Article
Reference
A two-way molecular dialogue between embryo and endosperm is
required for seed development
DOLL, N M, et al.
Abstract
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.
DOLL, N M, et al. A two-way molecular dialogue between embryo and endosperm is required
for seed development. Science, 2020, vol. 367, no. 6476, p. 431-435
DOI : 10.1126/science.aaz4131
PMID : 31974252
Available at:
http://archive-ouverte.unige.ch/unige:140497
Disclaimer: layout of this document may differ from the published version.
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Submitted Manuscript: Confidential
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Title: A two-way molecular dialogue between embryo and endosperm
required for seed development
Authors: N. M. Doll1, S. Royek2,†, S. Fujita3,†,‡, S. Okuda4,†, S. Chamot1, A. Stintzi2, T. Widiez1,
M. Hothorn4, A. Schaller2, N. Geldner3, G. Ingram1,*
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Affiliations:
1Laboratoire Reproduction et Développement des Plantes, University of Lyon, ENS de Lyon,
UCB Lyon 1, CNRS, INRA, F-69342, Lyon, France.
2Department of Plant Physiology and Biochemistry, University of Hohenheim, Stuttgart,
Germany.
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3Department of Plant Molecular Biology, University of Lausanne, 1015 Lausanne, Switzerland.
4Structural Plant Biology Laboratory, Department of Botany and Plant Biology, University of
Geneva, 1211 Geneva, Switzerland.
*Correspondence to: gwyneth.ingram@ens-lyon.fr.
†equal contribution
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‡current address: National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan.
Abstract (129 words): 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
20
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,
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triggering GASSHO-dependent cuticle reinforcement in the embryo. A bidirectional molecular
dialogue between embryo and endosperm thus safeguards cuticle integrity prior to germination.
Submitted Manuscript: Confidential
2
One Sentence Summary: Compartmentalized proteolytic activation of a signal peptide provides
spatial cues to ensure an intact embryo cuticle.
Main Text (2047 words): In Angiosperms, seeds comprise three genetically distinct
compartments, the zygotic embryo and the endosperm, and the maternal seed coat. Their
development must be tightly coordinated for seed viability. Here we have elucidated a bidirectional
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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).
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Formation of the embryonic cuticle was previously shown to depend on two Receptor-Like
Kinases (RLKs) GASSHO1/SCHENGEN3 (from here-on named GSO1) and GSO2, and on
ALE1, a protease of the subtilase family (SBTs) (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). Since SBTs have been implicated in
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the processing of peptide hormone precursors (7, 8, 9), we hypothesized that ALE1 may be
required for the biogenesis of the elusive inter-compartmental peptide signal required for GSO1/2-
dependent cuticle deposition.
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
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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
Submitted Manuscript: Confidential
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observed in this quadruple mutant (Fig. S1B-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. 1A-D, Fig. S2 A-D). These data suggest that a sulfated peptide may act as the ligand of
GSO1/2 during seed development.
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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 gso2-1
double 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. 1E-I) (Fig. S2F-J). This result supports the hypothesis that TPST and ALE1 act in
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parallel regarding their roles in embryonic cuticle formation, possibly through independent post-
translational modifications contributing to the maturation of the hypothetical peptide signal.
Identification of the peptide signal was facilitated by a study of TWISTED SEED1 (TWS1) (14),
that reported a loss-of-function phenotype strikingly similar to that of gso1 gso2 double mutants.
Because existing alleles of TWS1 are in the WS background, we generated new CRISPR alleles
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(tws1-3 to tws1-10) in the Col-0 background, and confirmed the phenotype of resulting mutants
(Fig. 1, Fig. S3). No additivity was observed when loss-of-function alleles of TWS1 and of other
pathway components (GSO1, GSO2, TPST 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
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in both the tws1 mutants and tpst mutants (Fig. 1 J-N, Fig. S5). Inspection of the TWS1 protein
sequence revealed a region with limited similarity to CIF peptides including a DY motif which
marks the N-terminus of the CIFs (Fig. 1O), and is the minimal motif required for tyrosine sulfation
Submitted Manuscript: Confidential
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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).
We tested whether TWS1 is a substrate of ALE1 by co-expression of ALE1:(His)6 and
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TWS1:GFP-(His)6 fusion proteins in tobacco (N. benthamiana) leaves. A specific TWS1 cleavage
product was observed upon co-expression 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 E. 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
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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). The data thus 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
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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.
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
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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
