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. 2017 Jan;173(1):326-337.
doi: 10.1104/pp.16.01219. Epub 2016 Dec 5.

Two SERK Receptor-Like Kinases Interact with EMS1 to Control Anther Cell Fate Determination

Zhiyong Li  1   2 , Yao Wang  1   2 , Jian Huang  1   2 , Nagib Ahsan  1   2 , Gabriel Biener  1   2 , Joel Paprocki  1   2 , Jay J Thelen  1   2 , Valerica Raicu  1   2 , Dazhong Zhao  3   4
Affiliations

Two SERK Receptor-Like Kinases Interact with EMS1 to Control Anther Cell Fate Determination

Zhiyong Li et al. Plant Physiol. 2017 Jan.

Abstract

Cell signaling pathways mediated by leucine-rich repeat receptor-like kinases (LRR-RLKs) are essential for plant growth, development, and defense. The EMS1 (EXCESS MICROSPOROCYTES1) LRR-RLK and its small protein ligand TPD1 (TAPETUM DETERMINANT1) play a fundamental role in somatic and reproductive cell differentiation during early anther development in Arabidopsis (Arabidopsis thaliana). However, it is unclear whether other cell surface molecules serve as coregulators of EMS1. Here, we show that SERK1 (SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1) and SERK2 LRR-RLKs act redundantly as coregulatory and physical partners of EMS1. The SERK1/2 genes function in the same genetic pathway as EMS1 in anther development. Bimolecular fluorescence complementation, Förster resonance energy transfer, and coimmunoprecipitation approaches revealed that SERK1 interacted biochemically with EMS1. Transphosphorylation of EMS1 by SERK1 enhances EMS1 kinase activity. Among 12 in vitro autophosphorylation and transphosphorylation sites identified by tandem mass spectrometry, seven of them were found to be critical for EMS1 autophosphorylation activity. Furthermore, complementation test results suggest that phosphorylation of EMS1 is required for its function in anther development. Collectively, these data provide genetic and biochemical evidence of the interaction and phosphorylation between SERK1/2 and EMS1 in anther development.

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Figures

Figure 1.
Figure 1.
SERK1/2 genetically interacts with EMS1 in anther development. Semithin sections show one anther lobe at stage 5. A, A mature wild-type anther lobe showing epidermis (E), endothecium (En), middle layer (ML), tapetum (T), and microsporocytes (M). B and C, Anther lobes from the ems1-1 strong mutant (B) and the serk1-1 serk2-1 double mutant (C) showing no tapetum but excess microsporocytes. D, The ems1-2 weak mutant anther lobe showing normal tapetum and microspocytes, which is similar to the wild type (A). E and F, Anther lobes from ems1-2 serk1-1 double (E) and ems1-1 serk1-1 serk2-1 triple (F) mutants showing no tapetum but excess microsporocytes, which resemble the ems1-1 single mutant (B) and the serk1-1 serk2-1 double mutant (C). G, The AtML1:TPD1 anther lobe exhibiting six layers of anther wall cells (indicated by red asterisks), which is two layers more than that of the wild type (A). H, The AtML1:TPD1 serk1-1 serk2-1 anther lobe showing a similar structure to that of serk1-1 serk2-1 (C). Bars = 10 µm.
Figure 2.
Figure 2.
BiFC results showing that SERK1 interacts with EMS1 via LRR and kinase domains. A, Schematic diagrams showing EMS1, SERK1, and SERK3 (control) primary structures as well as truncated versions for BiFC assays. SP, Signal peptide; LRRNT, LRR-containing N terminus; GAP, non-LRR gap region between two LRRs; OJM, outer juxtamembrane domain; TM, transmembrane domain; IJM, inner juxtamembrane domain. All constructs contain SP, OJM, TM, and IJM domains. Yellow ovals represent the N-terminal EYFP (nEYFP), and yellow diamonds represent the C-terminal EYFP (cEYFP). The size bar (aa = amino acids) only indicates sizes of EMS1, SERK1, and SERK3 but not nEYFP or cEYFP. B, Confocal image showing that the full-length EMS1 interacts with the full-length SERK1 at the plasma membrane. C and D, Confocal images showing that EMS1 interacts with SERK1 at the plasma membrane via their LRR domains (C) and kinase domains (D). E, Confocal image showing no EMS1-EMS1 interaction. F, Confocal image showing that SERK1 interacts with itself at the plasma membrane. G, No interaction between EMS1 and SERK3 (control). Bars = 10 µm.
Figure 3.
Figure 3.
Typical fluorescence images used to probe interaction between SERK1 and EMS1 using FRET in vivo. FRET assays show fluorescence images of the donor (EMS1-CFP) excited only at the 860-nm wavelength (A) and the acceptor (SERK1-YFP) following excitation at the 860-nm (B) and the 960-nm (C) wavelengths. Signals were obtained from spectral unmixing of images in cells coexpressing EMS1-CFP and SERK1-YFP. Bars = 10 µm.
Figure 4.
Figure 4.
co-IP results showing that SERK1 interacts with EMS1 in planta. A, Schematic diagrams showing construct structures of EMS1:EMS1-4xcMyc and SERK1:SERK1-YFP. B, SERK1 interacts with EMS1 in planta in a co-IP assay. Compared with wild-type, EMS1:EMS1-4xcMyc, and SERK1:SERK1-YFP single transgenic plants, both EMS1-4xcMyc and SERK1-YFP were detected using proteins from young buds of EMS1:EMS1-4xcMyc SERK1:SERK1-YFP double transgenic plants when immunoprecipitated (IP) by the anti-cMyc antibody.
Figure 5.
Figure 5.
In vitro transphosphorylation activities between EMS1 and SERK1/2. A and B, In vitro kinase assays were performed using EMS1-CD, SERK1-CD, and SERK2-CD in the presence of [γ-32P]ATP. Top gels, Input proteins stained with Coomassie Brilliant Blue. Bottom gels, Phosphorylation changes analyzed by autoradiography. EMS1-CDT930A and SERK1-CDK330E are inactive forms of EMS1 and SERK1 kinases, respectively. Consistent results were obtained from three independent repeats. C, Identified in vitro autophosphorylation (in black) and transphosphorylation (in blue) sites in the EMS1-CD via mass spectrometry. S, Ser; T, Thr. D, Relative phosphorylation level changes of specific residues in autophosphorylated and transphosphorylated EMS1-CD. Relative phosphorylation was calculated based on the ratio of spectral counts for total versus phosphorylated peptides identified by mass spectrometry analysis.
Figure 6.
Figure 6.
Effects of mutations in identified Ser and Thr residues on EMS1 autophosphorylation activities. A, Top gels, Coomassie Brilliant Blue staining showing equal amounts of the examined proteins. Bottom gels, Autoradiography showing EMS1 autophosphorylation activities. Consistent results were obtained from three independent repeats. B, Level changes of mutated EMS1 autophosphorylation activities. Relative phosphorylation activity for each mutated EMS1-CD was quantified based on comparison with the native form of EMS1-CD, set as 100%.
Figure 7.
Figure 7.
Functional analysis of EMS1 in vitro phosphorylation sites in planta. A, Schematic diagram showing the EMS1 gene structure and locations of mutated in vitro phosphorylation residues. TM, Transmembrane domain; AB, ATP-binding domain; AL, activation loop. B to D, Representative phenotypes found in T2 transgenic plants in the ems1-1 mutant background: fully fertile wild-type-like plants (B), partially sterile plants (C), and completely sterile plants (D). E to G, Pollen staining results showing viable pollen grains from wild-type-like plants (E), reduced viable pollen grains from partially sterile plants (F), and no pollen from completely sterile plants (G). H, Complementation effects of five mutated EMS1 versions in the ems1-1 mutant background. Values are means of 40 independent lines grown under the same condition.
Figure 8.
Figure 8.
Hypothetical model for the assembly of the active EMS1-SERK1/2 complex at the plasma membrane. Without the ligand TPD1, EMS1 and SERK1 remain as relaxed EMS1-SERK1 heterodimers and SERK1-SERK1 homodimers at the plasma membrane. Two TPD1 molecules consecutively bind to EMS1, inducing the assembly of an active SERK1-EMS1 heterodimer.
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