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已有 648 次阅读 2020-1-29 07:04 |个人分类:一周精读|系统分类:论文交流

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


The plant embryonic cuticle is a hydrophobic barrier deposited de novo by the embryo during seed development. At germination, it protects the seedling from water loss and is, thus, critical for survival. Embryonic cuticle formation is controlled by a signaling pathway involving the ABNORMAL LEAF SHAPE1 subtilase and the two GASSHO receptor-like kinases. We show that a sulfated peptide, TWISTED SEED1 (TWS1), acts as a GASSHO ligand. Cuticle surveillance depends on the action of the subtilase, which, unlike the TWS1 precursor and the GASSHO receptors, is not produced in the embryo but in the neighboring endosperm. Subtilase-mediated processing of the embryo-derived TWS1 precursor releases the active peptide, triggering GASSHO-dependent cuticle reinforcement in the embryo. Thus, a bidirectional molecular dialogue between embryo and endosperm safeguards cuticle integrity before germination.


In angiosperms, seeds comprise three genetically distinct compartments: the zygotic embryo, the endosperm, and the maternal seed coat. Their development must be tightly coordinated for seed viability. In this work, we have elucidated a bidirectional peptide-mediated signaling pathway between the embryo and the endosperm. This pathway regulates the deposition of the embryonic cuticle, which forms an essential hydrophobic barrier separating the apoplasts of the embryo and endosperm. After germination, the cuticle—one of the critical innovations underlying the transition of plants from their original, aqueous environment to dry land—protects the seedling from catastrophic water loss (1, 2).


Formation of the embryonic cuticle has previously been shown to depend on two receptor-like kinases (RLKs)—GASSHO1/SCHENGEN3 (hereafter named GSO1) and GSO2—and on ALE1, a protease of the subtilase family (2–5)gso1 gso2 and (to a lesser extent) ale1 mutants produce a patchy and highly permeable cuticle (2). Mutant embryos also adhere to surrounding tissues, causing a seed-twisting phenotype (6). Because subtilases have been implicated in the processing of peptide hormone precursors (7–9), we hypothesized that ALE1 may be required for the biogenesis of the elusive intercompartmental peptide signal required for GSO1/2-dependent cuticle deposition.

先前的研究显示植物胚胎表皮的形成依赖于两个GASSHO类受体激酶GSO1和GSO2,以及一个枯草蛋白酶家族的蛋白酶ALE1。gso1 gso2双突和ale1单突形成的胚胎表皮呈现拼凑状,且透水性很好。突变体的胚胎会粘着周围的组织,导致种子扭曲的异常表型。因为有研究显示类枯草杆菌蛋白酶作用于多肽激素前体的加工,作者假设ALE1可能会作用于不同组分间多肽信号的生物发生,而该信号对于依赖于GSO1/2的胚胎表皮沉积至关重要。

CASPARIAN STRIP INTEGRITY FACTORs (CIFs), a family of small sulfated signaling peptides, are ligands for GSO1 and GSO2 (10–12). CIF1 and CIF2 are involved in Casparian strip formation in the root endodermis (10, 11). The function of CIF3 and CIF4 is still unknown. To assess the role of CIF peptides in cuticle development, the quadruple mutant (cif1 cif2 cif3 cif4) was generated (fig. S1A). Neither cuticle permeability nor seed twisting phenotypes were observed in this quadruple mutant (fig. S1, B to E). However, reduction [in the leaky sgn2-1 allele (10)] or loss [in the tpst-1 mutant (13)] of tyrosyl-protein sulfotransferase (TPST) activity results in seed-twisting and cuticle-permeability phenotypes resembling those observed in ale1 mutants (Fig. 1, A to D, and fig. S2, A to D). These data suggest that a sulfated peptide may act as the ligand of GSO1/2 during seed development.

凯氏带完整因子CIFs是一类小的、硫酸化的信号多肽家族,是GSO1/2的配基。在植物根内皮层中,CIF1/2参与凯氏带的形成。而CIF3/4的功能还不清楚。为了研究CIF多肽在胚胎表皮发育过程中的作用,作者获得了cif1 cif2 cif3 cif4四突植株。作者发现cif1 cif2 cif3 cif4四突植株种子并未显示出表皮透水或种子扭曲的表型。然而,酪氨酰蛋白质磺基转移酶TPST活力的降低(sgn2-1突变)或丢失(tpst-1突变)导致了种子扭曲和表皮透水表型与ale1突变体中观察到缺陷表型类似(Fig. 1, A-D; Fig. S2, A-D)。这些结果表明可能存在一个硫酸化的多肽作为GSO1/2的配基在种子发育过程中发挥作用。

p.s. 甲苯胺蓝测试(toluidine‐blue test)检测植物组织表皮的完整性(Tanaka et al.,  2003, the plant journal, doi: https://doi.org/10.1046/j.1365-313X.2003.01946.x)

Consistent with the hypothesis that TPST acts in the same pathway as GSO1 and GSO2, no difference was observed between the phenotype of tpst-1 gso1-1 gso2-1 triple and gso1-1 gso2-1double mutants (fig. S2E). In contrast, TPST and ALE1 appear to act synergistically, as a phenotype resembling that of gso1 gso2 double mutants was observed in tpst-1 ale1-4 double mutants (Fig. 1, E to I, and fig. S2, F to J). This result supports the hypothesis that TPST and ALE1 act in parallel regarding their roles in embryonic cuticle formation, possibly through independent posttranslational modifications that contribute to the maturation of the hypothetical peptide signal.

作者发现tpst-1 gso1-1 gso2-1三突的表型与gso1-1 gso2-1双突的表型没有明显的差异(Fig. S2E),说明TPST与GSO1/2在同一个通路上发挥作用。相反,TPST与ALE1似乎协同发挥作用,因为在tpst-1 ale1-4双突变体中观测到了与gso1-1 gso2-1双突类似的缺陷表型(Fig 1, E-I; Fig. S2, F-J)。该结果表明TPST与ALE1平行作用于胚胎表皮的形成,可能通过独立的翻译后修饰途径来促进假想中的肽信号的成熟。

Identification of the peptide signal was facilitated by a study of TWISTED SEED1 (TWS1) (14), which reported a loss-of-function phenotype that was notably similar to that of gso1 gso2 double mutants. Because existing alleles of TWS1 are in the Wassilewskija (WS) background, we generated new CRISPR alleles (tws1-3 to tws1-10) in the Col-0 background and confirmed the phenotype of resulting mutants (Fig. 1 and fig. S3). No additivity was observed when loss-of-function alleles of TWS1 and of other pathway components (GSO1GSO2TPST, and ALE1) were combined, providing genetic evidence for TWS1 acting in the GSO signaling pathway (fig. S4). Furthermore, gaps in the cuticle of embryos and cotyledons, similar to those observed in ale1 and gso1 gso2 mutants (2), were detected in both the tws1 mutants and tpst mutants (Fig. 1, J to N, and fig. S5). Inspection of the TWS1 protein sequence revealed a region with limited similarity to CIF peptides, including a DY motif that marks the N terminus of the CIFs (Fig. 1O) and is the minimal motif required for tyrosine sulfation by TPST (15). Corroborating the functional importance of the putative peptide domain, the tws1-6 allele (deletion of six codons in the putative peptide-encoding region) and the tws1-5 allele (substitution of eight amino acids, including the DY motif) both showed total loss of function of the TWS1 protein (fig. S3).

对于TWS1的研究加速了多肽信号的鉴定,该研究报道了一个功能缺失突变体,而该突变体的表型与gso1-1 gso2-1双突的表型非常类似。由于现有的TWS1突变是在瓦斯莱(WS)生态型的拟南芥材料中,作者重新在Col-0背景下构建了一个CRISPR等位基因突变系(tws1-3tws1-10),并且确定了这些突变体的表型(Fig. 1)。TWS1的功能缺失突变与其它通路组分GSO1/2TPST以及ALE1之间并没有加性效应,说明TWS1也在GSO信号通路上发挥作用。另外,作者在tws1tpst突变体的胚胎和子叶表皮中鉴定到了间隙与ale1gso1 gso2突变体中所观察到的表型非常类似(Fig. 1, J to N)。对于TWS1蛋白序列的研究显示其存在一段与CIFs多肽相似度很高的区域,包括一个标记CIFs多肽N端的DY基序(Fig. 1O),并且该基序对于CIFs能够被TPST酪氨酸硫酸化非常重要tws1-6突变体在编码多肽区域删掉了6个密码子, tws1-5突变体替换了包含DY基序在内的8个氨基酸,而这两个突变体都表现出了TWS1蛋白的完全功能缺失,再一次证明了DY基序对于TWS1功能实现的重要性。

We tested whether TWS1 is a substrate of ALE1 by coexpression of ALE1:(His)6 and TWS1:GFP-(His)6 fusion proteins in tobacco (Nicotiana benthamiana) leaves. A specific TWS1 cleavage product was observed upon coexpression of ALE1 but not in the empty-vector control, suggesting that TWS1 is processed by ALE1 in planta (Fig. 1P). Likewise, recombinant TWS1 expressed as GST-fusion in Escherichia coli was cleaved by purified ALE1 in vitro. (Fig. 1Q). Mass spectroscopy analysis of the TWS1 cleavage product purified from tobacco leaves showed that ALE1 cleaves TWS1 between His54 and Gly55 (fig. S6). These residues are important for cleavage site selection, as ALE1-dependent processing was not observed when either His54 or Gly55 was substituted by site-directed mutagenesis (Fig. 1Q). His54 corresponds to the C-terminal His or Asn of CIF peptides (Fig. 1O). Thus, the data suggest that ALE1-mediated processing of the TWS1 precursor marks the C terminus of the TWS1 peptide. Because the CIF1 and CIF2 peptides are located at the very end of their respective precursors, C-terminal processing could represent a mechanism of peptide activation operating in the developing seed but not in the root. A summary of TWS1 modifications is provided in Fig. 1R.

作者通过在烟草叶片中共表达ALE1:(His)6和TWS1:GFP-(His)6融合蛋白测试TWS1蛋白是否是ALE1的底物。作者在共表达ALE1的烟草叶片中观察到了特定的TWS1剪切产物,而在空载对照中则没有,说明在植物体内ALE1能够加工TWS1(Fig. 1P)。在体外试验中,重组的GST-TWS1同样能够被纯化的ALE1蛋白所剪切(Fig. 1Q)。对于TWS1剪切产物的质谱分析显示ALE1将TWS1蛋白在His54与Gly55之间剪切开。通过定点突变这两个氨基酸残基能够阻止ALE1介导的TWS1蛋白剪切,说明这两个氨基酸残基对于TWS1剪切位点的选择至关重要(Fig. 1Q)。TWS1蛋白中His54对应着CIF多肽C端的His或Asn(Fig. 1O)。因此,这些数据说明ALE1介导的TWS1前体加工处于TWS1多肽的C端。CIF1/2多肽都定位于各自前体的末端位置,因此C端加工可能是发育中的种子特有的一种多肽激活机制,而不适用于根中。Fig 1R总结了TWS1蛋白的修饰位点。

通讯:G. Ingramhttp://www.ens-lyon.fr/RDP/spip.php?rubrique15=&lang=en


doi: 10.1126/science.aaz4131

Journal: Science

Published date: January 24, 2020



上一篇:Plant Biotechnol J:红麻基因组


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