FERONIA Receptor Kinase Controls Seed Size in Arabidopsis thaliana

Dear Editor, Plants have evolved elaborate mechanisms by which cell elongation regulation is coupled to the environmental signals. The plasma membrane receptor kinase FERONIA (FER) has emerged as an important regulatory node in controlling cell elongation and hormone crosstalk (Guo et al., 2009Guo H. Li L. Ye H. Yu X. Algreen A. Yin Y. Three related receptor-like kinases are required for optimal cell elongation in Arabidopsis thaliana.Proc. Natl Acad. Sci. U S A. 2009; 106: 7648-7653Crossref PubMed Scopus (246) Google Scholar; Deslauriers and Larsen, 2010Deslauriers S.D. Larsen P.B FERONIA is a key modulator of brassinosteroid and ethylene responsiveness in Arabidopsis hypocotyls.Mol. Plant. 2010; 3: 626-640Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar; Duan et al., 2010Duan Q. Kita D. Li C. Cheung A.Y. Wu H.M FERONIA receptor-like kinase regulates RHO GTPase signaling of root hair development.Proc. Natl Acad. Sci. U S A. 2010; 107: 17821-17826Crossref PubMed Scopus (399) Google Scholar; Yu et al., 2012Yu F. Qian L. Nibau C. Duan Q. Kita D. Levasseur K. Li X. Lu C. Li H. Hou C. et al.FERONIA receptor kinase pathway suppresses abscisic acid signaling in Arabidopsis by activating ABI2 phosphatase.Proc. Natl Acad. Sci. U S A. 2012; 109: 14693-14698Crossref PubMed Scopus (177) Google Scholar). Initially reported to inhibit pollen tube elongation during fertilization (Escobar-Restrepo et al., 2007Escobar-Restrepo J.M. Huck N. Kessler S. Gagliardini V. Gheyselinck J. Yang W.C. Grossniklaus U. The FERONIA receptor-like kinase mediates male–female interactions during pollen tube reception.Science. 2007; 317: 656-660Crossref PubMed Scopus (479) Google Scholar), FER has been since found to promote cell elongation in leaves and root hairs (Guo et al., 2009Guo H. Li L. Ye H. Yu X. Algreen A. Yin Y. Three related receptor-like kinases are required for optimal cell elongation in Arabidopsis thaliana.Proc. Natl Acad. Sci. U S A. 2009; 106: 7648-7653Crossref PubMed Scopus (246) Google Scholar; Duan et al., 2010Duan Q. Kita D. Li C. Cheung A.Y. Wu H.M FERONIA receptor-like kinase regulates RHO GTPase signaling of root hair development.Proc. Natl Acad. Sci. U S A. 2010; 107: 17821-17826Crossref PubMed Scopus (399) Google Scholar). A GEF–ROP/ARAC signaling network may lead FER to positively regulate auxin-induced cell elongation and negatively regulate ABA response in vegetative tissues (Duan et al., 2010Duan Q. Kita D. Li C. Cheung A.Y. Wu H.M FERONIA receptor-like kinase regulates RHO GTPase signaling of root hair development.Proc. Natl Acad. Sci. U S A. 2010; 107: 17821-17826Crossref PubMed Scopus (399) Google Scholar; Yu et al., 2012Yu F. Qian L. Nibau C. Duan Q. Kita D. Levasseur K. Li X. Lu C. Li H. Hou C. et al.FERONIA receptor kinase pathway suppresses abscisic acid signaling in Arabidopsis by activating ABI2 phosphatase.Proc. Natl Acad. Sci. U S A. 2012; 109: 14693-14698Crossref PubMed Scopus (177) Google Scholar). We report here that FER may also use the GEF–ROP/ARAC signaling network to inhibit cell elongation in seed development and thus control seed size. This provides intriguing diversity of FER functions in controlling cell sizes in different tissues. When conducting the phenotypic analysis of two FER-null mutants (fer-4 and srn; Yu et al., 2012Yu F. Qian L. Nibau C. Duan Q. Kita D. Levasseur K. Li X. Lu C. Li H. Hou C. et al.FERONIA receptor kinase pathway suppresses abscisic acid signaling in Arabidopsis by activating ABI2 phosphatase.Proc. Natl Acad. Sci. U S A. 2012; 109: 14693-14698Crossref PubMed Scopus (177) Google Scholar), we found that both fer-4 and srn mutants exhibited larger seed size compared with their wild-type control (Figure 1A), and larger seeds produced bigger cotyledons as shown in Figure 1B. We further confirmed this phenotypic difference using a quantitative assay using average weight of 100 seeds. Compared with its wild-type seeds, fer-4 was 38% larger and srn was 61.9% larger, respectively (Supplemental Figure 1). We performed reciprocal crosses between fer-4 mutant and wild-type and found that larger seeds resulted from the maternal effect of fer-4 mutation (Supplemental Figure 2). We next carried out a detailed analysis of the fer-4 mutant seeds at different developmental stages by examining cleared ovules under the microscope. We found that, before fertilization, the mutant and wild-type ovules were similar in size and structure (Supplemental Figure 3a and 3f). After fertilization, zygote, endosperm, and integument cells communicate with each other, leading to division of zygote and endosperm, and elongation of integument cells to determine the seed size (Garcia et al., 2005Garcia D. Fitz Gerald J.N. Berger F. Maternal control of integument cell elongation and zygotic control of endosperm growth are coordinated to determine seed size in Arabidopsis.Plant Cell. 2005; 17: 52-60Crossref PubMed Scopus (267) Google Scholar). Before the embryos reach the four-cell stage, the fer-4 mutant and wild-type were largely similar in their ovule size, with fer-4 ovules slightly longer than wild-type (comparing Supplemental Figure 3b with 3g). This difference became more obvious at the globular embryo stage when fer-4 clearly showed a larger seed volume than wild-type, whereas the embryo size has no significant difference (Supplemental Figure 3c and 3h). Following the globular stage, however, the mutant embryo gradually outgrew the wild-type embryo (Figure 1, Figure 1, and Supplemental Figure 3d, 3i, 3e, and 3j). We next examined the possible cause of a larger volume in the mutant seed by dissecting the various cells in the seeds. In order to conduct a detailed analysis of the mutant and wild-type seeds at the same development stage, mutant and wild-type flowers were artificially pollinated at the same time after emasculation. The cleared whole-mount ovules were analyzed 50h after pollination when most of the embryos reached globular stage. As shown in Figure 1C, the mutant ovule was much larger than the wild-type, but the embryo size remained the same. The larger volume was filled by more endosperm cells. We also examined two layers of abaxial integuments in wild-type and fer-4 ovules: oi2, the outer layer of the outer integument; and ii1, a layer of the inner integument that spans part of the embryo sac (Schruff et al., 2006Schruff M.C. Spielman M. Tiwari S. Adams S. Fenby N. Scott R.J The AUXIN RESPONSE FACTOR 2 gene of Arabidopsis links auxin signalling, cell division, and the size of seeds and other organs.Development. 2006; 133: 251-261Crossref PubMed Scopus (449) Google Scholar). We found no difference in cell number between the wild-type and fer-4 (Figure 1Cc). However, a significant difference was scored in the integument cell length between the wild-type and fer-4 ovules (Figure 1Cd). We thus concluded that larger ovules in the fer-4 mutant may result from more endosperm cell division and/or more elongated integument cells. If FER controls the integument cell size or endosperm division, we expected that it should be expressed in these cell types. Using the transgenic plants containing the proFER::FER–GFP construct, we found that FER was expressed in the synergid cell before fertilization (Figure 1, Figure 1 Df), consistently with the earlier observation (Escobar-Restrepo et al., 2007Escobar-Restrepo J.M. Huck N. Kessler S. Gagliardini V. Gheyselinck J. Yang W.C. Grossniklaus U. The FERONIA receptor-like kinase mediates male–female interactions during pollen tube reception.Science. 2007; 317: 656-660Crossref PubMed Scopus (479) Google Scholar). After fertilization and before the globular embryo stage, FER was highly expressed in the elongating integument cells, but no expression was detected in the embryo or endosperm (Figure 1Db, 1 Dc, 1Dg, and 1Dh). We also noted that FER protein was polarly localized in the abaxial integument cells, more abundant in the two ends of elongated cells (Figure 1, Figure 1 Dl). After the globular embryo stage, the expression of FER decreased rapidly and showed little signal after the cotyledon stage (Figure 1Dd, 1 De, 1Di, and 1Dj). This expression pattern aligns well with the function of FER in the control of integument cell elongation at specific stages of seed development. More endosperm cells in the mutant may result from larger space provided by longer integument. Because the loss-of-function fer-4 mutant seeds are larger than the wild-type seeds, we conclude that FER inhibits integument cell elongation and thereby puts constraints on seed size increase. To explore the molecular mechanism of action, we examined whether FER controls seed size by the GEF–ROP/ARAC signaling network that plays a role in controlling root hair elongation in auxin response (Duan et al., 2010Duan Q. Kita D. Li C. Cheung A.Y. Wu H.M FERONIA receptor-like kinase regulates RHO GTPase signaling of root hair development.Proc. Natl Acad. Sci. U S A. 2010; 107: 17821-17826Crossref PubMed Scopus (399) Google Scholar). We analyzed the gef1, gef4, gef10, gef1/4, and gef1/4/10 mutants (Yu et al., 2012Yu F. Qian L. Nibau C. Duan Q. Kita D. Levasseur K. Li X. Lu C. Li H. Hou C. et al.FERONIA receptor kinase pathway suppresses abscisic acid signaling in Arabidopsis by activating ABI2 phosphatase.Proc. Natl Acad. Sci. U S A. 2012; 109: 14693-14698Crossref PubMed Scopus (177) Google Scholar) and found no difference in seed size compared with wild-type. It is possible that functional redundancy among the many GEFs prevented the phenotypic changes in the mutants examined. We thus used the overexpression lines to check the possible relevance of GEF signaling to seed size control. Indeed, the seeds in the 35S–GEF1(GEF1–OX) plants (Duan et al., 2010Duan Q. Kita D. Li C. Cheung A.Y. Wu H.M FERONIA receptor-like kinase regulates RHO GTPase signaling of root hair development.Proc. Natl Acad. Sci. U S A. 2010; 107: 17821-17826Crossref PubMed Scopus (399) Google Scholar) were smaller than the control, and smaller cotyledons were also observed after germination (Figure 1E). As shown in Figure 1F, seeds of GEF1–OX plants were 27.8% smaller than those of wild-type (from 2.77±0.058mg/100 seeds to 2.0±0.058mg/100 seeds, P < 0.01). The cleared whole-mount ovules from GEF1–OX plants began to show obvious size difference after the globular embryo stage (Supplemental Figure 4a–4h), consistently with the observation on fer mutants. Furthermore, using the Pro-GEF1–GUS line (Yu et al., 2010), we found that GEF1 showed high expression especially in integument cells during seed development (around the globular embryo stage) (Figure 1G). Taken together, we conclude that integument cells express FER and GEF1 (and possibly other GEFs), forming a signaling pathway to negatively regulate the elongation of integument cells, leading to control of the seed size. The polarly localized GEF–ROP/ARAC network has an important role in regulating differential cell elongation as demonstrated in other cell types (Yang, 2008Yang Z. Cell polarity signaling in Arabidopsis.Annu. Rev. Cell Dev. Biol. 2008; 24: 551-575Crossref PubMed Scopus (207) Google Scholar; Xu et al., 2010Xu T. Wen M. Nagawa S. Fu Y. Chen J.G. Wu M.J. Perrot-Rechenmann C. Friml J. Jones A.M. Yang Z. Cell surface- and rho GTPase-based auxin signaling controls cellular interdigitation in Arabidopsis.Cell. 2010; 143: 99-110Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar; Cheung and Wu, 2011Cheung A.Y. Wu H.M THESEUS 1, FERONIA and relatives: a family of cell wall-sensing receptor kinases?.Curr. Opin. Plant Biol. 2011; 14: 632-641Crossref PubMed Scopus (116) Google Scholar). It is envisioned that FER, upon external signal recognition, will recruit the GEF1–ROP/ARAC network with time and space specificity and promote or inhibit cell elongation in different cell types. Supplementary Data are available at Molecular Plant Online. This work was supported by grants from the National Science Foundation of China (NSFC-31170229, 31371244), the Key Project of Hunan Education Commission (12A096), the Hunan Provincial Construct Program of the Key Discipline in Ecology (0713), the Cooperative Innovation Center of Engineering and New Products for Developmental Biology of Hunan Province (20134486), and a grant from the National Science Foundation (to S.L.).