Signaling of cell fate determination by the TPD1 small protein and EMS1 receptor kinase

Abstract

Sexual reproduction requires the specification of cells with distinct fates in plants and animals. The EMS1 (also known as EXS) leucine-rich repeat receptor-like kinase (LRR-RLK) and TPD1 small protein play key roles in regulating somatic and reproductive cell fate determination in Arabidopsis anthers. Here, we show that ectopic expression of TPD1 causes abnormal differentiation of somatic and reproductive cells in anthers. In addition, ectopic TPD1 activity requires functional EMS1. Yeast two-hybrid, pull-down, and coimmunoprecipitation analyses further demonstrate that TPD1 interacts with EMS1 in vitro and in vivo. Moreover, TPD1 induces EMS1 phosphorylation in planta. Thus, our results suggest that TPD1 serves as a ligand for the EMS1 receptor kinase to signal cell fate determination during plant sexual reproduction.

Fig. 1.

Ectopic expression of TPD1 causes abnormal anther cell differentiation, and TPD1 signaling requires EMS1. Semithin sections show one lobe from anthers. (Scale bars: 20 μm.) B, D, F–H, and K; C, E, I, and L have the same magnification. (A) Diagram of a mature wild-type anther shows epidermis (E), endothecium (En), middle layer (ML), tapetum (T), and microsporocytes (M). (B) A wild-type anther at stage 5. Enclosed by red dotted line are microsporocytes. (C) A wild-type anther at stage 6 shows strongly stained tapetal layer and isolated microsporocytes. (D) An ems1 anther at stage 5 lacks the tapetum layer but has excess microsporocytes (enclosed by red dotted line). (E) An ems1 anther at stage 6 lacks the tapetum layer. Microsporocytes are abnormally enlarged and not isolated. (F) A tpd1 anther at stage 5 has the same phenotype as that of ems1 (D). (G) An ems1 tpd1 double-mutant anther at stage 5 shows the same phenotypes as those of ems1 (D) and tpd1 (F). (H–J) Anthers of CaMV35S::TPD1 transgenic plants. (H) A stage-5 anther shows vacuolated cells in place of a normal tapetum (tapetum-positioned cells, Tpc) and degenerating cells instead of normal microsporocytes (microsporocyte-positioned cells, Mpc). (I) A stage-6 anther exhibits completely vacuolated Tpc and degenerating Mpc. (J) An anther after stage 6 shows the mix of completely vacuolated Tpc and Mpc. (K and L) Anthers from CaMV35S::TPD1 ems1 plants at stage 5 (K) and 6 (L) exhibit identical phenotypes to ems1 (D and E).

Fig. 2.

TPD1 interacts with EMS1 in yeast two-hybrid assays. (A) Diagram shows constructs for mapping the TPD1 interacting region (TIR) of EMS1. Bar with green color represents the TIR. (B) Yeast grown on synthetic complete (SC) medium without Leu, Trp, and His. (1) Positive control. (2) Negative control. (3) The entire EMS1 LRR domain and TPD1. (4) The entire EMS1 LRR domain. (5) TPD1 alone. (6) TIR and TPD1. (7) TIR alone. (8) Non-TIR and TPD1. (C) Verification of the interaction by filter lift assay showing blue color of yeast. (D) The amino acid sequence of TIR. The consensus LRR sequence is shown at the top.

Fig. 3.

In vitro and in vivo interaction between TPD1 and EMS1 and TPD1 induces the phosphorylation of EMS1. (A) TPD1 interacts with EMS1 in GST pull-down assay. Three micrograms of GST and GST fusion proteins were used to pull down the same amount of crude protein (200 μg) extracts containing HA-TPD1, respectively. (B and C) TPD1 interacts with EMS1 in planta in coimmunoprecipitation assay. (B) EMS1-cMyc (Upper) and TPD1-FLAG (Lower) were detected in EMS1::EMS1-cMyc TPD1::TPD1-FLAG double-transgenic plants, respectively, by Western blot. (1) Wild type. (2) EMS1::EMS1-cMyc TPD1::TPD1-FLAG double-transgenic plant. (C) TPD1-FLAG was detected when proteins from EMS1::EMS1-cMyc TPD1::TPD1-FLAG double-transgenic plants were immunoprecipitated with an anti-cMyc antibody (Upper). EMS1-cMyc was also detected when the same proteins were immunoprecipitated with an anti-FLAG antibody (Lower). (1) TPD1::TPD1-FLAG (Upper) and EMS1::EMS1-cMyc (Lower) single-transgenic plants. (2) EMS1::EMS1-cMyc TPD1::TPD1-FLAG double-transgenic plant. (D) TPD1 binding induces EMS1 phosphorylation. Lanes 1, 3, 4, and 6: EMS1::EMS1-cMyc transgenic plants; lanes 2 and 5: EMS1::EMS1-cMyc tpd1 transgenic plants, in which TPD1 is not present. Proteins in lanes 4 and 5 were treated with calf intestinal alkaline phosphatase (CIP). EMS1-cMyc protein from EMS1::EMS1-cMyc tpd1 plants (lane 2) migrated faster than those from EMS1::EMS1-cMyc plants (lanes 1 and 3). A shift in mobility of EMS1-cMyc did not occur (lane 4) after the CIP treatment (lanes 5 and 6 are controls).

Fig. 4.

A model for EMS1-TPD1 signaling in anther cell fate determination. In the wild-type anther, TDP1 small protein is secreted from microsporocytes or their precursors and then bind to EMS1 receptor kinases that are localized to tapetal precursors. EMS1-TPD1 signaling ensures specification of tapetal cell fate by activating the downstream signaling cascade. In the absence of TPD1 ligand or EMS1 receptor in the tpd1 or ems1 mutant, signals directing tapetum differentiation are blocked. Consequently, tapetal precursors adopt a microsporocyte fate, resulting in the formation of excess microsporocytes.