Research Article

Central Cell–Derived Peptides Regulate Early Embryo Patterning in Flowering Plants

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Science  11 Apr 2014:
Vol. 344, Issue 6180, pp. 168-172
DOI: 10.1126/science.1243005

Tripeptide Maternal Support

In flowering plants, fertilization involves multiple gametes. The diploid zygote, which will form the embryonic plant, is surrounded by the often triploid endosperm, which provides a supportive and nourishing function. Working in Arabidopsis, Costa et al. (p. 168; see the Perspective by Bayer) identified a trio of small signaling peptides that derive from the endosperm but that regulate growth of the embryo. RNA interference was used to down-regulate expression of all three peptides. Fertilization was not affected, but seed growth was. The peptides were critical for normal development of the suspensor, which tethers and nourishes the growing embryo.


Plant embryogenesis initiates with the establishment of an apical-basal axis; however, the molecular mechanisms accompanying this early event remain unclear. Here, we show that a small cysteine-rich peptide family is required for formation of the zygotic basal cell lineage and proembryo patterning in Arabidopsis. EMBRYO SURROUNDING FACTOR 1 (ESF1) peptides accumulate before fertilization in central cell gametes and thereafter in embryo-surrounding endosperm cells. Biochemical and structural analyses revealed cleavage of ESF1 propeptides to form biologically active mature peptides. Further, these peptides act in a non–cell-autonomous manner and synergistically with the receptor-like kinase SHORT SUSPENSOR to promote suspensor elongation through the YODA mitogen-activated protein kinase pathway. Our findings demonstrate that the second female gamete and its sexually derived endosperm regulate early embryonic patterning in flowering plants.

Double fertilization is unique to flowering plants and results in the production of a seed containing an embryo and an endosperm. Unlike in animals, plants can form zygotic and somatic embryos, which are mostly autonomously regulated by complex interactions involving intrinsic zygotic factors (13). Zygotic embryos undergo a series of conserved formative divisions to generate an embryo proper that later gives rise to the adult plant, and a stalk-like suspensor that acts as a nutritive conduit and growth regulator to sustain early embryogenesis (4). Although plants can regenerate via somatic embryogenesis, somatic embryos do not undergo some of the characteristic early divisions seen in zygotic embryos (5), suggesting that the external chemical and physical environment may influence certain formative divisions, such as the generation of a proper suspensor (6). Further evidence is provided by genetic analyses and ablation studies of endosperm and surrounding maternal tissues, which suggest the presence of additional extrinsic developmental cues in these tissues that are required for successful completion of embryogenesis (7). Such cues may be in the form of small mobile peptides, which have recently emerged as key signaling factors during plant reproduction and development (813).

Identification of Cysteine-Rich Peptides in Seeds

Previously, we identified a large class of small, cysteine-rich peptides specifically expressed in Arabidopsis reproductive tissues (14). Because of their abundant nature, we hypothesized that these could act as potential signals that direct and/or coordinate early embryogenesis. We therefore carefully surveyed their expression at six stages of seed development (0, 1, 3, 6, 12, and 21 days after fertilization) using a custom-designed microarray. From this initial analysis, we identified 180 peptide-encoding genes preferentially expressed in developing seeds at three distinct stages: prefertilization, early seed development, and seed maturation (Fig. 1A). Of these, one small gene family that shares homology with the maize MEG1 family (8) was highly expressed at the onset of fertilization (Fig. 1A). To investigate whether these Arabidopsis peptides played a similar pivotal role in seed development, we first assessed their temporo-spatial expression patterns (fig. S1 to S3) and identified three genes (At1g10747, At1g10745, and At1g10717) that were exclusively expressed in ovule embryo sacs and in early developing endosperms. To determine subcellular protein localization, we performed immunolocalization (fig. S4) and stably expressed translational gene fusions with a yellow fluorescent protein (YFP) reporter. Similar to our immunodetection data, YFP reporter analysis showed that these peptides were primarily expressed in the central cell gamete of nonfertilized ovules, which upon fertilization gives rise to the endosperm, and later in the micropylar endosperm, which surrounds the embryo (Fig. 1, B to G). Because of their specific expression profiles, we thereafter renamed the peptides expressed by At1g10747, At1g10745, and At1g10717: EMBRYO SURROUNDING FACTORS 1.1 to 1.3 (ESF1.1 to ESF1.3), respectively.

Fig. 1 ESF1 genes are expressed in the central cell and endosperm.

(A) Microarray analysis of small cysteine-rich peptide-encoding genes at different days after fertilization (DAF) showing those primarily expressed during early seed development (black box). (B to G) Confocal scanning laser microscopy (CSLM) images of ESF1.1-YFP (B and C), ESF1.2-YFP (D and E), and ESF1.3-YFP (F and G) transcriptional reporters showing fluorescent signal in central cell (cc) (B, D, and F) and embryo-surrounding cells (esc) (C, E, and G). Scale bars: 50 μm (B, D, and F) and 100 μm (C, E, and G). Microarray data are available in NCBI Gene Expression Omnibus (accession GSE36086).

ESF1 Are Required for Early Seed Development

The ESF1 genes are tandemly duplicated on Arabidopsis chromosome 1. Single-knockout mutants were obtained but did not display any visible phenotype, suggesting that ESF1.11.3 are functionally redundant. To elucidate ESF1 function, we generated RNA interference (RNAi) lines using an engineered two-component LhG4/pOp transactivation system (15) to simultaneously down-regulate expression of ESF1.1–1.3 genes specifically in central cell and embryo-surrounding cells by using the At4g23080/EBE2 promoter (see fig. S5 and supplementary materials and methods). Analysis of three randomly selected independent esf1_RNAi lines indicated that fertilization was not affected; however, 48% (n = 673) of mature seeds were significantly smaller and morphologically abnormal (Fig. 2, A to C, and fig. S6). Microscopic analysis of developing esf1_RNAi seeds revealed embryo abnormalities only, which included variable patterning defects in the embryo proper and a consistently reduced number of suspensor cells (Fig. 2, D to G, and table S1). However, when we expressed the esf1_RNAi hairpin in early developing embryos, we did not observe these defects (table S1), suggesting that ESF1 peptides may act in a non–cell-autonomous manner.

Fig. 2 ESF1 genes are essential for early embryonic patterning.

Seed progenies from wild-type (A) and esf1_RNAi plants (B) segregating smaller, abnormal-shaped seeds (arrowheads). (C) Seed size distribution in wild-type and three independent esf1_RNAi lines (Welch’s two-sample t test, **P < 0.001 and ***P < 0.0001). (D to G) Differential interference contrast (DIC) images of wild-type (D and E) and esf1_RNAi (F and G) developing embryos. Colors indicate embryonic (yellow and blue) and extraembryonic (red) cell lineages (H to K). CSLM images of globular embryos from wild-type (H and J) and esf1_RNAi (I and K) seeds carrying a WOX8-nGFP (H and I) and a PIN1:GFP/DR5:nVenus (J and K) reporter. Scale bars: 10 μm.

To further investigate the embryo defects, we first examined expression of WOX8, a homeobox gene that regulates apical-basal axis formation and suspensor development, using the WOX8-nGFP reporter (16). WOX8 expression was confined to suspensor cells in wild-type globular embryos, whereas esf1_RNAi embryos showed abnormal spatial expression of the reporter (Fig. 2, H and I, and fig. S7, A and B). Other suspensor markers, including WOX5 (17), ARF13, and IAA10 (18), showed similar abnormalities (fig. S7C to H), indicating that gene expression programs of the basal lineage are irregular in esf1_RNAi embryos.

Early embryonic cell identity in somatic and sexual embryos is mediated by the measured transport and distribution of the phytohormone auxin. Wild-type globular embryos exhibited accumulation of the auxin efflux carrier PIN1 (19) in embryonic cells outside the suspensor, and activation of the auxin-responsive DR5 reporter (20) in the uppermost suspensor cells and hypophysis (Fig. 2J). By contrast, we found that esf1_RNAi embryos displayed reduced expression of PIN1 and ectopic expression of the DR5 reporter in the basal-most portion of the embryo (Fig. 2K). Collectively, these data indicate that ESF1 peptides regulate suspensor development and correct auxin distribution in early developing embryos.

Structure-Function Characterization of ESF1

To further analyze the function of ESF1 in early embryo development, we individually overexpressed synthetic gene constructs (synESF1) encoding the three different ESF1 peptides in embryo-surrounding endosperm cells (see supplementary materials and methods). We found that lines overexpressing synESF1 wild-type peptides produced 49% (n = 578) mature seeds that were significantly smaller (fig. S8) and contained poorly developed proembryos with elongated or filamentouslike suspensors (Fig. 3, A to D, and table S2). In a complementary approach, we expressed ESF1.3 in BY-2 cell cultures and exogenously applied the purified peptide to fertilized ovule cultures (see supplementary materials and methods). Exogenous ESF1 peptide application could induce suspensor cell elongation at nanomolar concentrations only when the protein was folded correctly (Fig. 3, E and F). These data indicate that protein structure is essential for biological activity, as shown for other similar peptides, such as STOMAGEN (21).

Fig. 3 Secreted ESF1 peptides positively regulate suspensor elongation.

(A to D) DIC images of wild-type (A) and ESF1 overexpression embryos with elongated suspensors and patterning defects (B to D). Colors indicate embryonic (yellow) and extraembryonic (red) cell lineages. Scale bars: 50 μm. (E) Confocal images of pGRP-GUS:GFP seeds grown in culture with and without 30 μM purified ESF1.3. (F) Graph showing ESF1.3 dose-dependent suspensor elongation response. Red, BY-2 purified ESF1.3 (n = 240); blue, heat-denatured BY-2 purified ESF1.3 (n = 240); green, bacterial purified recombinant ESF1.3 (n = 160) (E). Error bars represent the standard deviation.

We then resolved the three-dimensional structure of ESF1 by solution nuclear magnetic resonance (NMR) and found that the mature ESF1 peptide, containing 68 residues, consists of four loops and a scaffold supported by four disulfide bonds (Fig. 4, A and B, figs. S9 to S11, and table S3). We analyzed the electrostatic potential of the molecular surface of ESF1 and identified one edge of the peptide as being relatively hydrophobic, containing a negatively charged region and enclosing two aromatic residues that are conserved among the ESF1 peptides (Fig. 4C and fig. S12). To test the structure-function relationship of ESF1, we generated stable transformants containing mutated synthetic ESF1 gene constructs (synESF1) encoding protein variants lacking individual disulfide bonds and/or both conserved aromatic residues and assessed their effects on suspensor elongation. Whereas overexpression of the wild-type synESF1 gave rise to elongated suspensor formation, either removal of any of the disulfide bonds or both tryptophan residues abolished this effect (Fig. 4D). We then analyzed the NMR spectra of wild-type and mutant synESF1 peptides and found that whereas ΔSS mutants disrupted protein topology, mutations in both tryptophan residues had no effect on protein structure (Fig. 4E). Collectively, our data indicate that ESF1 biological activity depends on structural topology, which is stabilized by the disulfide bonds and is characteristic of other plant and animal cysteine-rich peptides (8, 12, 13, 2123). Similarly, the aromatic residues are also a conserved feature of regulatory plant peptides involved in a variety of cellular processes (8, 12, 13, 24, 25) and may be important for anchoring to carbohydrates or proteins at the membrane interface (26, 27).

Fig. 4 ESF structure-function relationship.

(A) Amino acid sequence of secreted ESF1.3 peptide with disulfide bonds indicated by connecting lines. Blue arrow and red cylinder indicate β strand and α helix, respectively. (B) Ribbon model of the ESF1.3 structure showing the location of four pairs of disulfide bonds and two conserved aromatic residues. (C) Electrostatic potential distribution on the molecular surface of ESF1.3 showing negative (red), positive (blue), and hydrophobic (gray) residues. (D) Graph showing suspensor length of embryos from plants expressing wild-type and mutant synESF1 constructs (unpaired Student’s t test, **P < 0.001). Error bars represent the standard deviation. (E) 1H-NMR spectra of wild-type ESF1 (top), four ΔSS mutants (middle), and W35A/W48A mutant (bottom) showing no, severe, and minor disruptions to ESF1 structure, respectively. Abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

ESF1 Act with SSP and YDA to Regulate Embryogenesis

To position ESF1 in a possible genetic pathway, we performed crosses with plants carrying mutations for the YODA (YDA) mitogen-activated protein kinase (MAPK) gene or the Pelle/IL-1R (interleukin-1 receptor)–associated kinase (IRAK)-like kinase gene SHORT SUSPENSOR (SSP), both of which exhibit suspensor abnormalities (28, 29). Homozygous esf1_RNAi lines were crossed with recessive loss-of-function YDA mutants (yda-2991) to generate esf1_RNAi/esf1_RNAi; yda-2991/+ plants, which were self-pollinated. Of the segregating sibling seeds, 46.7% (n = 278) displayed embryos with severely reduced suspensors that resembled YDA mutants, and 30.1% displayed other embryonic abnormalities (fig. S13 and table S4). Because YDA loss-of-function causes severe embryonic defects that could mask the esf1_RNAi phenotype, we also crossed esf1_RNAi lines with plants carrying a constitutively active variant of YDA (CA-YDA) (29). Self-pollinated heterozygous CA-YDA plants produced 48.1% (n = 528) embryos with elongated suspensors (table S4); a phenotypic class that was also prevalent in CA-YDA; esf1_RNAi plant progeny (Fig. 5, A to D, and table S4). These data indicate that the hyperactive YDA can suppress the effects of esf1 down-regulation. By contrast, when we crossed homozygous esf1_RNAi plants with heterozygous plants segregating the paternal effect ssp-2 mutation, we observed an additional phenotype consisting of a cone-shaped embryo with short suspensor (Fig. 5, E and F, and table S4). Collectively, these results implicate ESF1s as additional components in the SSP/YDA-dependent signaling pathway that regulates early embryo development. This creates an intriguing scenario whereby early embryogenesis is regulated not only by unique paternal factors (SSP), but also by extraembryonic maternal factors contributed by the sister egg cell gamete, the central cell.

Fig. 5 ESF1 peptides act in the YDA pathway to regulate suspensor development and are required maternally.

(A to F) Whole-mount DIC images of wild-type (A), esf1_RNAi (B), CA-YDA (C), CA-YDA/esf1_RNAi (D), ssp-2 (E), and ssp-2/+;esf1_RNAi (F) embryos. Scale bars: 10 μm. (G to J) Wild-type (G), esf1_RNAi (H), +/cdka;1 (I), esf1_RNAi; +/cdka;1 (J) seeds. Scale bars: 100 μm. Colors indicate cells attributed to embryonic (yellow), hypophysis (blue), and extraembryonic (red) lineages.

ESF1 Are Maternally Required Factors

Therefore, to investigate the extent to which maternally contributed ESF1 peptides (i.e., from the central cell and prior to fertilization) are required for early embryonic patterning, we crossed esf1_RNAi plants with one of two mutant lines that only undergo single fertilization events, cdka;1 (30) and kokopelli (kpl) (31), to generate seeds containing an embryo that lacks an endosperm (see supplementary materials and methods). In control experiments, wild-type plants were used as females and crossed to cdka;1/+ heterozygous plants to produce mixed seed progeny resulting from either double or single fertilization events. In single-fertilized seeds, 98% (n = 250) of embryos developed normally up until the globular stage (30, 32). However, when esf1_RNAi plants were crossed as females with cdka;1 pollen, 23% (n = 280) of single-fertilized globular embryos exhibited severe patterning defects and short suspensors (Fig. 5, G to J, and table S5). Similar results were obtained in crosses using kpl-1 pollen (table S5). Collectively, our data strongly indicate that maternally contributed central cell ESF1 peptides play an important role in suspensor formation and proembryo development, which is likely reinforced by the contribution of the embryo-surrounding endosperm after fertilization.


Since the discovery of double fertilization and the notable appearance of a second female gamete, questions have been raised about its importance and participation in sexual plant reproduction (33). Here, we provide evidence for a hitherto unrecognized signaling role of the central cell and its ephemeral endosperm derivative in regulating the apical-basal developmental program of its neighboring embryo (34). The pivotal structure-function relationship of ESF1 is consistent with other cysteine-rich peptides, such as those involved in cell signaling of the stomatal cell lineage (24), and further supports their signaling role during early embryogenesis. Moreover, we argue that ESF1 regulation of early suspensor growth arose from parental conflict (35) and provides a maternal advantage over embryo growth at a critical stage when parental investment determines the fate of the offspring. ESF1 peptides are not imprinted like the related maternally expressed MEG1 peptides in maize (8, 36), but are maternally deposited in the central cell gamete and early in the endosperm, which may be indicative of their immediate requirement at the onset of fertilization. Thus, plants appear to have evolved multiple independent strategies in the form of embryonic and extraembryonic factors, including mobile RNAs and peptides, to maternally control early embryogenesis.

Methods Summary

All plant material used in this study was derived from the wild-type Columbia (Col-0) accession and mutant alleles cdka;1 (30), kpl-1 (31), ssp-2 (28), CA-YDA, and yda-2991 (29). Details of transgenic lines can be found in the supplementary materials and methods. Microarray analysis was performed on wild-type developing siliques collected after manual pollination (14). Quantitative polymerase chain reaction analysis was performed as described in the supplementary materials and methods, with oligonucleotides listed in table S6. Details of histochemical and structural biochemical analyses of ESF1 peptides are fully described in the supplementary materials and methods. Sequence of the codon-optimized synthetic ESF1 gene constructs is listed in table S7.

Supplementary Materials

Materials and Methods

Figs. S1 to S13

Tables S1 to S7

References (3752)

References and Notes

  1. Acknowledgments: We thank H. Prescott, A. Sarmiento, D. Garcia, and E. Caamaño for technical assistance. We also thank T. Laux, W. Lukowitz, I. Moore, B. Scheres, A. Schnittger, M. Bayer, and D. Weijers for seed stocks and valuable suggestions. We acknowledge funding from the Royal Society, ESF/RTD Framework COST action (FA0903), and Biotechnology and Biological Sciences Research Council grants (BB/F008082, BB/L003023/1 and BB/L003023). The work at the University of Minnesota was supported by NSF award IOB-0516811. NMR experiments were partly supported by Nanotechnology Platform Program of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. K.S. and M.T. acknowledge the Minnesota Supercomputing Institute for computational resources and systems support used in microarray analysis. M.T. and K.S. performed microarray experiments and data analysis. E.M validated the array data, generated most transgenic lines, and conducted genetic analyses. L.M.C designed and performed genetic experiments, generated the LhG4 transactivation lines, and performed immunodetection. S.L.O. and R.P. performed the ovule culture experiments. K.B. identified the suspensor-specific At2g30560 gene and generated pGRP-GUS:GFP lines. M.M. expressed ESF1 peptides. M.M., Y.U. and S.O. purified synthetic wild-type and mutant ESF1 peptides and performed solution NMR structural analysis. J.G-M., K. V., S.O., and M.M. conceived and supervised the study. J.G-M. and L.M.C. wrote the paper with input from S.O. All authors have reviewed and approved the paper, and the authors disclose no conflicts of interest.
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