TURAN and EVAN Mediate Pollen Tube Reception in Arabidopsis Synergids through Protein Glycosylation

Heike Lindner,Sharon A Kessler,Lena M Müller,Hiroko Shimosato-Asano,Aurélien Boisson-Dernier,Ueli Grossniklaus,Zhenbiao Yang

doi:10.1371/journal.pbio.1002139

Abstract

Pollen tube (PT) reception in flowering plants describes the crosstalk between the male and female gametophytes upon PT arrival at the synergid cells of the ovule. It leads to PT growth arrest, rupture, and sperm cell release, and is thus essential to ensure double fertilization. Here, we describe TURAN (TUN) and EVAN (EVN), two novel members of the PT reception pathway that is mediated by the FERONIA (FER) receptor-like kinase (RLK). Like fer, mutations in these two genes lead to PT overgrowth inside the female gametophyte (FG) without PT rupture. Mapping by next-generation sequencing, cytological analysis of reporter genes, and biochemical assays of glycoproteins in RNAi knockdown mutants revealed both genes to be involved in protein N-glycosylation in the endoplasmic reticulum (ER). TUN encodes a uridine diphosphate (UDP)-glycosyltransferase superfamily protein and EVN a dolichol kinase. In addition to their common role during PT reception in the synergids, both genes have distinct functions in the pollen: whereas EVN is essential for pollen development, TUN is required for PT growth and integrity by affecting the stability of the pollen-specific FER homologs ANXUR1 (ANX1) and ANX2. ANX1- and ANX2-YFP reporters are not expressed in tun pollen grains, but ANX1-YFP is degraded via the ER-associated degradation (ERAD) pathway, likely underlying the anx1/2-like premature PT rupture phenotype of tun mutants. Thus, as in animal sperm–egg interactions, protein glycosylation is essential for the interaction between the female and male gametophytes during PT reception to ensure fertilization and successful reproduction.

Highlights

In flowering plants, male and female gametes are constituents of the male and female gametophytes (FG, embryo sac)
To gain more insight into the molecular mechanisms involved in pollen tube (PT) reception in Arabidopsis, we conducted a forward genetic screen that yielded several mutants showing a fer-like PT overgrowth phenotype
The PT continued to grow inside the FG, failed to arrest its growth, and did not rupture to release the sperm cells (Fig 1A–1C and S1C Fig)

Summary

Introduction

Male and female gametes are constituents of the male (pollen) and female gametophytes (FG, embryo sac). The synergids flank the egg cell at the micropylar end of the FG and secrete LUREs, small defensin-like proteins (DEFLs), which form a subgroup of cysteine-rich polypeptides (CRPs) [4,5,6]. Transduction of these female signals in the PT involves two receptor-like cytoplasmic kinases, LOST IN POLLEN TUBE GUIDANCE1 (LIP1) and LIP2 [7]. After arrival of the PT at the micropyle, it grows beyond the filiform apparatus (FA), a membrane-rich structure at the micropylar pole of the synergids, enters the receptive synergid, and ruptures to release the sperm cells [8]. The first being PT reception at the FA, where PT growth is temporally slowed down or arrested, and the second involving rapid growth towards the PT entry site, PT rupture, and release of the two sperm cells with the concomitant death of the receptive synergid [9]

Methods

Results

Discussion

Conclusion