A two-way molecular dialogue between embryo and endosperm is required for seed development

Author: N. M. Doll,  S. Royek,  S. Fujita, et al.

Corresponding author: G. Ingram.

Affiliations: Laboratoire Reproduction et Développement des Plantes, University of Lyon, ENS de Lyon, UCB Lyon 1, CNRS, INRAE, F-69342, Lyon, France, et al.

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    To understand how the elements of the signaling pathway cooperate to ensure the formation of a functional cuticle, we analyzed their spatial organization. In silico data indicate that the TPST gene is expressed in all seed tissues (fig. S11) (19, 20). 

    为了探究这个信息通路中各组分的合作究竟是如何保证表皮的功能正常，我们分析了它们的空间构成。计算机模拟表达分析（in silico expression analysis）的数据显示TSPT在种子的所有组织中都会表达。如下图。B为前球形胚期，C为球形胚早期，D为球形胚晚期，E为心形胚期，F为鱼雷型胚期，G为鱼雷型胚解剖。箭头指胚体。

    To investigate in which compartment TPST [which acts cell autonomously (13)] is required for TWS1 maturation, reciprocal crosses and complementation assays using tissue-specific promoters were performed. No cuticle permeability defects were observed when homozygous mutants were pollinated with wild-type pollen, confirming their zygotic origin. (Fig. 3, A to C). 

    为了探究tws1形成过程中TPST究竟在哪一个区域发挥作用，我们在互交试验中运用了组织特异性启动子（tissue-specific promoters）。当我们用野生型与纯合突变体杂交后，没有观察到任何表皮破损的表型。这证明了合子的纯合性。

    Expressing TPST under the ubiquitously active RPS5A promoter (21) or the PIN1 promoter [which is embryo-specific in seed (fig. S12)] complements tpst-1 cuticle defects. 

    而当我们运用全能活性（ubiquitously active）的启动子RPS5A或者胚胎特异性启动子PIN1时，tpst-1的表皮缺陷得到完善。下图是球形胚、心形胚、鱼雷形胚时期运用PIN1启动子表达TPST的结果。

    In contrast, no complementation was observed using the endosperm-specific RGP3 promoter (22), indicating that TPST activity is required for TWS1 sulfation specifically in the embryo to ensure cuticle integrity (Fig. 3D and fig. S13). 

    与此相反，当用到胚乳特异性启动子RGP3表达TPST时，表皮缺陷并没有得到改善。这个结果说明，TWS1的硫酸化只在胚胎中需要TPST来完善表皮的完整性。见下图。

    Consistent with this observation and with a previous report (14), the TWS1 promoter was found to drive expression specifically in the developing embryo from the early globular stage onwards (Fig. 3E and fig S14). 

    这些观察结果与与之前的报道相一致，从球形胚开始之后的每个阶段，TWS1的自身启动子都会一直只在胚胎中特异性表达。如下图。

    The TPST promoter (10) drove expression throughout the embryo proper at the onset of embryo cuticle establishment (the globular stage) before becoming restricted to the root tip (fig. S11). We conclude that the TWS1 peptide is both sulfated and secreted specifically in the embryo.

    TPST自身启动子会在整个胚胎发育过程中（从球形胚开始直到被根尖束缚）启动TPST表达。因此我们可以得到结论，TWS1多肽只会特异性地在胚胎中分泌以及硫酸化。

    However, production of mature TWS1 requires a C-terminal cleavage event that we have shown to be mediated by ALE1. ALE1 is expressed only in the endosperm (4, 23), on the opposite side of the nascent cuticle to the GSO1 and GSO2 receptors, which are localized on the membranes of the epidermal cells that produce the cuticle (figs. S15 to S17) (2). 

    然而，前文提到过，TWS1需要进行由ALE1介导的羧基端分裂过程。而在前人的研究中，ALE1只在胚乳中表达，也就是在初生表皮以及GSO1/2受体的反方向，其中，GSO1/2是被定位在产生表皮的epider cells的膜上的。

    下图为GSO1的定位结果。A-G分别为八分体期/球形期/过渡期/心形期/鱼雷期/弯曲子叶期/弯曲子叶期的放大。

    下图为GSO2的定位。A-H分别为八分体期/球形期/过渡期/心形期/鱼雷期/弯曲子叶期/成熟子叶的表面/成熟胚轴表面/成熟胚轴表面的放大。

    下图为GSO2自身启动子启动GSO2表达对gso1/2双突突变体的种子表型的完善。BCD分别为野生型/双突突变体/双突突变体中GSO2自身启动子表达。

    Our data therefore support a model in which activation of the GSO signaling pathway depends on the diffusion of the TWS1 peptide precursor to the endosperm, where it is cleaved and activated by ALE1 before diffusing back to the embryo to trigger GSO1/2-dependent cuticle deposition. An intact cuticle would separate the subtilase from its substrate, terminating signaling.

    因此，我们的实验结果证明了一个完整的工作通路——GSO信号通路中，首先TWS1多肽从胚胎扩散到胚乳中，并通过ALE1介导进行分裂及活化，然后再次扩散回胚胎中去启动GSO1/2来完成表皮沉积，最后当完整的表皮形成后会分裂枯草杆菌蛋白酶，从而终止信号通路的运作。

    Expressing ALE1 in the embryo, under the control of the TWS1 promoter, provided support for this model. Multiple transformants were obtained in tws1 mutants, but not in the wild-type background. When tws1 plants from four independent plants carrying the pTWS1: ALE1 transgene were pollinated with wildtype pollen—introducing a functional TWS1 allele into the zygotic compartments and thus inducing colocalization of TWS1 precursors with ALE1, GSO1, and GSO2 in the embryo— premature embryo growth arrest was observed in all seeds. This leads to severe shriveling of all seeds at maturity (Fig. 3, F to M, and figs. S18 and S19).

    在胚胎中利用TWS1启动子控制的ALE1的表达实验结果，也进一步证实了这个工作通路。我们获得了非常多的转基因植株。我们利用野生型花粉给pTWS1: :ALE1转基因植株授粉，这样就可以将功能性的ALE1等位基因引入合子区室，这样就可以进行TWS1前体以及ALE1以及GSO1/2在胚胎中的共时空（共同在胚胎中）表达——最后所有的种子中都观察到了胚胎发育未成熟的提前终止，以及种子严重皱缩。见下图。

    D-F,J-L：Chloral hydrate clearing on 9DAP (Days After Pollination) seeds

    （博主注：这里逻辑应该是，共时空表达后，胚胎发育速度跟不上表皮沉积速度，在表皮沉积完成后通路被停止，所以胚胎在未成熟时发育即被叫停）

    A proportion of seeds could, nonetheless, germinate to give developmentally normal plants (fig. S20), indicating that coexpression of all signaling components in the embryo—although detrimental to embryo development—does not lead to a complete loss of viability. 

    我们发现，一部分的种子甚至仍然可以发芽（germination）并长成和野生型无异的植株。这表示即使在胚胎中，共时空表达信息通路上的所有组分（当然是）对胚胎发育不利，但胚胎并不会完全失去生存活性。

    Growth arrest may be due to constitutive embryonic activation of the GSO1/GSO2 signaling pathway, and stressresponsive genes shown to require GSO1/GSO2 signaling for expression in the seed (2) were upregulated in seeds coexpressing GSO1, GSO2, TWS1, and ALE1 in the embryo (fig. S21). We thus postulate that the spatial separation of the TWS1 precursor and the GSO receptors from the activating protease by cuticle is required for signaling attenuation.

    发育停止可能是因为GSO1/2信号通路控制的胚胎发育过程，因为在胚胎中所有组分共时空表达时，GSO1/2信号的有关种子发育的受体基因上调了。如下图（图中的几个基因是已经被证实是GSO的靶向受体，且与种子发育有关）。我们因此推测，TWS1前体、GSO与ALE1激活蛋白酶的空间分离是为了等待GSO信号衰减。

    We next tested if CIF1, CIF2, and TWS1 could complement tws1 and ale1 mutants when expressed in the endosperm (under the RGP3 promoter). All three peptides complemented tws1 mutants, confirming that retrograde peptide movement from endosperm to embryo is sufficient to allow integrity monitoring (Fig. 3N and fig. S22). 

    接着我们测试了CIF1/2以及TWS1（利用RGP3启动子）在胚乳中特异性表达时，是否能够补充tws1以及ale1突变体的缺陷。最后发现全部都可以弥补tws1的缺陷。这证实了从胚乳到胚胎的逆向的多肽运动也可以一定程度上完成通路。见下图。

    Lack of full complementation could reflect suboptimal N-terminal processing or sulfation in the endosperm. CIF1 and CIF2 (lacking C-terminal extensions) complemented ale1 mutants much more efficiently than TWS1 (fig. S23). Weak complementation of ale1 by TWS1 may reflect the presence of redundantly acting subtilases in the endosperm, as suggested by the weak phenotype of ale1 mutants.

    对突变体缺陷的弥补效果并不完整说明，在胚乳中并没有完整地进行氨基端的硫酸化。在ale1突变体中，CIF1/2（缺少羧基端的延伸）对缺陷的弥补比TWS1要有效很多。如下图。ale1中TWS1低效弥补可能是因为在胚乳中枯草杆菌蛋白酶的冗余活性，正如之前ale1突变体只有微弱的表型。

    The proposed bidirectional signaling model allows efficient embryo cuticle integrity monitoring. The sulfated TWS1 precursor is produced by the embryo and secreted (probably after N-terminal cleavage of the pro-peptide) to the embryo apoplast. In the absence of an intact cuticular barrier, it can diffuse to the endosperm and undergo activation by ALE1 (and potentially other subtilases). Activated TWS1 peptide then leaks back through cuticle gaps to bind the GSO1 and GSO2 receptors and activate local gap repair (Fig. 3O). When the cuticle is intact, proTWS1 peptides are confined to the embryo where they remain inactive.

    这个假定的双向信号模型使胚胎表皮完整性的监控变得有效。被硫酸化的TWS1前体在胚胎中生成，随后被分泌至胚胎质外体（可能在前肽的氨基端分裂之后）。当完整的胚胎表皮障碍不存在时，TWS1可以扩散至胚乳并且被在胚乳中表达的ALE1（甚至还会有其他的枯草杆菌蛋白酶）激活。活化之后的TWS1随后再通过胚胎表皮孔洞返回到胚胎中，结合GSO1/2受体然后再激活存在于胚胎中的“孔洞修复者（gap repair）”。如下图。当表皮发育完整后，TWS1多肽就会被封锁在胚胎中，一直保持非活性状态（inactive）。

    Our results demonstrate a role for a subtilase in providing spatial specificity to a bidirectional peptide signaling pathway. In contrast, the related CIF1-, CIF2-, and GSO1-dependent signaling pathway controlling Casparian strip integrity is unidirectional, negating the need for C-terminal cleavage-mediated peptide activation (10, 12). Both pathway components and their spatial organization differ between the two systems, suggesting an independent recruitment of the GSO receptors to different integrity monitoring functions within the plant.

    我们的实验证明了一种枯草杆菌蛋白酶ALE1在一个双向多肽信号通路中提供空间特异性功能。与此相反，CIF1/2以及GSO决定的凯氏带形成通路是单向的，且并不需要羧基端分裂带来的多肽活性。在这两个信号通路系统中，各环节组分以及它们的空间表达都不一样，这表明植物中存在对受体GSO的两种不同的“完整性监管（integrity monitor）”应用。

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