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已有 2589 次阅读 2020-1-31 06:38 |个人分类:一周精读|系统分类:论文交流|关键词:学者

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

First author: N. M. Doll; Affiliations: Laboratoire Reproduction et Développement des PlantesLyon, France

Corresponding author: G. Ingram

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). 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). 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. 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). 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). 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.

为了理解该信号通路上不同组分如何协同作用于胚胎表皮的正确形成,作者分析了这些组分在空间上的组织情况。生信数据显示TPST基因在所有的种子组织中都有表达。为了研究TPST是否作用于TWS1的成熟,作者利用组织特异性的启动子进行了互交和互补试验。当利用野生型花粉杂交到tpst纯合突变体中时并未发现明显的胚胎表皮发育缺陷,说明该信号是来自于合子的(Fig. 3A-C)。利用组成型RPS5A启动子或是胚胎特异性PIN1启动子来表达TPST均能够互补tpst-1的表皮缺陷表型。相反,利用一个胚乳特异性RGP3启动子表达TPST并不能互补tpst-1的表皮缺陷表型,说明胚胎中的TPST活性对于TWS1的硫酸化是非常重要的,从而保证表皮的完整性(Fig. 3D)。与先前的研究一致,作者发现TWS1的启动子特异性的在胚胎球形胚时期开始表达(Fig. 3E)。而TPST启动子在胚胎表皮建起之处,即球形胚时期驱动基因在整个胚胎的表达,直到后来局限于根尖表达。作者认为TWS1多肽在胚胎中被硫酸化以及分泌。

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). 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.


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 wild-type 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). 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. Growth arrest may be due to constitutive embryonic activation of the GSO1/GSO2 signaling pathway, and stress-responsive 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.

利用TWS1基因的启动子驱动ALE1基因在胚胎中的表达证实了上述的模型。作者在tws1突变体背景下获得了很多的遗传转化植株,但没有获得以野生型为背景的遗传转化株。因此,作者用野生型的花粉杂交到4个带有pTWS1:ALE1转基因的tws1突变体株系,用来向合子中引入功能性的TWS1等位基因,获得了TWS1前体、ALE1以及GSO1/2都定位于胚胎的种子,结果发现所有的种子都存在胚胎生长提前停止。这导致了种子在成熟时期严重萎缩(Fig. 3F-M)。尽管如此,还是存在一定比例的种子可以发芽,进而形成发育正常的植株,说明尽管所有的信号通路组分同时存在于胚胎对于胚胎的发育存在有害影响,但并不会导致完全的生存障碍。生长停滞可能是由于GSO1/2信号通路的组成型胚胎激活,某些需要GSO1/2信号才能在种子中表达的胁迫响应基因在GSO1/2、TWS1以及ALE1共定位于胚胎的种子中上调表达了。因此,作者提出胚胎表皮介导的、TWS1前体和GSO受体与其激活蛋白酶的空间隔离对于衰减信号是必须的。

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). 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.

接着,作者测试了利用RGP3启动子驱动CIF1、CIF2和TWS1在胚乳中的表达是否能够互补tws1和ale1突变体。结果显示,这三个多肽均能互补tws1突变体,确定了从胚乳逆行到胚胎的多肽能够确保胚胎表皮沉积的完整性(Fig. 3N)。但不能完全互补,可能是由于在胚乳中的N端加工或是硫酸化并不是最优状态。CIF1/2能够比TWS1更加有效的互补ale1突变体。TWS1在胚乳中表达只能部分互补ale1突变体可能是由于胚乳中存在功能冗余的类枯草杆菌蛋白酶,这也能够解释为何ale1突变体的缺陷表型相对微弱。

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.

作者提出的双向信号转导模型能够保证胚胎完整性的有效检测。硫酸化的TWS1前体在胚胎中产生,可能经过了N端的剪切,然后被分泌到胚胎的质外体中。由于缺少完整的胚胎表皮屏障,硫酸化的TWS1能够扩散到胚乳中,而后被胚乳中的ALE1激活,亦或是其它潜在的类枯草杆菌蛋白酶。激活后的TWS1多肽通过胚胎表皮的间隙再次返回到了胚胎中,然后结合GSO1/2受体,激活胚胎表皮间隙的修复(Fig. 3O)。经过不断来回的信号,胚胎表皮越加完整,最终,当胚胎表皮完整后,TWS1前体不能够越过障碍,被限制在了胚胎内部,保持未激活的状态。

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.


doi: 10.1126/science.aaz4131

Journal: Science

Published date: January 24, 2020





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