢（包括生物合成和分解代谢）和信号传递途径的调控^{[12-13,17-19]},环境因子对种子休眠和萌发的影响也是通过ABA和GA起作用^[20]。然而,许多证据表明GA主要是在种子休眠被解除后起促进萌发的作用,而不是参与解除种子休眠^[17,20-21]。本文主要综述ABA代谢与信号传递的研究进展,ABA在种子发育、休眠与萌发中的作用,以及种子休眠基因DOG1（DELAY OF GERMINATION1）和ABA信号组分的关系;此外,我们提出了该领域需要进一步研究的问题,试图为深入研究ABA调控种子休眠与萌发的分子机理提供新的参考。

1 ABA代谢与信号传递

1.1 ABA生物合成和分解代谢

1.1.1 ABA生物合成 ABA的生物合成和分解代谢决定细胞中的ABA水平,从而决定ABA信号的强度^[18,22]。在陆生植物中,C40环氧类胡萝卜素（epoxycarotenoid）是ABA生物合成的前体,是由质体中甲基赤藓糖醇磷酸（methylerythritol phosphate,MEP）途径合成的异戊烯二磷酸（isopentenyl diphosphate,IPP）产生的（图1）。β-类胡萝卜素生物合成基因的突变体由于缺乏叶绿素表现为多效性的ABA缺乏表型,包括幼苗致死和光漂白（photobleaching）。玉米黄质环氧化酶（zeaxanthin epoxidase,ZEP）催化全反式玉米黄质（all-trans-zeaxanthin）环氧化成为全反式紫黄质（all- trans-violaxanthin）^[22]。全反式紫黄质被转化为9-顺式紫黄质（9-cis-violaxanthin）,或者被转化为全反式新黄质（all-trans- neoxanthin）。拟南芥（Arabidopsis thaliana）ABA4催化全反式紫黄质转化成为全反式新黄质^[23]。然而,转化全反式环氧类胡萝卜素（all-trans- epoxycarotenoid）、紫黄质和新黄质形成其相应的9-顺式异构体的异构酶仍然不清楚^[13]。尽管环氧类胡萝卜素和紫黄质的9-顺式异构体可能是9-顺式-环氧类胡萝卜素二加氧酶（9-cis-epoxycarotenoid dioxygenase,NCED）的底物,但9'-顺式-新黄质（9'-cis-neoxanthin）被认为是NCED的主要底物^[23]。

图1

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图1ABA生物合成和分解代谢途径（根据DEJONGHE等^[13]修改）

ABA前体是由甲基赤藓糖醇磷酸（MEP）途径合成的。酶用红色表示。ZEP：玉米黄质环氧化酶;NSY：新黄质合酶;NCED：9-顺式-环氧类胡萝卜素双加氧酶;XD：黄氧素脱氢酶;ABAO：脱落醛氧化酶;CYP707A：ABA 8'-羟化酶;ABH1：红花菜豆酸还原酶1;ABAGT：ABA葡糖基转移酶;βG：β-葡糖苷酶。酶的抑制剂用蓝色表示。(+)-9'-AABA：(+)-9'-乙炔-ABA;AHI4：ABA 8'-羟化酶抑制剂4;(+)-8'-MABA：(+)-8'-次甲基-ABA;NDGA：去甲二氢愈创木酸;SLCCD13：类倍半萜类胡萝卜素裂解双加氧酶抑制剂13
Fig. 1ABA biosynthetic and catabolic pathways (Modified from DEJONGHE et al.^[13])

ABA precursor is synthesized from the methylerythritol phosphate (MEP) pathway. Enzymes are shown in red colour. ZEP: Zeaxanthin epoxidase; NSY: Neoxanthin synthase; NCED: 9-cis-epoxycarotenoid dioxygenase; XD: Xanthoxin dehydrogenase; ABAO: Abscisic aldehyde oxidase; CYP707A: ABA 8'-hydroxylase; ABH1: Phaseic acid reductase 1; ABAGT: ABA glucosyltransferase; βG: β-glucosidase. Enzyme inhibitors are shown in blue colour. (+)-9'-AABA: (+)-9'-acetylene-ABA; AHI4: ABA-8'-hydroxylase inhibitor 4; (+)-8'-MABA: (+)-8'-methylidyne-ABA; NDGA: Nordihydroguaiaretic acid; SLCCD13: Sesquiterpene-like carotenoid cleavage dioxygenase inhibitor 13


NCED将9-顺式-环氧类胡萝卜素氧化裂解为黄氧素（xanthoxin）是ABA生物合成的关键调控步骤^[22]。因此,内源ABA水平的变化与NCED的表达密切相关。在陆生植物中,NCED酶由多基因家族编码,不同的家族成员在植物发育过程和胁迫反应中起独特的作用^[24]。AtABA2编码一个短链脱氢酶/还原酶,该酶催化黄氧素转化为脱落醛（abscisic aldehyde）（图1）^[25,26],脱落醛经脱落醛氧化酶（abscisic aldehyde oxidase）转化为ABA^[22]。醛氧化酶的活性需要一个钼辅因子（molybdenum cofactor,Moco）。因此,Moco生物合成缺陷的突变体也表现出ABA缺乏的表型^[27]。

1.1.2 ABA合成代谢抑制剂 ABA是由类胡萝卜素合成的,类胡萝卜素生物合成抑制剂能够降低内源ABA水平。氟啶酮（fluridone）和氟草敏（norflurazon）是八氢番茄红素去饱和酶（phytoene desaturase）的抑制剂,用这些化合物处理也能降低内源ABA水平;同时由于叶绿素的光氧化引起植物发生漂白（图1）^[28]。去甲二氢愈创木酸（nordihydroguaiaretic acid,NDGA）是一种脂氧合酶的抑制剂,脂氧合酶催化多聚不饱和脂肪酸的脱氧作用（deoxygenation）,在受到渗透胁迫的大豆（Glycine max）悬浮细胞中抑制ABA的积累^[29]。研究证明,NDGA抑制脂肪的合成和植物生长^[30],因此,需要研发更专一的NCED抑制剂。Abamine及其类似物abamineSG是NCED的专一性抑制剂,在拟南芥渗透胁迫处理过程中可抑制ABA的积累和ABA诱导基因的表达,但这些化合物对植物生长没有负面影响^[31,32]。类倍半萜类胡萝卜素裂解双加氧酶（sesquiterpene-like carotenoid cleavage dioxygenase,SLCCD）抑制剂13（SLCCD13）阻止拟南芥中由渗透胁迫诱导的ABA积累和抑制ABA反应基因（图1）^[33]。

1.1.3 ABA分解代谢 ABA的分解代谢包括羟基化作用和与葡萄糖结合（图1）,其中,8'-羟基化作用是ABA分解途径中的关键步骤。ABA C-8'位置上的羟基化被CYP707A家族催化,产物8'-羟基-ABA是不稳定的,能自发地异构化成为红花菜豆酸（phaseic acid,PA）^[34,35]。CYP707A家族属于Cyt P450单加氧酶（monooxygenase）,在高等植物中被多基因家族编码^[36],家族中的每一个成员在不同的生理或者发育过程中起作用^[37,38]。PA是一种弱的ABA作用类似物^[39,40],被PA还原酶转化成为生物学活性丧失的二氢红花菜豆酸（dihydrophaseic acid,DPA）。

ABA的羧基（C-1）和羟基及其氧化分解产物是与葡萄糖结合的潜在靶点^[18]。ABA葡糖酯（ABA glucosyl ester,ABA-GE）是一种最常见的结合物,被认为是ABA的一种储存或者远距离运输形式^[18,22]。葡糖基转移酶（glucosyltransferase）催化ABA羧基的葡糖基化。ABA-GE被β-葡糖苷酶水解后释放ABA,从而调节细胞内局部的ABA浓度^[22]。

1.1.4 ABA分解代谢抑制剂 影响ABA分解代谢的化合物包括唑类抑制剂（azole-type inhibitor）和ABA类似物（ABA analog）,它们作用于Cyt P450单加氧酶CYP707A（图1）。唑类抑制剂烯效唑（uniconazole）和烯效醇（diniconazole）抑制CYP707A的活性,增加内源ABA水平^[41,42]。烯效唑和烯效醇不但抑制CYP707A,而且抑制其他的Cyt P450单加氧酶,对植物的生长发育有负面影响^[43]。Abscinazole-E3M选择性地抑制CYP707A,从而增加内源ABA水平,提高水分胁迫耐性,但对植物的生长影响较小（图1）^[44]。与唑类抑制剂相反,ABA类似物能够作为专一的ABA分解代谢抑制剂起作用。ABA分解代谢的第一步是CYP707A酶对环上8'和9'甲基的羟基化^[18]。ABA类似物(+)-8'-次甲基-ABA （(+)-8'-methylidyne-ABA）和(+)-9'-乙炔-ABA（(+)-9'-acetylene-ABA）不可逆地抑制CYP707A活性（图1）,但这些化合物保留了ABA类似物的活性^[45]。ABA 8'-羟化酶抑制剂4（ABA 8'-hydroxylase inhibitor 4,AHI4）不表现出ABA活性（例如停滞生长和抑制种子萌发）,但强烈地抑制CYP707A活性^[46]。

1.2 ABA信号传递

核心ABA信号传递组分主要由PYR/PYL/RCAR（pyrabactin resistance 1/pyrabactin resistance 1-like/ regulatory components of ABA receptor）蛋白、A组2C类蛋白磷酸酶（group A type 2C protein phosphatase,PP2C）、亚类Ⅲ蔗糖非发酵-1-相关蛋白激酶2（subclass Ⅲ sucrose nonfermenting-1-related protein kinase2,SnRK2）和ABF（ABA-responsive element (ABRE)- binding factor）/AREB（ABRE-binding protein）转录因子组成（图2和图3）^{[12-13,19,47-49]}。ABA通过与PYR/PYL/ RCAR蛋白中高度保守的氨基酸进行直接的和水介导的接触,被结合进疏水的配体结合的ABA受体（结合）口袋中。结合口袋（binding pocket）含有类似于一只折叠的手的7个β折叠,以及1个大的和2个较小的α螺旋^[50,51]。ABA的结合促进了包含β3和β4之间的一个门环（gate-loop）的构象变化,这种构象变化关闭结合口袋,形成与ABA的接触。除了PYR/PYL/RCAR12和PYR/PYL/RCAR13分别含有序列-SDLPA-和-SGFPA-外,在所有的PYR/PYL/ RCAR蛋白的门环中都含有序列-SGLPA-^[13]。β5和β6含有不变的序列-HRL-,它们之间的第二个“门闩（latch）”环也发生构象改变;这种改变使受体-配体复合物对接和抑制PP2C。PP2C含有一个高度保守的、定位于A组专一识别环中的色氨酸残基,该残基能插入到由门环关闭所产生的小口袋中,并与ABA的酮基产生水介导的接触。这个水分子位于ABA、门的脯氨酸（-SGLPA-）、门闩的精氨酸（-HRL-）和PP2C的色氨酸锁之间的H-键网络中心（图2）^[12,50,52]。

图2

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图2ABA诱导的受体构象变化（引自CUTLER等^[12]）

在ABA缺乏时,PYR/PYL（pyrabactin resistance 1/pyrabactin resistance 1-like）蛋白具有一个开放的门和门闩环的构型（分别为红色和绿色）,它们位于ABA结合口袋的侧面。ABA的结合诱导门和门闩的关闭,依次产生相互作用的表面,使2C类蛋白磷酸酶（PP2C）对接到结合ABA的受体上。门中的一个保守的脯氨酸（对应于PYR1中的脯氨酸88残基,用蓝色表示）在对接位点与PP2C形成直接的接触,这解释了用PYR1^P88观察到的PP2C结合的缺陷
Fig. 2ABA-induced changes in receptor conformation (From CUTLER et al.^[12])

In the absence of ABA, PYR/PYL (pyrabactin resistance 1/pyrabactin resistance 1-like) proteins possess an open conformation of the gate and latch loops (red and green, respectively) that flank the ABA-binding pocket. Binding of ABA induces closure of the gate and latch, which in turn creates the interaction surface that recruits docking of type 2C protein phosphatases (PP2C) onto the ABA-bound receptors. A conserved proline in the gate (which corresponds to the residue to proline 88 in PYR1 and is shown in blue) forms a direct contact with the PP2C at the docking site, which explains the PP2C-binding defect observed with PYR1^P88

图3

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图3ABA信号传递途径和DOG1调控种子休眠的新模型（根据NONOGAKI^[19]修改）

在ABA感受和信号传递途径中（左）,ABA受体（PYR/PYL/RCAR）与ABA不敏感1（ABI1）亚家族2C类蛋白磷酸酶（PP2C）包括ABI1、ABI2、ABA过敏感1（HAB1）和HAB2结合,并使PP2C失活,从而导致激酶例如蔗糖非发酵-1相关的蛋白激酶2（SnRK2）的去抑制和活化。这些激酶然后磷酸化和活化转录因子（TF）,TF与靶启动子（Pro）结合,诱导下游的ABA反应基因。对于种子休眠的调节（右）,DOG1与ABA过敏感萌发1（AHG1）和AHG3结合,PP2C主要在种子中起作用。DOG1被认为是通过束缚这些ABA信号传递的负调控因子和增加种子对ABA的敏感性而引起种子休眠
Fig. 3ABA signalling pathway and emerging model of seed dormancy regulated by DOG1 (Modified from NONOGAKI ^[19])

In the ABA perception and signaling pathway (left), ABA receptors (PYR/PYL/RCARs) bind to and inactivate the ABA INSENSITIVE1 (ABI1) subfamily protein phosphatases 2C (PP2Cs), including ABI1, ABI2, HYPERSENSITIVE TO ABA1 (HAB1) and HAB2, which results in de-repression and activation of kinases, such as sucrose nonfermenting1-related protein kinase 2 (SnRK2). These kinases then phosphorylate and activate transcription factors (TF), which bind to the target promoters (Pro), to induce ABA-responsive genes downstream. For seed dormancy regulation (right), DOG1 binds to ABA HYPERSENSITIVE GERMINATION1 (AHG1) and AHG3, PP2Cs primarily functioning in seeds. The DOG1 is thought to cause seed dormancy by sequestrating these negative regulators of ABA signaling and increasing ABA sensitivity in seeds


PYR/PYL/RCAR受体能够间接地控制亚类Ⅲ SnRK2的活性,SnRK2在对ABA的反应中磷酸化许多胁迫活化的靶点^[53,54]。在对发育信息或者环境胁迫的反应中,当ABA在细胞中积累时,ABA与PYR/PYL/RCAR受体结合,以及触发受体的构象变化,从而使受体-ABA复合物与PP2C结合并抑制其活性（图2和图3）^[12]。因而,亚类Ⅲ SnRK2被释放,SnRK2磷酸化和控制下游因子的活性以激活生理反应。SnRK2的靶点主要有2种类型,包括膜通道蛋白和转录因子。膜通道蛋白包括慢阴离子通道1（slow anion channel 1,SLAC1）、拟南芥钾通道1（potassium channel in Arabidopsis thaliana 1,KAT1）和NADPH氧化酶呼吸爆发氧化酶同源物F（NADPH oxidase respiratory burst oxidase homolog F,RBOHF）,它们是质膜蛋白以及能被SnRK2磷酸化^[12,22]。转录因子包括含有碱性亮氨酸拉链（basic leucine zipper,bZIP）结构域的转录因子,例如ABF、AREB和ABI5,它们能够在ABA诱导的基因启动子中与ABRE结合（图3）^[12,22]。另外,ABA诱导的基因还可以调控与相容性溶质生物合成、晚期胚胎发生丰富（late embryogenesis abundant,LEA）蛋白和热休克蛋白（heat shock protein）有关基因的表达,从而增加脱水耐性。ABI5与植物专一的VP1/ABI3转录因子结合,可以控制种子休眠^[55]。

被子植物的PYR/PYL/RCAR受体分为3个亚家族（Ⅰ、Ⅱ和Ⅲ）^[47,56]。亚家族Ⅰ和Ⅱ存在于除苔藓植物外的所有陆生植物中（苔藓植物似乎仅仅含有亚家族Ⅰ）;亚家族Ⅲ几乎仅存在于被子植物中^[56,57]。大多数亚家族Ⅲ受体形成同源二聚体,它们对ABA的敏感性比亚家族Ⅰ/Ⅱ受体低;而亚家族Ⅰ/Ⅱ为单体,对ABA具有较高的亲和性。在拟南芥原生质体中表达不同PYR/PYL/RCAR受体的试验表明,亚家族Ⅰ和Ⅱ受体介导非胁迫植物中对低水平的ABA起反应,而亚家族Ⅲ受体需要较高水平的ABA来启动信号传递;这些差异与它们具有不同内在的ABA亲和力相一致^[58]。然而,遗传分析表明拟南芥受体亚家族之间存在广泛的冗余,模拟ABA缺失突变体的表型需要去除所有3个亚家族受体^[49]。

在拟南芥中,有9个A组PP2C参与ABA的信号传递。一些拟南芥PP2C突变体表现出ABA过敏感表型,PP2C的活性被ABA受体选择性地抑制^[58,59]。在A组PP2C中,仅仅ABA过敏感萌发1（ABA hypersensitive germination 1,AHG1）缺乏保守的色氨酸锁残基,在受体-配体-PP2C复合物中这个残基是与ABA的环已烯酮上的氧形成水介导的氢键必需的^[58,59]。尽管AHG1对ABA受体介导的抑制是抗性的,但在种子萌发分析中ahg1突变体的种子对ABA过敏感,表明AHG1参与了ABA反应^[59,60]。

拟南芥中有9个SnRK2,其中3个亚类Ⅲ SnRK2（SRK2D/SnRK2.2、SRK2E/SnRK2.6/OST1和SRK2I/SnRK2.3）被ABA诱导。SnRK2三重突变体在种子萌发、植物生长、气孔关闭和ABA反应基因表达中几乎表现出完全ABA不敏感^[53,61-63]。因此,ABA的作用是由亚类Ⅲ SnRK2介导的底物（靶）蛋白的磷酸化所触发,尽管只有少量SnRK2的直接底物被鉴定。野生型或者用ABA处理的SnRK2三重突变体的磷酸化蛋白质组分析鉴定了新的SnRK2候选底物,包括与开花、核苷酸结合、转录调控、信号传递和叶绿体形成有关的蛋白质^[64,65,66]。此外,SnRK2似乎是一个信号中枢,能够被多种途径调控;例如,拟南芥OST1（SnRK2.6）以不依赖于ABA的方式被冷胁迫活化,调节冷诱导的基因表达^[67]。另外,被调控的ABA受体和PP2C的蛋白水解、以及受体与不同靶点之间的相互作用已经被报道,表明核心ABA途径的复杂调控作用^{[68,69,70,71]}。

2 ABA在种子发育、休眠解除和萌发中的作用

2.1 种子发育

ABA在种子发育过程中逐渐积累,在种子成熟中后期达到峰值。拟南芥种子成熟中期积累的ABA主要由母体组织（包括种皮）合成^[72];在拟南芥和皱叶烟草（Nicotiana plumbaginifolia）中,这种母体来源的ABA有助于胚的发育^[73]。拟南芥种子初生休眠的诱导和成熟需要合子组织中合成的ABA,这些ABA在成熟后期积累^[74]。拟南芥中有5个NCED,其中NCED6和NCED9在种子发育过程中起促进ABA的积累和诱导休眠的作用^[75,76];CYP707A1是种子成熟中期ABA失活的主要同源异构体,而CYP707A2则是成熟后期ABA失活的主要类型^[37]。成熟中期（未成熟）的种子分解代谢活性较高,使母体来源的ABA失活。在未成熟和成熟种子中,cyp707a1突变体比cyp707a2突变体积累更多的ABA,但cyp707a2突变体由于种子吸胀后ABA的缓慢下降表现出更强的休眠特性^[37]。在大麦（Hordeum vulgare）种子发育过程中和吸胀后,ABA的代谢和敏感性受环境条件调控^[16]。

拟南芥哥伦比亚生态型（Col）干种子中大量储存的mRNA的启动子富含ABRE,同时也含有丰富的ABI3和ABI4等目标顺式元件^[77],这些结果表明,在种子成熟后期ABA可能会影响含有这些目标元件的储存mRNA的组成。

2.2 种子后熟

后熟（after-ripening）是指成熟种子离开母体植株后,需要经过一系列的生理生化变化才能具备萌发能力的一个生理过程。后熟能够解除许多物种的种子休眠,完成后熟所需要的时间与种子的休眠类型和休眠程度密切相关^[78]。许多试验表明,在禾谷类植物中,后熟不改变干燥种子中ABA的含量或者ABA代谢;而当休眠种子和后熟种子经历吸胀作用时,ABA含量（或者代谢）发生显著的变化。刚收获的成熟小麦和大麦干燥种子仍然是休眠的,其ABA含量与经过3—4个月后熟已解除休眠的种子类似;尽管干燥的休眠种胚和后熟种胚中的ABA含量没有差异,但后熟种子的萌发时间要比休眠种子短很多^[79,80,81]。在吸胀初期的几个小时,休眠种子和后熟种子中的ABA含量迅速下降;但在随后的几个小时后熟种子中的ABA含量较低,而在休眠种子中保持稳定,在一些品种中甚至增加^{[16, 79-80]}。

在大麦种子中,后熟对ABA生物合成基因表达的影响还不明确,但它显著地增加ABA分解代谢基因HvABA8'OH1的表达。在吸胀的最初几个小时内,在后熟种子和休眠种子中HvABA8'OH1的表达都增加,随后降低;然而,在后熟种胚中的表达比休眠种胚中强很多^[16,79]。HvABA8'OH2的表达水平在后熟种子和休眠种子的吸胀过程中都非常低;因此,与休眠种胚比较,在吸胀的最初几个小时,后熟大麦种胚中ABA含量的显著下降是由于通过增加HvABA8'OH1的表达,从而促进ABA的分解代谢造成的。在吸胀的后熟种子和休眠种子的 HvABA8'OH1原位定位显示,在后熟种胚中,仅仅在胚根鞘（包围禾谷类种子胚初生根的组织）中检测到HvABA8'OH1的表达,而在休眠种胚中没有被检测到^[16]。目前已经发现,胚根鞘在种子休眠的调控中起重要作用,调控的机制可能是基于高水平的ABA阻止胚根鞘的弱化和生长,导致胚根伸长受阻,而增加的ABA分解代谢则可解除这一限制^[16,82]。

2.3 种子萌发

种子萌发表现出3个阶段的水分吸收。第一阶段是由于干种子的被动吸水,其次是少量的水分吸收（第二阶段）,进一步的水分吸收是与萌发完成和随后的幼苗生长有关（第三阶段）^[1]。一系列证据表明尽管在萌发早期阶段的ABA含量也起一些作用,但萌发完成前（第二阶段后期）种子中的ABA含量是种子萌发的决定因素^[17]。

干种子中存留的ABA会在种子吸胀后下降,这在休眠（佛得角群岛生态型（Cape Verde Islands ecotype,Cvi））和非休眠（Col）的拟南芥种子中都有发生,主要取决于CYP707A2的活性^{[16,37,83-84]}。在Col和Cvi种子中,CYP707A2在种子吸胀开始后的2—3 h被诱导^[84],导致ABA的迅速下降,结果表明与CYP707A2蛋白的从头合成（de novo synthesis）有关^[83]。这种早期诱导能被一些因素例如硝酸盐^[85]、一氧化氮（nitric oxide,NO）^[83]和后熟^[16]调控。就整个萌发过程而言,虽然休眠的Cvi种子和热抑制的Col种子在吸胀早期表现出ABA含量下降,但此后会有所增加^[86]。

此外,在一些物种中胚的周围组织（胚乳或者外胚乳和种皮）是胚根伸出的物理障碍^[78]。在大麦种子中,这些组织也会引起缺氧,从而抑制ABA的分解代谢和增加对ABA的敏感性^[87]。一些物种例如莴苣（Lactuca sativa）、番茄（Lycopersicum esculentum）和拟南芥的胚乳都是由活细胞组成的,它们的弱化对于萌发的完成是必需的。尽管在番茄种子萌发过程中ABA不抑制珠孔端胚乳（micropylar endosperm）内-β-甘露聚糖酶活性的增加^[88],但阻止细胞壁降解酶的合成可能是ABA的一个重要功能^[89]。研究已经发现,ABA调控细胞壁松弛酶的表达或者活性氧（reactive oxygen species,ROS）的积累,ROS可能氧化细胞壁多糖^[78,90]。

3 ABA信号传递和DOG1与种子休眠

种子的休眠与萌发不仅与核心ABA信号途径有关,而且受DOG1-AHG1/AHG3途径的调控（图3）^[19,91],这两条途径在下游组分PP2C产生重叠^[19,48,92]。NONOGAKI^[19]认为近年来种子休眠研究的突破是在一定程度上揭示了DOG1的生化功能。下面主要介绍该领域的近期进展,包括DOG1对种子休眠的作用,DOG1表达和功能的调控,以及DOG1与ABA信号途径的关系。

3.1 DOG1对种子休眠的作用

利用拟南芥自然变异的数量性状位点（quantitative trait locus,QTL）分析已经鉴定了种子休眠的特异位点,包括DOG1^[93,94,95]。DOG1转录物在种子成熟阶段积累,授粉后14—16 d达到峰值^[95],在刚收获的种子中减少到约20%,在种子吸胀过程中消失^[96]。DOG1蛋白也在种子成熟阶段积累,但在种子接近成熟时并未降低,故刚收获的种子含有相对高水平的DOG1蛋白;即使在后熟13周,种子的休眠状态已经被解除,蛋白水平仍然保持相对较高^[96]。因此,在后熟种子中,DOG1蛋白的数量和休眠水平之间缺乏相关性。研究发现,DOG1保持种子休眠特性最重要的是其化学性质而不是数量,在后熟过程中,DOG1蛋白变化成为非功能形式,从而引起种子萌发;事实上,在种子后熟之前和后熟之后,DOG1蛋白的等电点（pI）发生了变化^[96]。dog1突变体种子缺乏休眠是由于DOG1主要在成熟阶段起作用,在成熟种子中含有的DOG1蛋白可能是其残留物^[96]。

3.2 DOG1表达和功能的调控

DOG1在种子成熟过程中表达,其表达受可变剪接（alternative splicing）^[95,97]和可变多腺苷酸化（alternative polyadenylation）的调控^[91,98]。DOG1的几个剪接变体（splicing variant）^[95]可产生5种转录物变异体（transcript variant,α、β、γ、δ、ε）和3种不同的蛋白（图4-A）^[97];其中DOG1-ε不是一个真正意义上的剪接变体,但是拟南芥发育种子中的主要形式^[97];可变剪接可以构成不同功能的蛋白,包括它们的亚细胞定位和增加的休眠潜能。这3种蛋白（α、（β、γ、ε）、δ）都能被转运到细胞核^[97],因此,可以认为DOG1是作为一种调控蛋白以同源二聚体的形式起作用^[96,97]。过表达分析结果表明,这3种同源异构体可以诱导种子休眠,且共表达时更为稳定。因此,异源二聚体的形成不能解释DOG1蛋白具有更好的稳定性。对于DOG1的稳定性,同源异构体共表达的正作用机制尚不够清楚^[91]。当酵母剪接体组分19号复合物相关蛋白1（nineteen complex- related protein 1）的拟南芥直系同源物（AtNTR1）被突变时,它引起DOG1中内含子保留和外显子跳跃（intron retention and exon skipping）的主要缺陷。这种DOG1剪接的错误能调控种子减少休眠,但这种表型不会由可变剪接本身引起,而可能是这种突变体中DOG1表达水平降低的结果^[99]。研究已经发现,转录延伸的效率（transcription elongation efficiency）对于DOG1的表达和种子休眠是一种重要的因子^[7,100-102],认为AtNTR1是在剪接位点控制着RNA聚合酶Ⅱ（PolⅡ）的作用,并可作为转录延伸的校正点（checkpoint）^[99]。

图4

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图4DOG1表达和功能的调节（引自NONOGAKI^[91]）

A：DOG1的结构。顶部：具有外显子（E1、E2、E3）和内含子（I1、I2）的DOG1基因组DNA。可变剪接区域用粉红色和橙色作标记。表明dog1突变（dog1-3、dog1-4和dog1-5中的T-DNA,以及dog1-1中的单个碱基缺失（-C））的大致位置。中部：可变的DOG1转录物（α、β、γ、δ、ε）和相应的蛋白。注意DOG1-ε不是一个真正意义上的可变剪接产物。底部：可变多腺苷酸化的短DOG1（shDOG1,与DOG1-ε相同）和长DOG1（lgDOG1,包括DOG1-α、DOG1-β、DOG1-γ和DOG1-δ）转录物。转录起始（TSS）和终止（TTS）位点被表明。反义DOG1（asDOG1）的大致位置和方向用蓝色箭头标明。B：AsDOG1功能的可能机制。相对稳定的asDOG1 RNA可能以一种序列专一的方式或者通过它的二级结构作为一种调节RNA起作用,用于RNA介导的染色质重塑（右图,反式调节）。然而,等位基因专一的asDOG1的表达已经表明asDOG1在顺式调节中起作用（左图）。转录本身的“行为”而不是转录产物（RNA）发挥asDOG1的表达对DOG1表达和休眠的负面作用。反义表达可能引起转录干扰和影响转录延伸,这对DOG1表达和种子休眠是重要的;而转录介导的染色质重塑也是可能的。AS：可变剪接;APA：可变多腺苷酸化;Dist：远端;Prox：近段;Prot：蛋白;Tran：转录物
Fig. 4Regulation of DOG1 expression and function (From NONOGAKI^[91])

A: Structures of the DOG1 gene. Top: DOG1 gDNA with exons (E1, E2, E3) and introns (I1, I2). Alternatively spliced regions are highlighted in pink and orange. Approximate positions of the dog1 mutations (T-DNAs in dog1-3, dog1-4, dog1-5 and a single-base deletion [-C] in dog1-1) are also indicated. Middle: Alternative DOG1 transcripts (α, β, γ, δ, ε) and the corresponding proteins. Note that DOG1-ε is not exactly an alternative splicing product. Bottom: Alternatively polyadenylated short DOG1 (shDOG1), which is identical to DOG1-ε and long (lgDOG1) transcripts, which comprises DOG1-α, -β, -γ and -δ. The transcriptional start (TSS) and termination (TTS) sites are indicated. Approximate position and the orientation of antisense DOG1 (asDOG1) are shown as a blue arrow. B: Possible mechanisms of asDOG1 function. Relatively stable asDOG1 RNA could function as a regulatory RNA, in a sequence-specific manner or through its secondary structure, for RNA-mediated chromatin remodeling (right panel, trans regulation). However, allele-specific asDOG1 expression has indicated that asDOG1 functions in cis (left panel). The “act” of transcription itself, rather than its product (RNA), exerts the negative effects of asDOG1 expression to DOG1 expression and dormancy. Antisense expression could cause transcriptional interference and affect transcription elongation, which is known to be important for DOG1 expression and seed dormancy while transcription-mediated chromatin remodeling is also possible. AS: Alternative splicing; APA: Alternative polyadenylation; Dist: Distal; Prox: Proximal; Prot: Protein; Tran: Transcription


产生转录变异体的另一种机制是可变多腺苷酸化,在3'端产生不同的转录物^[98,103]。现已发现可变多腺苷酸化能产生2种形式的DOG1转录物——短DOG1（shDOG1）和长DOG1（lgDOG1）^[98]。shDOG1终止于近端转录终止位点（proximal transcription termination site,pTTS）,而lgDOG1可延伸至远端转录终止位点（distal transcription termination site,dTTS）。shDOG1和lgDOG1（第2个内含子/第3个外显子）的DOG1基因组区域含有一个反义方向的启动子,它可驱动反义DOG1 RNA（antisense DOG1 RNA,asDOG1）的表达^[104]。试验已经证明,从终止子区域（terminator region）的反义RNA表达可能是一种常见的现象^[105,106],DOG1中asDOG1的表达可能通过转录控制负调控正义DOG1的表达^[91]和减少种子休眠^[104]。shDOG1与DOG1-ε相同,而lgDOG1包括DOG1-α、DOG1-β、DOG1-γ和DOG1-δ（图4-A）。在许多物种中,DOG1蛋白的C末端是缺失的或者是不保守的,因此,C末端对于DOG1的功能不是必需的。事实上,shDOG1对于补偿dog1突变和恢复种子休眠是足够的^[98]。尽管在已发表的研究结果中关于lgDOG1转录本的重要性存在一些分歧,但一致认为由2个外显子组成的shDOG1是有功能的,是种子休眠所必需的主要蛋白^[97-98,104]。

根据编码的多肽序列,DOG1基因组DNA的第3外显子区域几乎没有保守性。相反,这一区域在DNA水平上是高度保守的,它延伸到内含子2^[104]。在DNA水平,DOG1序列的保守性与相同区域蛋白序列的低进化压力是矛盾的,暗示DOG1基因组区域可能是一个调控长链非编码RNA（long non-coding RNA,lncRNA）的产生位点。事实已证明lncRNA从这个区域（和附近）反义方向表达（asDOG1）（图4-A）^[104];试验也证实其表达不是假的转录噪音,而是被一个转录活性启动子反义方向调控^[104]。AsDOG1的表达负影响shDOG1的表达,表明asDOG1是DOG1表达和种子休眠的负调控因子。（正义链）DOG1启动子（dog1-3（T-DNA））、外显子1（dog1-4（T-DNA））和外显子2（dog1-1（1 bp删去））的突变导致减少或者几乎没有种子休眠^[95,98,104]。相反,外显子3（asDOG1启动子）区域（dog1-5（T-DNA））的突变反而增加种子休眠（图4-A）^[98,104],这为asDOG1作为种子休眠负调控因子起作用提供了令人信服的证据。

反义DOG1是一种相对稳定的RNA（半衰期约为46 min）^[104],也是典型的调节RNA,因此,asDOG1可能在转录后水平依赖于RNA分子。然而,asDOG1表达分析显示,asDOG1不能反式、但能顺式起作用^[104];即asDOG1转录的产物（RNA分子本身）可能不重要,但转录本身的“行为”^[107,108]可能是DOG1抑制的原因^[104]。研究已经发现,反义表达的共转录作用而不是反义RNA分子的转录后调控引起转录干扰^[108]。转录干扰可能被不同的机制介导,包括RNA聚合酶不直接结合到启动子序列上和启动子竞争（图4-B）^{[108,109,110]}。在酵母中,反义介导的转录干扰阻断IME4的转录延伸^[108,111]。因此,asDOG1的表达可能影响DOG1的转录延伸,这是种子休眠的一个关键因子（图4-B）^[7,100-102]。由于在种子吸胀过程中DOG1和asDOG1的表达量降低,因此,asDOG1的作用可能被局限在种子成熟阶段^[104],DOG1蛋白功能的修饰而不是它的转录控制对于后熟诱导种子萌发可能是关键的^[96,112]。asDOG1在种子成熟阶段的表达水平可能决定成熟种子的休眠深度,但为了理解asDOG1在种子休眠生物学中的确切作用还有待于进一步研究。

3.3 DOG1与ABA信号传递

目前,已经报道DOG1能与AHG1/AHG3和PP2C相互作用^[48,92],而AHG1^[60]和AHG3^[113]是ABA信号传递和种子休眠的负调控因子。AHG1（ahg1-5）或AHG3（ahg3-2）的功能缺失突变将增强种子休眠,双突变体ahg1-5 ahg3-2的种子休眠特性增强^[92],在ahg1-1 ahg3-1双突变体种子中也观察到类似现象^[48]。这些结果表明,这2个AHG在种子休眠中起冗余的作用。当非休眠突变dog1-2与单一ahg突变的任何一个组合时,双突变体种子（dog1-2 ahg1-5和dog1-2 ahg3-2）是完全非休眠的^[92],这表明DOG1对AHG1和AHG3的拮抗作用。相反,三重突变体dog1-2 ahg1-5 ahg3-2种子像双突变体ahg1-5 ahg3-2种子一样仍然是深休眠的^[92],表明AHG1和AHG3对DOG1是上位性的（epistatic）。这些遗传分析表明AHG1和AHG3在DOG1的下游起促进萌发的作用,DOG1通过束缚它们维持种子休眠^[19,48]。

AHG1和AHG3的PP2C功能缺失增加种子对ABA的敏感性^[48,92]。DOG1被认为通过结合和抑制AHG1和AHG3增加对ABA的敏感性。这种机制类似于ABA感受和信号途径中通过ABA受体PYR/PYL/RCAR对ABI1亚家族PP2C的抑制（图3）^[12,47,49]。PP2C活性的抑制（ABA受体的主要作用）也在DOG1蛋白中观察到^[48],尽管在不同的实验室也观察到一些相互矛盾的结果,这可能是由于在体外试验中缺失一些因素所致^[92]。对于种子休眠,PP2C的失活可能是DOG1功能的一个重要部分。

根据PP2C抑制激酶的典型作用,与种子休眠有关的PP2C的一个显著特征是AHG1（但不是AHG3）对ABA受体的抑制作用是抗性的^[59]。在体外试验中当激酶与PP2C一起孵育时,激酶的自体磷酸化作用被PP2C抑制;而ABA受体和ABA一起加入到同一反应体系时则抑制PP2C的活性和恢复激酶的自体磷酸化。相反,在ABA和激酶存在下ABA受体和AHG1的共孵育不表现出自体磷酸化,表明AHG1仍然能够抑制激酶活性,即PP2C对ABA受体的抑制作用是抗性的^[59]。AHG1的另一特征表明即使在高水平的ABA存在下,这些种子表达的和与休眠相关的PP2C仍然能够负调控ABA信号传递。这些研究结果表明只要通过这个开放的AHG1窗口,种子萌发程序仍然能够在含有高水平ABA的发育种子中运行^[19]。因此,为了完全暂停萌发程序,必须关闭拮抗ABA抑制的AHG1途径。DOG1的主要生物学作用可能是阻止AHG1/AHG3途径以确保种子休眠^[19]。DOG1似乎是作为ABA作用的“看门狗（watchdog）”,直到它在种子后熟过程中被化学修饰和失活^[19],从而允许种子恢复萌发程序。

另一个种子特异性的ABA信号是DOG1被认为是一种血红素结合蛋白（heme-binding protein）。血红素不是DOG1与AHG1相互作用所必需的,AHG1通过DOG1 N端区域中的6个残基DSYLEW（位置13—18）介导。然而,在DOG1蛋白的His 245和His 249结合的血红素对于DOG1在种子休眠中的功能是必需的^[48]。由于血红素结合蛋白是作为O₂和NO传感器起作用^[114],但至今血红素怎样与ABA信号体系相联系仍不够清楚。研究发现,种子的氧化还原状态变化显著地影响种子的休眠与萌发^[112]。NO触发ABI5的S-亚硝酰化（S-nitrosylation）,从而引起萌发抑制因子ABI5的稳定性降低^[91,115]。血红蛋白是一种NO的清除剂以及负影响ABI5的S-亚硝酰化作用,从而稳定ABI5^[91];DOG1可能参与了这个过程^[19]。

4 展望

ABA是调控种子发育、休眠与萌发以及脱水耐性的重要激素,也显著地影响种子的产量与质量和幼苗形成^[1,2];因此,自1960年以来ABA的代谢、生理作用和信号传递就得到了广泛的研究^{[12,48,116-117]}。尽管近年来这些领域已取得了重要的突破,但仍然有一些重要的科学问题尚不清楚。例如,ABA代谢中ABA被CYP707A家族催化成为8'-羟基-ABA,然后自发地异构化成为PA;ABA葡糖基转移酶能将ABA转化成为ABA-GE,作为ABA的储存池;ABA-GE又能被β-葡糖苷酶水解成为ABA和葡萄糖（图1）。那么,这些酶及其基因怎样响应发育和环境变化以维持正常的ABA浓度是不清楚的。

图3总结了种子中ABA信号途径的新模型。PYR/PYL/RCAR蛋白与ABA结合,并抑制A组PP2C的活性;PP2C使SnRK2去磷酸化和阻止活性SnRK2的积累;SnRK2又参与bZIP转录因子的直接磷酸化;从而负调控ABA介导的转录反应^[12,19,48]。尽管这个模型能够解释核心ABA信号传递途径,但不能解释ABA生理的许多重要调控因子的作用,包括第二信息^[12]。因此,研究已知因子例如Ca²⁺或者ROS与PYR/PYL/RCAR信号机制的相互关系将是今后的任务之一。此外,DOG1和AHG1/AHG3结合,调控下游组分包括SnRK2和ABI5,组成与核心ABA信号途径并行的种子休眠与萌发调控系统;这两条途径在组分PP2C重叠^[19,48,92]。值得注意的是,在整合生理条件或者环境信号时PP2C优先响应哪一条途径,这两条途径怎样协调,以及PP2C还有哪些新的靶组分都还不够清楚。

组学（-omics）技术已经应用于种子休眠与萌发的研究^{[118,119,120]},结合种子ABA生物合成和信号组分突变体,利用相应的专一性抑制剂,构建新的种子休眠与萌发的组学研究体系,包括转录组、翻译组、蛋白质组、代谢组和环境组可能产生一些新的知识和有助于更全面地理解种子休眠的机制。

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