Figure 1.
A phylogenetic tree of paralogous DEFL genes of A. thaliana and a close relative A. lyrata.
The tree includes 13 subtrees, which belong to 13 CRP subgroups. Each subtree contains four or more paralogous DEFL genes of A. thaliana (blue filled circle) and their orthologs from A. lyrata (red filled circle). The scale shows the number of substitutions per site. The CRP90 subtree contains AtPDF1 genes [11]. The CRP700 subtree contains A. thaliana trypsin inhibitor (ATTI) genes [75]. The CRP580 and CRP860 subtrees contain genes that form the largest gene cluster of LCR (low molecular weight, cysteine-rich) and SCRL (SCR-like), respectively [76]. The CRP810 subtree branched into A. thaliana and A. lyrata gene clusters. Also see Table S1 for sequences of these DEFL genes.
Figure 2.
Phylogenetic and syntenic relationship between the CRP810_1 genes of A. thaliana and A. lyrata.
(A) Phylogenetic tree of the CRP810_1 (blue) and AlCRP810_1 (red) genes based on the coding region of their genomic sequences, with At4g08869 and At4g08875 (the closest related genes to CRP810_1 genes) as the outgroup. Only bootstrap values ≥90 are indicated. The scale shows the number of substitutions per site. (B) Representations of syntenic regions containing CRP810_1 and AlCRP810_1 genes in the A. thaliana (Col-0) and A. lyrata genomes. Blue and red arrows represent CRP810_1 and AlCRP810_1 genes, respectively. Gray arrows show flanking genes. Dashed lines indicate syntenic genes between A. thaliana and A. lyrata. The genes with asterisks in (A and B) are probably nonfunctional. Also see Figure S1. (C) Full-length amino acid sequences of CRP810_1.1 to _1.5 and CRP810_1.6 (Ψ). The arrow indicates the position of the predicted cleavage sites. Asterisks mark conserved cysteine residues. Also see Figure S2 for comparison with AlCRP810_1.
Figure 3.
Expression pattern and localization of CRP810_1 peptides.
(A) Real-time qRT-PCR analysis of CRP810_1 genes (CRP810_1.1 to _1.6) in seedlings 10 d after germination (Sd), stems (St), leaves (Le), open flowers without the pistil (Fl), pistils (Pi), and siliques 2 d after pollination (Si). Relative quantities are expression levels relative to that in the pistil. Each expression level was normalized to that of ACT2. The data are the means and standard errors of three independent samples. (B) Absolute gene expression levels of CRP810_1 genes. Absolute quantity represents the copy number of cDNA per that of MYB98 cDNA. The means and standard deviations of three independent experiments are shown. (C) Schematic of the ovule (left) and part of the synergid cell (sy) (right) in A. thaliana. The filiform apparatus (fa) is formed by the thickened cell walls of the synergid cells. f, funiculus; mp, micropyle. (D) Confocal laser scanning microscopic (CLSM) image of a pCRP810_1.2::GFP ovule. Scale bar, 20 µm. (E) Fluorescence microscopic images of pCRP810_1::CRP810_1-GFP ovules. Scale bar, 20 µm. (F) A CLSM image of an ovule after immunostaining with anti-CRP810_1.2 antibodies. Green Alexa Fluor fluorescence (for CRP810_1 peptides) was observed at the micropyle and funicular surface. Magenta indicates autofluorescence of the ovule. The synergid cell is delineated by the dashed line. Scale bar, 20 µm. Also see Figure S3. (G) Immunostaining with anti-CRP810_1.2 antibodies for the female gametophytic mutants myb98/myb98, myb98/MYB98, ccg/CCG, and maa3/MAA3. Representative fluorescence microscopic images of ovules on the septum are shown. Arrows indicate fluorescence around the micropylar end of the ovules. Scale bars, 50 µm. (H) The rate of immunostained ovules to the total number (n) of ovules in the wild type and mutants.
Figure 4.
Knockdown analyses of the defects in micropylar guidance.
(A) Representative fluorescence microscopic image of immunostained ovules on the septum with anti-CRP810_1.2 antibodies for the vector control and RNAi line. Arrows indicate fluorescence around the micropylar end of the ovules. Scale bar, 50 µm. (B–E) Aniline blue staining for pollen tubes inside the pistil. Asterisks indicate the micropylar opening. The ovule is delineated by the dashed line. f, funiculus. (B) The pollen tube was attracted normally to the wild-type ovule. (C) The pollen tube went past the micropylar opening and then turned back and grew into the micropyle. (D) The pollen tube growing on the funiculus (left side) went back down (right side) near the micropylar opening and then grew on the funiculus again (center) and reached the micropyle. (E) The wandering pollen tube failed to grow into the micropyle. Scale bar, 50 µm for (B–E). (F) Summary of abnormal pollen tube guidance around the micropylar opening in wild-type and transgenic ovules. Sums of class I and class II abnormalities in pollen tube guidance are shown. The data are averages and standard deviations of the frequencies per pistil. The total numbers of counted ovules are 339, 230, 183, 389, 135, and 173 for wild-type, vector control #1, #2, RNAi #1, #2, and #3 ovules, respectively. The frequencies of RNAi lines (#1–3) are significantly different from the wild type (asterisks, p<0.01). (G and H) In vitro comparative pollen tube attraction assay. (G) An arrow indicates the tip of the pollen tube that was initially between the wild-type and RNAi ovules (upper panel). The pollen tube was preferentially attracted to the wild-type ovule (lower panel). An arrowhead indicates the pollen tube entering the wild-type micropyle. (H) Summary of attraction frequencies in this assay. The attraction frequencies for the wild-type ovule (black boxes) or competing ovules (gray boxes) are represented collaterally. The numbers (n) are the total number of pollen tubes competitively attracted to the ovules. Asterisks indicate significant differences from a 1∶1 ratio in the binomial test (p<0.01). Also see Figure S4.
Figure 5.
In vitro pollen tube attraction assay using recombinant proteins.
(A) Pollen tube attraction toward gelatin beads containing 50 µM histidine-tagged CRP810_1.2. Arrowheads mark the position of the pollen tube tips when the gelatin beads (asterisk) were placed (0 min). Arrows indicate the tips of pollen tubes growing toward the beads 30 and 60 min after placement. At 60 min, the upper pollen tube was spontaneously disrupted and the lower pollen tube was trapped at the bead. Scale bar, 20 µm. (B) Concentration-dependent pollen tube attraction activity of CRP810_1.2 (AtLURE1.2). The data are the frequencies for the total number of pollen tubes (n) in at least three assays. Pollen tubes growing toward beads with a >30° change were designated as attracted pollen tubes. (C) Representative samples of attracted or non-attracted pollen tubes to recombinant TfLURE1 and CRP810_1. Arrowheads mark the position of the pollen tube tip when the gelatin beads (asterisk) were placed. Arrows indicate the tips of the pollen tubes. Scale bar, 20 µm. (D) Summary of the rates of attraction of the pollen tubes to each recombinant protein. The data are the frequencies for the total number of pollen tubes (n) in at least three assays per protein. An asterisk and double asterisks indicate significant differences compared with buffer alone (0 M) (Figure 5B) using Fisher exact test (*p<0.03; **p<0.01). Also see Figure S5.
Figure 6.
Pollen tube attraction of A. lyrata and species preferentiality of AtLURE1 and AlLURE1 peptides.
(A) A representative example of an attracted pollen tube of A. lyrata (Al PT) to recombinant AlCRP810_1.3 (AlLURE1.3). Arrowheads mark the position of the pollen tube tip when a gelatin bead (asterisk) was placed. An arrow indicates the tip of the pollen tube. Scale bar, 20 µm. (B) Attraction activity of AtLURE1.2 (5 µM) and AlLURE1.3 (50 µM) to A. thaliana and A. lyrata pollen tubes. The data represent the frequencies of the total number of attracted pollen tubes (n) in at least three assays. An asterisk indicates a significant difference according to Fisher exact test (*p = 0.033). Note that the effective rate for each recombinant peptide is unknown because of differing efficiencies in peptide refolding.
Figure 7.
Interspecific pollen tube attraction assay using transgenic T. fournieri ovules and A. thaliana pollen tubes.
(A) Schematic of the sequences introduced into T. fournieri. The TfLURE2 promoter (pTfLURE2) was used to express both AtLURE1.2 and GFP genes in synergid cells. The sequence of the AtLURE1.2 gene is a genomic sequence from the translation initiation codon to the putative 3′ untranslated region. Gray boxes indicate nopaline synthase terminators. (B) GFP expression in two synergid cells of the transgenic T. fournieri ovule. Scale bar, 50 µm. (C) Immunostaining of transgenic T. fournieri ovules with anti-AtLURE1.2 (CRP810_1.2) antibodies. Red Alexa Fluor fluorescence for AtLURE1.2 (arrow) was observed at the micropylar surface of the embryo sac of the ovule labeled with GFP (left panel). In the ovule without GFP labeling (right panel), a weaker pseudo-signal was observed at the micropylar surface of the embryo sac. Scale bars, 50 µm. (D) An overview of the pollen tube attraction assay. Pollen tubes of A. thaliana (At PTs) are observed emerging from a cut style and growing across the medium. A manipulated T. fournieri ovule (Tf OV) was placed near the out-growing pollen tubes. Scale bar, 200 µm. (E) Pollen tube attraction toward the synergid cell of a transgenic T. fournieri ovule. Arrowheads indicate the position of the tip of the A. thaliana pollen tube. At 0 min, an ovule was placed in front of the tip of the pollen tube using a glass needle. The ovule expressed GFP in the synergid cell, indicating co-expression of the AtLURE1.2 peptide. Green fluorescence in the pollen tube indicates pollen-expressed pLAT52::GFP. The pollen tube was attracted to the ovule (20 min). After shifting the position of the transgenic ovule (22 min), the pollen tube redirected itself once again toward the micropyle (80 min). The arrow indicates the ovular micropyle penetrated by the attracted pollen tube. Scale bar, 50 µm. (F) A confocal laser scanning microscopic (CLSM) image of the transgenic T. fournieri ovule shown in (E), demonstrating that the tip of the A. thaliana pollen tube has fully penetrated the transgenic embryo sac (arrowhead). The pollen tube within the embryo sac is delineated by a dashed line. Note that discharge of sperm cells (arrows, sperm nuclei labeled with pHTR10::HTR10:mRFP) did not occur. (G) A projection of a 3-D reconstruction from CLSM images of cytosolic GFP-expressing synergid cells in the transgenic T. fournieri ovule and an attracted A. thaliana pollen tube (pLAT52::GFP×pHTR10::HTR10:mRFP). The arrowhead, double arrowheads, and arrow indicate the tip of the pollen tube, two synergid cells, and sperm nuclei, respectively. The projection is an oblique perspective viewed from the micropylar end of the embryo sac. The pollen tube entered the embryo sac at the filiform apparatus, which is surrounded by two synergid cells and an egg cell (left third part lacking GFP staining, adjacent to the two synergid cells).