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 Plantes: Lyon, France

Corresponding author: G. Ingram

To test the biological activity of TWS1, the predicted peptide encompassing the conserved N-terminal DY motif and the C terminus defined by the ALE1 cleavage site was custom-synthesized in tyrosine-sulfated form. As synthetic TWS1 cannot easily be applied to developing embryos, a root bioassay for CIF activity was used. In wild-type roots, TWS1 induced ectopic endodermal lignification, as previously observed for the CIF1 and CIF2 peptides (12). TWS1 activity was GSO1-dependent, suggesting that processed TWS1 peptide can replace CIF1 and CIF2 as a ligand for GSO1 during Casparian strip formation (Fig. 2A and fig. S7). Supporting this, TWS1 application complemented the cif1 cif2 mutant, albeit with reduced activity compared with CIF2 (Fig. 2B and fig. S8). TWS1 activity in this assay was reduced when sulfation on the DY motif was missing (Fig. 2B). Versions of TWS1 in which Y33 was mutated to either F or T only partially complemented the mutant phenotype of tws1-4 (fig S9), consistent with a residual but weak activity for nonsulfated TWS1 in vivo and with the weak loss-of-function phenotype of the tpst-1 mutant.

为了测试TWS1的生物活性，作者定制合成了包含保守N端DY基序和包含ALE1剪切位点C端的多肽，并且是以酪氨酸硫酸化的形式。合成的TWS1蛋白并不能直接施用到正在发育的胚胎中，因此作者采用了用于CIF活性检测的根生物测定试验。在野生型的根组织中，TWS1会诱导异位的根内皮层木质化，与之前CIF1/2多肽的表型类似。TWS1的活性依赖于GSO1，说明加工后的TWS1多肽可以代替CIF1/2作为GSO1的配体作用于凯氏带的形成（Fig. 2A）。与此一致的是，施加TWS1能够互补cif1 cif2双突的表型，尽管相比于CIF2的活性可能有所下降（Fig. 2B）。当DY基序上的硫酸化去除会导致TWS1的活性完全丢失。TWS1蛋白中Y33突变成F或T均只能够部分互补tws1-4的突变表型，这与非硫酸化TWS1蛋白在体内的残留、微弱活性结果一致，同时也与 tpst-1突变体的功能缺失表型一致。

To confirm TWS1 as a ligand of GSO1 and GSO2, the interaction of the synthetic peptide with the leucine-rich repeat (LRR) ectodomains of the receptors was analyzed in grating-coupled interferometry binding assays. GSO1 bound sulfated TWS1 with a KD (dissociation constant) of ~30 nM (Fig. 2C). The observed binding affinity is ~1/10 that of the CIF2 peptide (KD = 2.5 nM) (fig. S10), which is consistent with the reduced ability of TWS1 to complement the root phenotype of the cif1 cif2 double mutant (Fig. 2B). Sulfated TWS1 also bound to the LRR domain of GSO2, albeit with slightly reduced affinity (KD ~ 100 nM) (Fig. 2D). As previously shown for other CIF peptides (11), tyrosine sulfation was critical for the interaction of TWS1 with GSO1 and GSO2 in vitro (Fig. 2, E and F). Technical issues at high peptide concentrations may explain discrepancies between in vitro binding assays and the in vivo activity of nonsulfated TWS1. In vivo activities for nonsulfated versions of other normally sulfated peptides, including CIF2, have been reported (11, 16–18). Adding a 3AA C-terminal extension to the sulfated TWS1 peptide reduced binding affinity to both GSO1 and GSO2 (fig. S10), consistent with the need for ALE1-mediated C-terminal processing for efficient signaling.

为了确认TWS1是GSO1/2的配基，作者通过光栅耦合干涉法（GCI，https://www.creoptix.com/technology）结合试验分析了合成多肽与受体的富含亮氨酸重复LRR胞外域的互作情况。GSO1与硫酸化的TWS1结合的KD值约为30 nM（Fig. 2C）。GSO1与CIF2之间结合的KD值约为2.5 nM，大概是GSO1与硫酸化TWS1之间结合的亲和力的十倍，这与之前TWS1只能部分互补cif1 cif2双突表型的结果一致（Fig. 2B）。硫酸化的TWS1同时还会结合到GSO2的LRR结构域上，尽管亲和力稍有降低，KD值约为100 nM（Fig. 2D）。与之前对于CIF多肽的研究一样，酪氨酸硫酸化对于TWS1与GSO1/2在体内的互作至关重要（Fig. 2E, F）。高浓度多肽的技术问题可能解释了非硫酸化TWS1在体内结合试验与体外活性试验的差异。已有很多研究报道了包括CIF2在内的其它正常硫酸化多肽的非硫酸化形式具有体内活性。硫酸化TWS1多肽的C端多添加一个3AA会降低其与GSO1/2的结合亲和力，说明TWS1需要ALE1介导的C端加工才能够顺利参与下游的信号通路。

p.s. K_d（dissociation constant）与K_D（equilibrium dissociation constant）

Kd，单位为S^-1，表示单位时间内解离了的AB占解离前初始AB的比例，Kd表示的是解离反应的快慢：Kd越大，解离越快；Kd越小，解离越慢；

KD，单位为M（mol/L），表示出处于平衡状态时AB的解离程度：KD越大，解离越多，AB之间亲和力越弱；KD越小，解离越少，AB间亲和力越强。

所以这里应该是“GSO1 bound sulfated TWS1 with a KD (equilibrium dissociation constant) of ~30 nM (Fig. 2C).”

Taken together, our results suggest the sulfated TWS peptide as the missing link in the intercompartmental signaling pathway for embryonic cuticle formation. The activities of ALE1 and TPST both contribute to the formation of the bioactive peptide (Fig. 1R), which is perceived by GSO1 and GSO2 to ensure appropriate cuticle deposition.

综上，作者的试验结果说明了硫酸化的TWS多肽是作用于胚胎表皮形成过程中种子内部不同组分之间信号通路的隐秘联系。ALE1和TPST的活力都作用于生物活性多肽的形成，但哪一个才是GSO1/2作用于正确的胚胎表皮沉积必不可少的？

doi: 10.1126/science.aaz4131

Journal: Science

Published date: January 24, 2020