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. 2015 Apr 28;13(4):e1002139.
doi: 10.1371/journal.pbio.1002139. eCollection 2015 Apr.

TURAN and EVAN mediate pollen tube reception in Arabidopsis Synergids through protein glycosylation

Heike Lindner  1 , Sharon A Kessler  1 , Lena M Müller  1 , Hiroko Shimosato-Asano  1 , Aurélien Boisson-Dernier  1 , Ueli Grossniklaus  1
Affiliations

TURAN and EVAN mediate pollen tube reception in Arabidopsis Synergids through protein glycosylation

Heike Lindner et al. PLoS Biol. .

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. tun and evn ovules display pollen tube overgrowth and increased callose accumulation at the filiform apparatus.
(A–C) Aniline Blue staining of callose in PT cell walls 2 d after pollination (DAP). (A) PT reception in a wild-type FG. Arrowhead indicates site of PT growth arrest. (B–C) PT overgrowth in tun-1 (B) and evn-1 mutant FGs (C). Asterisks indicate PT overgrowth. (D–F) β-glucuronidase (GUS) staining of synergid marker ET2634 2 d after emasculation (DAE) in wild-type (D), tun-1 (E), and evn-1 mutant FGs (F). Arrow indicates abnormal structure at the FA. (G–I) Chloral hydrate clearings of ovules 2 DAE in wild-type (G), tun-1 (H), and evn-1 mutants (I). Arrows indicate abnormal structure at the FA. (J–L) Aniline Blue staining of callose in 6 μm sections of wild-type (J), tun-1 (K), and evn-1 ovules 2 DAE (L). Boxes represent close-ups of indicated regions, whereby mutant close-ups in (K) and (L) were captured with reduced exposure time compared to the wild type (J). Scale bars in A–F and J–L = 20 μm; scale bars in G–I = 10 μm.
Fig 2
Fig 2. Distinct pollen defects in tun and evn mutants.
(A) Pollen in vitro germination assay of qrt/qrt pollen grains. (B) Pollen in vitro germination assay of tun-1/TUN;qrt/qrt pollen. Arrows indicate PT bursting. (C) Graph of PT bursting counts in qrt/qrt and tun-1/TUN;qrt/qrt pollen. (D) Pollen in vitro germination assay of evn-1/EVN;qrt/qrt mutant pollen. Arrowheads indicate degenerated pollen grains. (E) Graph of degenerating pollen grain counts at different stages of evn-1/EVN mutants after DAPI staining. Stage four refers to the bicellular and early tricellular, stage three to the tricellular, stage two to the late tricellular and early mature, and stage one to the mature pollen stage. Scale bars: 20 μm. (F–G) Gene model of TUN (F) and EVN (G) with mutant alleles. Ethane methyl sulfonate (EMS) single nucleotide polymorphisms (SNPs) are indicated by lines, T-DNA insertions by triangles.
Fig 3
Fig 3. ConA reveals altered glycoprotein patterns in TUN(RNAi) and EVN(RNAi) seedlings.
Lectin blot using ConA of a wild-type, three independent TUN(RNAi) lines, two independent EVN(RNAi) lines, and two ost3/6-2 control plants. Arrowheads indicate wild-type bands with differential abundance and/or mobility in knockdown lines, marked by asterisks. 55kDa fraction represents a Commassie-Brilliant Blue stained loading control.
Fig 4
Fig 4. TUN(RNAi) lines show fer-like vegetative dwarf phenotype.
(A–D) Plant size of 30-d-old seedlings of wild-type (A), fer-2/fer-2 (B), TUN(RNAi) (C), and EVN(RNAi) lines (D). Asterisks indicate TUN(RNAi) seedlings, that accumulate athocyanins and degenerate without further growth. (E–H) Plant size of adult wild-type (E), fer-2/fer-2 (F), TUN(RNAi) (G), and EVN(RNAi) (H) plants. (F) Left plant is at the same developmental stage as wild-type, TUN(RNAi), and EVN(RNAi) individuals. Scale bars: 1 cm. All lines are in the Col-0 background.
Fig 5
Fig 5. NTA, FER and LRE show proper localization in tun and evn mutant embryo sacs.
(A–I) Confocal microscope analysis of fluorescently labeled proteins. (A–C) Vesicle-associated NTA-GFP localization in the cytoplasm of a wild-type (A), tun-2 (B), and evn-3 FG (C). (D–F) FER-GFP at the FA and in membranes of sporophytic tissue of a wild-type (D), tun-2 (E), and evn-3 FG (F). (G–I) Extracellular localization of LRE-Citrine in a wild-type (G), tun-2 (H), and evn-3 FG (I). Scale bars: 20 μm.
Fig 6
Fig 6. ANX1-YFP fluorescence is not detectable in tun mutant pollen grains.
(A–D) Confocal microscope analysis of ANX1-YFP expression under a pollen-specific promoter. (A) ANX1-YFP expression in TUN/TUN;qrt/qrt;ANX1-YFP/ANX1-YFP (wild-type segregants homozygous for the reporter gene). (B) ANX1-YFP expression in TUN/TUN;qrt/qrt;ANX1-YFP/- (wild-type segregants hemizygous for the reporter gene). (C) ANX1-YFP expression in tun-2/TUN;qrt/qrt;ANX1-YFP/ANX1-YFP mutant tetrads. Arrowheads indicate missing fluorescence in tun pollen grains. (D) ANX1-YFP expression in tun-2/TUN;qrt/qrt;ANX1-YFP/ANX1-YFP mutant tetrads after Kifunensine (Kif) treatment. (E) ANX1-YFP expression in tun-2/TUN;qrt/qrt;ANX1-YFP/ANX1-YFP mutant tetrads after mock treatment for fluorescence intensity decrease comparison. (F–H) ANX1-YFP expression in tun-2/TUN;qrt/qrt;ANX1-YFP/ANX1-YFP mutant tetrads after Eeyarestatin I (EerI) treatment. (F) 10 μm EerI recovers ANX1-YFP fluorescence in tun pollen grains. (G) Higher concentrations of EerI can lead to cytosolic inclusions (asterisk) or pollen grain burst (arrow). (H) ANX1-YFP fluorescence recovery in several tun-2/TUN;qrt/qrt;ANX1-YFP/ANX1-YFP mutant tetrads after EerI treatment. Residual fluorescence signal from the pollen coat is autofluorescence. Scale bars: 20 μm.
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