Research Progress on the Mechanism of GA and ABA during Seed Germination

Zuo Yue　Xu Yonghua ^*

Jilin Agricultural University, College of Traditional Chinese Medicine, National Engineering Research Center of Ginseng Breeding and Application, Changchun, 130118

* Corresponding author, xuyonghua777@yeah.net

Abstract　The dormancy and germination of seeds are determined by the balance between the embryo growth potential and the binding force imposed by the seed coat. The germination of different seeds is not synchronized, and the stimulus required to promote germination varies greatly. Before germination, the seeds need to undergo water absorption , reactivate metabolic activities and redifferentiate embryonic tissues to mobilize nutrients stored in seeds and initiate meristematic activities. The transition from dry seeds to seedlings is highly sensitive to different environmental conditions, especially light, temperature and water. This response to environmental signals is regulated by one or more hormones. Various plant hormones regulate seed germination through highly complex interactions. Among them, the role of GA and ABA in regulating seed germination is particularly critical. This article reviewed the mechanisms by which GA and ABA control seed dormancy at the molecular level, and discussed the way they interact with other hormones. Finally, the development direction of plant hormone research on seed germination is prospected.

Keywords　GA, ABA, seeds, Dormancy, Germination

植物激素是天然存在的信号分子，其在调节植物生理，发育和适应环境刺激中起关键作用，具有广泛的物理化学性质。随着现代生化技术的发展，更多的植物激素被发现和认识，目前包括(但不限于)脱落酸、生长素(AX, auxin)、油菜素类固醇(BR, brassinosteroid)、细胞分裂素(CTK, cytokinin)、赤霉素、乙烯(ethylene)、茉莉酸(JA, jasmonic acid)、水杨酸(SA, Salicylic acid)和独脚金内酯(Strigolactone)，在调节植物对广泛的生物和非生物胁迫响应的发育过程和信号传导网络中，起着举足轻重的作用。植物生命始于种子形成，是植物生命周期下一代的载体，种子萌发受到许多因素的影响，例如外界的温湿条件，内部的激素变化，对种子萌发过程中的激素研究将有利于了解植物的生长发育过程，从而指导植物的育种及栽培方法。

1 ABA在种子萌发中的作用

ABA在保持种子休眠和调节种子萌发方面起着重要的作用，它通过控制胚根的萌发，抑制细胞壁的松动和膨胀来控制种子的萌发(Gimeno-Gilles et al.,2009)。NCED(9-cis-epoxycarotenoid dioxygenase)和ABA8’OH(ABA 8′-hydroxylase)是ABA代谢中的关键酶，其中NCED主要参与ABA的合成，而ABA8’OH是ABA分解代谢中的关键酶，由CYP707A编码，NCED的过量表达会延缓种子的萌发，拟南芥(Arabidopsis)cyp707a2突变体种子中的ABA增加且发芽减少(Zheng et al.,2015)。ABA既可以在休眠种子中合成，也可以在非休眠种子中合成，但是在非休眠种子中ABA的分解代谢较强，而休眠种子更有利于ABA的合成，即休眠诱导ABA的代谢过程发生了变化(Millar et al.,2006)。ABA的信号传导主要由ABA受体蛋白PYR (pyrabactin resistence)/PYL (PYR1-Like)/RCAR (regulatory component of ABA receptor)、PP2C (phosphatase 2C)、SnRK2 (SNF1-related protein kinase 2)、转录因子ABFs (ABRE binding factors)/AREBs (ABA responsive element binding proteins)和ABA响应原件ABRE (ABA-responsive element)参与(图1) (Klingler et al., 2010)。ABA可以增强许多与休眠有关的基因的表达，例如AtPr1参与了提高种子的休眠能力，atper1-1突变体表现为抑制种子休眠，CYP707A基因的缺失可降低atper1-1种子的休眠抑制(Chen et al., 2019)。

2 GA在种子萌发中的作用

GA是一类广泛存在于高等植物中的二萜羧酸类化合物,在已发现的100多种GA中，只有小部分具有生物活性，可促进器官的扩张和发育变化,最常见的有GA1、GA3和GA4，由GA12通过GA20ox和GA3ox催化反应合成(Hedden and Thomas, 2012)。众多关于GA的生物合成缺失型突变体的研究也证明了GA对种子萌发是必不可少的。DELLA蛋白是GA反应的抑制因子，也是一类生长抑制因子，在很多植物中存在，属于GRAS (GAI, RGA和SCARECROW)转录因子家族。不同的DELLA蛋白在种子萌发中扮演着不同的作用，例如AtEXPA2是拟南芥种子萌发中特异表达的一种扩张素基因，该基因的突变体发芽延迟，DELLA突变体显示，RGL1、RGL2、RGA、GAI均参与抑制AtEXP2的表达，而RGL1在控制AtEXP2的表达中起主导作用(Yan et al., 2014)。GA利用GID1 (GA-INSENSITIVE DWARF1)-GA-DELLA复合物的形成分解DELLA蛋白缓解DELLA对GA反应的抑制，并促进植物的生长(图2)。

3 ABA/GA在种子萌发过程中的相互作用

3.1 ABA和GA在代谢上的相互作用

大量关于ABA和GA生物合成和信号突变的遗传研究表明，这两种激素在休眠和萌发中具有重要的拮抗作用，GA促进种子休眠的释放，抵消ABA的作用。ABA和GA的比值是植物种子休眠状态的主要调节器。在适宜的生长条件下，GA的生物合成相关途径被激活，促使ABA的抑制作用释放。如在棉籽中，ABA水平自后熟开始持续下降，当含量降低到较低水平后GA开始起作用，且在种子萌发期间变化明显(Wang et al., 2019)。

ABA缺陷突变体aba2-2显示GA3ox1和GA3ox2的表达高于野生型，种子发育过程中，aba2-2突变体的GA生物合成也被激活，表明ABA参与了抑制GA生物合成的过程(Seo et al., 2006; Toh et al., 2008)。aba2-2还显示高温下种子吸胀过程中SPY(SPINDLY)的表达降低，表明ABA可促进GA信号的负调控基因SPY表达(Toh et al., 2008)。ABA和GA在调节种子发育过程上还存在组织特异性和时空差异，发育中的大麦籽粒中胚乳的GA20ox3，GA3ox1和GA2ox5有相对较高的表达，而GA3ox2在胚胎中的表达较高；ABA生物合成基因NCED1和NCED2在胚乳组织中转录，而在种子成熟后期胚乳和胚中皆有ABA分解代谢基因CYP707A1的转录。NCED9对GA生物合成有负面影响，经过多效唑和GA生物合成抑制剂处理的nced9突变体的发芽率高于野生型，说明ABA的生物合成调节了种子的GA合成途径(Seo et al., 2016)。ABA和GA除了在代谢上存在相互作用外，还可通过多种转录因子和休眠基因相互影响。

3.2 ABA和GA在信号传导上的相互作用

DELLA可通过促进ABA的积累来抑制植物生长，DELLA-Q突变体的ABA含量明显低于野生型(Guo et al., 2014)。RGL2和RGL3均可抑制种皮破裂，且ABA可促进rgl2和rgl3的转录，rgl3受ABA的影响较大，RGL2在抑制种皮破裂过程中起主导作用(Piskurewicz and Lopez-Molina, 2009)。ABI3 (ABA-INSENSITIVE3)、ABI4和ABI5均是ABA信号传导中的转录因子，在休眠种子中的表达高于休眠水平较低的种子，可抑制GA的生物合成(Shu et al., 2013; Zhao et al., 2018)。RGL2和ABI5可共同作用抑制种子萌发，RGL2在胚乳中促进ABA的生物合成，然后ABA被释放到胚中，确保ABI5的表达，从而抑制种子萌发(Lee et al., 2010)。

3.3休眠基因在ABA/GA中的作用

DOG1 (DELAY OF GERMINATION1)基因的表达决定了种子的休眠深度，新收获的种子DOG1表达较高，随着后熟的进行，休眠的逐渐接触，DOG1的转录水平降低(Nakabayashi et al., 2012)。低温会促使DOG1基因的表达，DOG1促进赤霉素分解代谢基因GA2ox6的表达，影响种子中的ABA/GA值，且DELLAs和ABA的缺失会导致DOG1表达明显下降(Kendall et al., 2011)。高温可诱导SOM(SOMNUS)转录，它编码一种CCH型锌指蛋白，该蛋白可同时调控ABA和GA的代谢，导致ABA的积累和GA的减少，使ABA/GA上升并，从而抑制拟南芥种子萌发，且ABI3，ABI5和DELLAs可共同作用，通过正向调节SOM表达来推迟种子萌发(Lim et al., 2013)。MFT (MOTHER-OF-FT-AND-TFL1)是种子休眠的强促进因子，在小麦(Triticum aestivum L.)种子发育过程中，低温或启动子突变导致MFT表达上调，种子休眠增强(Chono et al., 2015)。ABA通过ABI3和ABI5调控MFT的表达，前者作为抑制因子，后者作为启动子，MFT通过直接抑制ABI5，对ABA信号通路进行负反馈调节；另一方面，GA通过DELLA蛋白下调MFT的表达，抑制ABA的合成(Xi et al., 2010)。

3.4其他转录因子在ABA/GA中的作用

光敏色素作用因子PIL5 (PHYTOCHROME-INTERACTING FACTOR3-LIKE5)又称PIF1，是调节GA和ABA信号传导一个重要转录因子。PIF1抑制活性GA生物合成基因GA3ox1和GA3ox2并间接激活GA分解代谢基因GA2ox2，同时对ABA生物合成基因具有相反的作用，激活ABA生物合成基因ABA1、NCED6和NCED9，并抑制ABA分解代谢基因CYP707A2，PIF1还促进编码DELLA蛋白GAI和RGA的基因的表达(Finkelstein et al., 2008)。许多植物的种子萌发是由日光触发的，红光可促使转录因子PIF1降解，而PIF1可促进SOM表达(Vaistij et al., 2018)。SPT (SPATULA)是一种多功能转录因子，控制萌发对冷、光的响应，能够抑制GA3ox1和GA3ox2的表达，并同时调节了五个转录因子编码基因的表达：ABI4、ABI5、RGA、RGL3和MFT，其中ABI4、RGA和MFT被SPT抑制，ABI5和RGL3被诱导(Vaistij et al., 2013)。SPT、SOM、PIF1、ABI5、DELLA和MFT可共同通过调控ABA和GA信号传导途径完成对种子萌发在红光下的响应(Vaistij et al., 2018)。

4其他激素在种子萌发中的作用

很多激素(包括油菜素, 乙烯, 生长素, 细胞分裂素和独角金内酯)都与ABA存在着相互作用，并通过这些相互作用控制着种子的萌发。外源生长素可增强ABA对种子萌发的抑制作用，生长素响应因子ARF10增强了ABA的敏感性，MIR160可下调生长素信号中三个转录因子ARF10、ARF16和ARF17的表达，ARF10和ARF16通过维持ABI3的表达，增强ABA对种子休眠的促进作用(Liu et al., 2013)。BR可拮抗ABA对种子萌发的抑制，并调节植物生殖发育来促进种子发芽，MFT在ABA和BR调节种子萌发中发挥了作用(Xi and Yu, 2010)。BIN2 (BRASSINOSTEROID INSENSITIVE 2)是BR信号的关键抑制因子，在种子萌发和发芽后生长期间正调控ABA反应，在ABA存在下，BIN2可以磷酸化和稳定ABI5，而活性BR则抑制了BIN2对ABI5的调节(Hu and Yu, 2014)。乙烯可调节胚乳帽的软化和破裂，从而抵消ABA的作用，SNL1 (SIN3-LIKE1)和SNL2是拟南芥中组蛋白脱乙酰基复合物的两个成员，通过同时调节种子中的乙烯和ABA含量来促进种子休眠，双突变体snl1 snl2种子休眠减少，同时乙烯生物合成(ACO1, ACO4)基因和ABA分解代谢基因(CYP707A1, CYP707A2)表达增加(Linkies et al., 2009; Wang et al., 2013)。CTK促进种子萌发，而ABA可通过减少CTK的生物合成来抑制这种作用，AAR (Arabidopsis response regulator)是CTK信号传导中的重要组成部分，其中A型ARR4、ARR5和ARR6可负调控ABI5的表达，使CTK抵抗ABA的抑制作用，而ABI4可直接与启动子结合，对ARR6、ARR7和ARR15的转录产生负调控作用(Wang et al., 2011; Huang et al., 2016)。另外一些比较新发现的激素也均被发现对种子的萌发具有调控作用，独脚金内酯可通过调节ABA生物合成酶来降低ABA/GA，从而缓解种子萌发的热抑制作用；KAR (Karrikin)可通过促进GA的生物合成基因GA3ox1和GA3ox2的表达来促进种子萌发(Toh et al., 2012; Meng et al., 2017)。

5展望

植物内源激素在种子萌发过程中起着多功能化学调节的作用。从分子水平上看，他们的代谢途径和信号传导途径都受到各种转录因子的调控。ABA/GA认为是影响种子休眠的一个重要组成因素，GA促进种子萌发需要破坏DELLA蛋白，然而ABA对ABI类转录因子和DELLA蛋白均有正向调控作用，DELLA蛋白反过来抑制GA的生物合成，使ABA和GA形成一个反馈环。ABA/GA受种子发育阶段和环境的控制，在这个过程中多种转录因子参与了种子休眠与萌发的调控，影响了种子的萌发进程。在种子发育早期，GA敏感性低，ABA敏感性高，随着萌发进程的加深这种情况将会改变。

另外在种子萌发的过程中，其它激素也会通过各种方式影响种子的萌发能力，其中大部分激素对种子萌发起到促进作用，而生长素却可与ABA产生协同作用，共同抑制种子萌发。目前在对其他植物激素对种子萌发影响的研究上还多有不足，虽然突变体研究能发现相关基因与表型的关系，但其分子层面具体机制还不清晰，对多种激素信号交叉的分子机制研究，有助于构建植物发育和环境适应中激素信号整合的新图谱。

种子的休眠与萌发是一个复杂的生理过程，在不同种类的植物中，其种子休眠调节机制多有不同，目前对种子休眠萌发的研究多集中在模式植物上，对其他种类植物的研究相对较少，对与种子休眠萌发相关的植物激素的作用机制有更好的理解，有助于获得具有适当种子休眠水平的新品种。

作者贡献

佐月是本论文的主要执行者，完成数据收集和论文初稿的写作；许永华是论文的构思者及负责人，指导论文写作与修改。两位作者阅读并同意最终的文本。

致谢

本研究由国家重点研发计划课题(2017YFC1702101)和吉林省重点科技研发项目(20180201006YY)共同资助。

参考文献

Chen H., Ruan J., Chu P., Fu W., Liang Z., Li Y., Tong J., Xiao L., Liu J., Li C., and Huang S., 2019, AtPER1 enhances primary seed dormancy and reduces seed germination by suppressing the ABA catabolism and GA biosynthesis in Arabidopsis seeds, Plant J., 101(2): 310-323

Chono M., Matsunaka H., Seki M., Fujita M., Kiribuchi-Otobe C., Oda S., Kojima H., and Nakamura S., 2015, Molecular and genealogical analysis of grain dormancy in Japanese wheat varieties, with specific focus on MOTHER OF FT AND TFL1 on chromosome 3A, Breeding Science, 65(1): 103-109

Finkelstein R., Reeves W., Ariizumi T., and Steber C., 2008, Molecular aspects of seed dormancy, Annual Review of Plant Biology, 59(1): 387-415

Gimeno-Gilles C., Lelièvre E., Viau L., Malik-Ghulam M., Ricoult C., Niebel A., Leduc N. and Limami A.M., 2009, ABA-Mediated inhibition of Germination is related to the inhibition of genes encoding Cell-Wall Biosynthetic and Architecture: Modifying enzymes and structural proteins in Medicago truncatula Embryo Axis, Mol. Plant, 2(1): 108-119

Guo W., Cong Y., Hussain N., Wang Y., Liu Z., Jiang L., Liang Z., and Chen K., 2014, The Remodeling of Seedling Development in Response to Long-Term Magnesium Toxicity and Regulation by ABA-DELLA Signaling in Arabidopsis, Plant Cell Physiol., 55(10): 1713-1726

Hedden P., and Thomas S.G., 2012, Gibberellin biosynthesis and its regulation, Biochem. J., 444(1): 11-25

Hu Y., and Yu D., 2014, BRASSINOSTEROID INSENSITIVE2 interacts with ABSCISIC ACID INSENSITIVE5 to mediate the antagonism of brassinosteroids to abscisic acid during seed germination in Arabidopsis, Plant Cell, 26(11): 4394-4408

Huang X., Zhang X., Gong Z., Yang S. and Shi Y., 2017, ABI4 represses the expression of type-A ARRs to inhibit seed germination in Arabidopsis, Plant J., 89(2): 354-365

Kendall S.L., Hellwege A., Marriot P., Whalley C., Graham I.A. and Penfield S., 2011, Induction of Dormancy in arabidopsis summer annuals requires Parallel Regulation of DOG1 and Hormone Metabolism by low temperature and CBF transcription factors, Plant Cell, 23(7): 2568-2580

Klingler J.P., Batelli G., and Zhu J.K., 2010, ABA receptors: the START of a new paradigm in phytohormone signalling, Journal of Experimental Botany, 61(12): 3199-3210

Lee K.P., Piskurewicz U., Turečková V., Strnad M., and Lopez-Molina L., 2010, A seed coat bedding assay shows that RGL2-dependent release of abscisic acid by the endosperm controls embryo growth in Arabidopsis dormant seeds, Proceedings of the National Academy of Sciences, 107(44): 19108-19113

Lim S., Park J., Lee N., Jeong J., Toh S., Watanabe A., Kim J., Kang H., Kim D.H., Kawakami N., and Choi G., 2013, ABA-INSENSITIVE3, ABA-INSENSITIVE5, and DELLAs interact to activate the expression of SOMNUS and other High-Temperature-Inducible genes in imbibed seeds in Arabidopsis, Plant Cell, 25(12): 4863-4878

Linkies A., Müller K., Morris K., Turečková V., Wenk M., Cadman C.S.C., Corbineau F., Strnad M., Lynn J.R., Finch-Savage W.E., and Leubner-Metzger G., 2009, Ethylene interacts with abscisic acid to regulate endosperm rupture during Germination: A comparative approach using Lepidium sativum and Arabidopsis thaliana, The Plant Cell, 21(12): 3803-3822

Liu X., Zhang H., Zhao Y., Feng Z., Li Q., Yang H.Q., Luan S., Li J., and He Z.H., 2013, Auxin controls seed dormancy through stimulation of abscisic acid signaling by inducing ARF-mediated ABI3 activation in Arabidopsis, Proceedings of the National Academy of Sciences, 110(38): 15485-15490

Meng Y., Shuai H., Luo X., Chen F., Zhou W., Yang W., and Shu K., 2017, Karrikins: Regulators involved in phytohormone signaling networks during Seed Germination and Seedling Development, Front. Plant Sci., 7: 2021

Millar A.A., Jacobsen J.V., Ross J.J., Helliwell C.A., Poole A.T., Scofield G., Reid J.B., and Gubler F., 2006, Seed dormancy and ABA metabolism in Arabidopsis and barley: The role of ABA 8'-hydroxylase, Plant J., 45(6): 942-954

Nakabayashi K., Bartsch M., Xiang Y., Miatton E. Pellengahr S., Yano R., Seo M., and Soppe W.J., 2012, The time required for Dormancy release in Arabidopsis is determined by DELAY OF GERMINATION1 Protein levels in freshly harvested seeds, Plant Cell, 24(7): 2826-2838

Piskurewicz U., and Lopez-Molina L., 2009, The GA-signaling repressor RGL3 represses testa rupture in response to changes in GA and ABA levels, Plant Signaling and Behavior, 4(1): 63-65

Seo M., Hanada A., Kuwahara A., Endo A., Okamoto M., Yamauchi Y., North H., Marion-Poll A., Sun T., Koshiba T., Kamiya Y., Yamaguchi S., and Nambara E., 2006, Regulation of hormone metabolism in Arabidopsis seeds: phytochrome regulation of abscisic acid metabolism and abscisic acid regulation of gibberellin metabolism, Plant Journal, 48(3): 354-366

Seo M., Kanno Y., Frey A., North H.M. and Marion-Poll A., 2016, Dissection of Arabidopsis NCED9 promoter regulatory regions reveals a role for ABA synthesized in embryos in the regulation of GA-dependent seed germination, Plant Science, 246: 91-97

Shu K., Zhang H., Wang S., Chen M., Wu Y., Tang S., Liu C., Feng Y., Cao X. and Xie Q., 2013, ABI4 Regulates Primary Seed Dormancy by Regulating the Biogenesis of Abscisic Acid and Gibberellins in Arabidopsis, PLoS Genetics, 9(6): e1003577

Toh S., Imamura A., Watanabe A., Nakabayashi K., Okamoto M., Jikumaru Y., Hanada A., Aso Y., Ishiyama K., Tamura N., Iuchi S., Kobayashi M., Yamaguchi S., Kamiya Y., Nambara E. and Kawakami N., 2008, High Temperature-Induced Abscisic Acid Biosynthesis and Its Role in the Inhibition of Gibberellin Action in Arabidopsis Seeds, Plant Physiology, 146(3): 1368-1385

Toh S., Kamiya Y., Kawakami N., Nambara E., McCourt P., and Tsuchiya Y., 2011, Thermoinhibition uncovers a role for strigolactones in arabidopsis seed germination, Plant and Cell Physiology, 53(1): 107-117

Vaistij F.E., Barros-Galvão T., Cole A.F., Gilday A.D., He Z., Li Y., Harvey D., Larson T.R., and Graham I.A., 2018, MOTHER-OF-FT-AND-TFL1 represses seed germination under far-red light by modulating phytohormone responses in Arabidopsis thaliana, Proceedings of the National Academy of Sciences, 115(33): 8442-8447

Vaistij F.E., Gan Y., Penfield S., Gilday A.D., Dave A., He Z., Josse E.M., Choi G., Halliday K.J., and Graham I.A., 2013, Differential control of seed primary dormancy in Arabidopsis ecotypes by the transcription factor SPATULA, Proceedings of the National Academy of Sciences, 110(26): 10866-10871

Wang L.R., Yang X.N., Gao Y.S., Zhang X.Y., Hu W., Zhou Z., and Meng Y.L., 2019, Investigating seed dormancy in cotton (Gossypium hirsutum L.): understanding the physiological changes in embryo during after-ripening and germination, Plant Biology, 21(5): 911-919

Wang Y., Li L., Ye T., Zhao S., Liu Z., Feng Y.Q. and Wu Y., 2011, Cytokinin antagonizes ABA suppression to seed germination of Arabidopsis by downregulating ABI5 expression, The Plant Journal, 68(2): 249-261

Wang Z., Cao H., Sun Y., Li X., Chen F., Carles A., Li Y., Ding M., Zhang C., Deng X., Soppe W.J.J., and Liu Y.X., 2013, Arabidopsis paired Amphipathic Helix Proteins SNL1 and SNL2 redundantly regulate primary seed dormancy via Abscisic Acid–Ethylene Antagonism Mediated by Histone Deacetylation, The Plant Cell, 25(1): 149-166

Xi W., and Yu H., 2010, MOTHER OF FT AND TFL1 regulates seed germination and fertility relevant to the brassinosteroid signaling pathway, Plant Signaling and Behavior, 5(10): 1315-1317

Xi W., Liu C., Hou X., and Yu H., 2010, MOTHER OF FT AND TFL1 regulates seed Germination through a negative feedback loop modulating ABA Signaling in Arabidopsis, Plant Cell, 22(6): 1733-1748

Yan A., Wu M., Yan L., Hu R., Ali I., and Gan Y., 2014, AtEXP2 is involved in seed Germination and Abiotic stress response in Arabidopsis, PLoS One, 9(1): e85208

Zhao X., Dou L.R., Gong Z.Z., Wang X.F., and Mao T.L., 2018, BES1 hinders ABSCISIC ACID INSENSITIVE5 and promotes seed germination in Arabidopsis, New Phytologist, 221(2): 908-918

Zheng C., Halaly T., Acheampong A.K., Takebayashi Y., Jikumaru Y., Kamiya Y., and Or E., 2015, Abscisic acid (ABA) regulates grape bud dormancy, and dormancy release stimuli may act through modification of ABA metabolism, Journal of Experimental Botany, 66(5): 1527-1542