Three MYB transcription factors control pollen tube differentiation required for sperm release

Abstract

In flowering plants, immotile sperm cells develop within the pollen grain and are delivered to female gametes by a pollen tube. Upon arrival at the female gametophyte, the pollen tube stops growing and releases sperm cells for successful fertilization. Several female signaling components essential for pollen tube reception have been identified; however, male components remain unknown. We show that the expression of three closely related MYB transcription factors is induced in pollen tubes by growth in the pistil. Pollen tubes lacking these three transcriptional regulators fail to stop growing in synergids, specialized cells flanking the egg cell that attract pollen tubes and degenerate upon pollen tube arrival. myb triple-mutant pollen tubes also fail to release their sperm cargo. We define a suite of pollen tube-expressed genes regulated by these critical MYBs and identify transporters, carbohydrate-active enzymes, and small peptides as candidate molecular mediators of pollen tube-female interactions necessary for flowering plant reproduction. Our data indicate that de novo transcription in the pollen tube nucleus during growth in the pistil leads to pollen tube differentiation required for release of sperm cells.

Figure 1. MYB97, MYB101, and MYB120 localize to the growing pollen tube nucleus

(A) Phylogeny of the S18 group of MYBs (bootstrap values at branches). (B) Expression heatmap of S18 MYBs. Pollen development: U, Unicellular; B, Bicellular, T, Tricellular [29]; D, Dry Pollen. Pollen tube: 30′, 30′ in vitro grown pollen; 4h - 4 hour in vitro grown pollen; S, Semi in vivo grown pollen [19]. Ov, Ovary; St, Stigma [32], M, maximum sporophytic expression from 7 tissues; A, Anther [31]. (C, H, M) Hydrated pollen grains of plants hemizygous for MYB:GFP reporter constructs. HTR10:RFP homozygous plants were transformed with MYB101:GFP, qrt1-2 plants were transformed with MYB97:GFP or MYB120:GFP. (D, I, N) Pollen tubes grown for 6 hours in vitro. (E, J, O) DIC and (F, K, P) Epifluorescence images of semi-in vivo grown MYB:GFP pollen. (G, L, Q) Confocal micrographs of semi-in vivo grown MYB:GFP pollen tubes. (R) DAPI staining of MYB101:GFP, HTR10:RFP pollen tubes grown in vitro.

Figure 2. MYB97, MYB101, and MYB120 are required for pollen tube burst, sperm release and synergid degeneration

ms1 pistils were pollinated with wild-type LAT52:GFP or myb triple mutant LAT52:GFP pollen 12 hours prior to dissection and imaging by confocal microscopy. (A) Wild-type GFP+ pollen tubes target the micropyle (m) and burst in the female gametophyte yielding a diffuse green signal (arrow). (B) myb triple mutant GFP+ target ovules, overgrow (arrowhead) and fail to burst, with a contained GFP signal inside the pollen tube (inset). Scale bars, 50μm. (C) Quantitative analysis of pollen tube burst by confocal microscopy. The percentage of ovules receiving a pollen tube with a normal burst or a coiling phenotype is shown as a percentage (+/− sd). P-value <0.005, student’s t-test. (D) Wild-type ACT11:MSI1:GFP ovule with position of micropyle (m) and funiculus (fn) indicated. Inset: sn, synergid nucleus; ecn, egg cell nucleus; ccn, central cell nucleus. (E–G) First panel, GFP and DsRed channels merged; middle panel, DsRed channel; last panel, GFP Channel. (E) Wild-type RFP+ sperm nuclei (arrows) undergo double fertilization. (F,G) myb triple mutant RFP+ sperm nuclei remain condensed and associated (arrowheads). (G) Multiple pairs of unfused sperm (arrowheads) were observed myb triple mutant pollinations. Scale bars, 50μm. (H) Quantitative analysis of double fertilization. 1 UFP, ovules with one pair of unfused sperm; 2 UFP, two pairs of unfused sperm; 1 UFP 1 FP, one unfused pair and one fused pair of sperm; 1 FP, one fused pair of sperm. (I–L) Wild-type ACT11:MSI1:GFP pistils were pollinated with wild-type LAT52:DsRed or myb triple mutant, LAT52:DsRed and allowed to grow for 12h. (I) Wild-type, LAT52:DsRed pollen tubes targeting wild-type, ACT11:MSI1:GFP ovules (dsn, degenerated synergid; sn, persisting synergid nucleus), Inset is GFP channel alone. (J) Coiling myb triple pollen tubes with two intact synergid nuclei, (K) one intact synergid or, (L) two degenerated synergids. Scale bars, 50μm. (M) Quantitative analysis of synergid degeneration. Percent of ovules targeted by pollen tubes with; 2 SN, two persisting synergid nuclei; 1 SN, one persisting synergid nucleus; 0 SN, no persisting synergid nuclei; N, total targeted ovules.

Figure 3. MYB97, MYB101, and MYB120 regulate gene expression during pollen pistil interactions

(A) Expression heat map of genes identified as differentially expressed by microarray analysis. ms1 pistils were left un-pollinated (Un-Poll), or pollinated by either wild-type (W) or myb triple mutant (M) pollen and grown for 8 hours. The genes identified (Locus Identifier) are indicated with the gene name or predicted function (Gene Title). †, Array ID identifies two. The fold change (FC) between wild-type and myb triple mutant pollinations is shown, with asterisks (*) indicating fold changes that were statistically significant (P<0.05). Previously published data from pollen grain development [29], pollen tube growth [19], sperm cells [30], and sporophytic tissues [31] are included for comparison. Pollen development: U, Unicellular; B, Bicellular; T, Tricellular; D, Dry Pollen. Pollen tube: 0.5, 30′ in vitro grown pollen; 4, 4 hour in vitro grown pollen; S, Semi in vivo grown pollen tubes; Sperm, Isolated sperm cells, M - Max sporophytic expression from 7 tissue types.