Cysteine-rich peptides promote interspecific genetic isolation in Arabidopsis

Abstract

Reproductive isolation is a prerequisite for speciation. Failure of communication between female tissues of the pistil and paternal pollen tubes imposes hybridization barriers in flowering plants. Arabidopsis thaliana LURE1 (AtLURE1) peptides and their male receptor PRK6 aid attraction of the growing pollen tube to the ovule. Here, we report that the knockout of the entire AtLURE1 gene family did not affect fertility, indicating that AtLURE1-PRK6-mediated signaling is not required for successful fertilization within one Arabidopsis species. AtLURE1s instead function as pollen tube emergence accelerators that favor conspecific pollen over pollen from other species and thus promote reproductive isolation. We also identified maternal peptides XIUQIU1 to -4, which attract pollen tubes regardless of species. Cooperation between ovule attraction and pollen tube growth acceleration favors conspecific fertilization and promotes reproductive isolation.

Fig. 1.. AtLURE1.7 and −1.8 are newly identified members of the AtLURE1 family.

(A) Phylogenetic tree of A. thaliana AtLURE1.1 to AtLURE1.8 (in blue, except for AtLURE1.6) and their A. lyrata orthologs (in black) on the basis of protein sequences. The closely related At5g18403 was used as an outgroup. The scale bar indicates the average number of amino acid substitutions per site. (B) GUS signals were predominantly concentrated in synergid cells. (C) Images of ovules with both AtLURE1.7–green fluorescent protein (GFP) and AtLURE1.8-GFP located in the filiform apparatus of synergid cells. Scale bars [(B) and (C)], 50 μm. (D to F) Semi–in vivo pollen tube attraction assay tracing guided pollen tube growth toward recombinant AtLURE1.2, AtLURE1.7, and AtLURE1.8 embedded in gelatin beads (indicated by asterisks). (D) A. thaliana WT pollen tubes; (E) A. lyrata pollen tubes; (F) prk6-2 pollen tubes. Arrowheads indicate the direction of pollen tube growth. Asterisks indicate the center of the beads. Scale bars, 25 μm. (G) Statistical analysis of the pollen tube responding ratio shown in (D) to (F). Three repeats of the pollen tube attraction assays with 9 to 20 pollen tubes each were conducted for each assay. Data are mean values ± SD. ***P < 0.01 (Students’s t test).

Fig. 2.. atlure1 null and prk6-2 mutants show a full seed set.

(A) Schematic diagram of AtLURE1 protein structures containing six conserved cysteine residues (C’s). CRISPR-Cas9–edited mutant structures (m) for each AtLURE1 peptide in the atlure1 null mutant are shown below the WT protein structure (s). AtLURE1.6 is a pseudogene because of a premature stop codon. SP, signal peptides. Gray boxes indicate missense sequences due to frameshift mutations. (B) Western blots of AtLURE1.2 (designated AtLURE1.2e) and mutated AtLURE1 peptides with His tags (designated AtLURE1me) expressed in E. coli. AtLURE1.2PM represents E. coli–expressed mutated AtLURE1.2 with its C-terminal three conserved cysteines (C⁷⁵, C⁸², and C⁸⁴) mutated to alanines (A⁷⁵, A⁸², and A⁸⁴, respectively). (C) Statistical analysis of the pollen tube (PT) responding ratio with E. coli–expressed peptides. AtLURE1.2PM served as a negative control, and buffer 1 (tris-HCl, pH 8.0) as an empty control. Three repeats of the pollen tube attraction assays with 8 to 20 pollen tubes each for each expressed peptide were conducted. Six repeats of the pollen tube attraction assays with three to five pollen tubes each were conducted for positive, negative, and empty controls. Data are mean values ± SD. (D) Statistical analysis of the pollen tube responding ratio with chemically synthesized mutated peptides (designated AtLURE1ms). Pollen germination medium (buffer 2) served as the empty control. Three repeats of the pollen tube attraction assays with 8 to 20 pollen tubes each for each synthesized peptide were conducted. Six repeats of the pollen tube attraction assays with three to five pollen tubes each were conducted for controls. Data are mean values ± SD; P values are >0.05 (Student’s t test). (E) Dissected siliques of WT, atlure1 null, and prk6-2 mutant plants, as well as products of a cross between atlure1 null and prk6-2 plants. Scale bars, 1 mm. (F) Statistical analysis of seed set in WT, atlure1 null, and prk6-2 self-fertilized siliques and siliques from a cross of atlure1 null plants with prk6-2 plants. Three or four repeats of silique examination were conducted, and 8 to 18 siliques were examined each time. Data are mean values ± SD; P values are >0.05 (Student’s t test).

Fig. 3.. Pollen tube emergence onto the septa and ovules of atlure1 null mutants is strongly delayed compared with emergence in A. thaliana WT pistils.

(A to D) Aniline blue staining and statistical analysis of A. thaliana WT and prk6-2 mutant pollen tube (PT) emergence onto the septum and ovule in WT and atlure1 null mutant pistils as indicated, (A and B) 4.5 HAP and (C and D) 8 HAP. Three to five repeats of the pollen tube targeting experiments were conducted for each combination, each with 6 to 12 siliques being examined. White asterisks indicate ovules targeted by pollen tubes. Data are mean values ± SD. ***P < 0.01 (Student’s t test). Scale bars, 50 μm. (E) GUS staining of WT and atlure1 null mutant pistils pollinated with either a mixture of WT and LAT52pro:GUS (WT) pollen grains or a mixture of LAT52pro:GUS (WT) and prk6-2 mutant pollen grains, respectively. Asterisks indicate ovules that are targeted by pollen tubes without GUS staining. Scale bars, 50 μm. (F) Statistical analysis of (E). Three repeats of the pollen tube competition experiments were conducted for each combination, each with seven or eight siliques being examined. Data are mean values ± SD. ***P < 0.01 (Student’s t test). (G) Genetic analysis of male (M) transmission efficiency (TE) of the prk6-2 allele in WT, atlure1.1 to −1.5 quintuple mutants, and atlure1 null septuple mutants.

Fig. 4.. Ovule targeting by pollen tubes after interspecific pollination between A. thaliana and A. lyrata is strongly delayed, resembling the atlure1 null mutant phenotype.

(A to D) Aniline blue staining and statistical analysis of the total numbers of A. lyrata pollen tubes (PT) emerging onto the septum and ovule in A. thaliana WT and atlure1 null mutant pistils as indicated, (A and B) 4.5 HAP and (C and D) 8 HAP. Three or four repeats of the pollen tube targeting experiments were conducted for each combination, each with 7 to 11 siliques being examined. White asterisks indicate ovules targeted by pollen tubes. Data are mean values ± SD; P values were >0.05 (Student’s t test). Scale bars, 50 μm. (E) GUS staining of A. thaliana WT and atlure1 null mutant pistils pollinated with a mixture of A. lyrata and LAT52pro:GUS (A. thaliana WT) pollen grains at 24 HAP. Asterisks indicate ovules that were targeted by pollen tubes without GUS staining. Scale bars, 50 μm. (F) Statistical analysis of the percentage of ovules targeted by A. lyrata pollen tubes per silique shown in (E). Three repeats of the pollen tube competition experiments were conducted for each combination, each with seven or eight siliques being examined. Data are mean values ± SD. ***P < 0.01 (Student’s t test). (G) Siliques of the WT or atlure1 null mutant pistils pollinated with a mixture of A. lyrata and A. thaliana WT pollen grains. Asterisks indicate aborted seeds, and arrowheads indicate white unfertilized ovules, both of which are counted as aborted seeds. Scale bars, 50 μm. (H) Statistical analysis of aborted seeds in the siliques shown in (G). Seven to nine siliques were examined for each line for three repeats. Data are mean values ± SD. ***P < 0.01 (Student’s t test). (I) Pollen tube targeting in vivo at 36 HAP in A. thaliana WT and atlure1 null septuple-mutant pistils crossed with A. lyrata pollen. Scale bars, 200 μm. (J) Statistical analysis of the A. lyrata pollen tube targeting ratio shown in (I). Three repeats of the pollen tube targeting experiments were conducted for each combination, each with seven or eight siliques being examined. Data are mean values ± SD. The P value was >0.05 (Student’s t test).

Fig. 5.. XIUQIU peptides attract pollen tubes of A. thaliana and A. lyrata in similar manners and contribute to fertility.

(A) Expression levels of XIUQIU genes and related genes in WT and myb98 mature ovules. RNA-seq data are shown. RPKM, reads per kilobase per million reads. (B) Quantitative reverse transcription polymerase chain reaction analysis of XIUQIU genes and related genes in WT and myb98 mature ovules. Three biological replicates, each with three technical repeats, were conducted for each gene in each sample. Data are mean values ± SD. (C) GUS signals from XIUQIU1, −2, −3, and −4 promoters were predominantly concentrated in synergid cells. Scale bars, 50 μm. (D) Images of ovules with XIUQIU1-, −2–, −3–, and −4–GFP fusion proteins localized in the filiform apparatus of synergid cells. Scale bars, 50 μm. (E to G) Semi–in vivo pollen tube attraction assays revealed guided growth of A. thaliana (E), A. lyrata (F), and prk6-2 (G) pollen tubes toward recombinant XIUQIU1, −2, and −4 embedded in gelatin beads (indicated by asterisks). Arrowheads indicate pollen tube tips. Scale bars, 50 μm. (H) Statistics of the ratio of attraction by XIUQIU1 to −4, comparing A. thaliana, A. lyrata, and prk6-2 pollen tubes. For each assay, 8 to 24 pollen tubes were examined, and the assay was repeated three times for each condition. Data are mean values ± SD. The P value was >0.05 (Student’s t test).

Fig. 6.. Mutant generation and phenotypic analysis of hendecuple mutants.

(A) Schematic diagram of protein structures of mutated (m) XIUQIU1 to −4 in hendecuple-1 and hendecuple-2 mutants. SP, signal peptide; C’s, conserved cysteine residues; gray box, missense sequence due to frameshift mutation. (B) Statistical analysis of the pollen tube responding ratio with chemically synthesized mutated peptides (designated XIUQIUms). XIUQIU1PM represents E. coli–expressed mutated XIUQIU1 with its C-terminal three conserved cysteines (C⁸⁵, C⁹⁰, and C⁹²) mutated to alanines (A⁸⁵, A⁹⁰, and A⁹², respectively) to serve as a negative control. Pollen germination medium (buffer) served as the empty control. Three repeats of the pollen tube attraction assays with 6 to 10 pollen tubes for each synthesized peptide were conducted. Six repeats of the pollen tube attraction assays with three to five pollen tubes each were conducted for positive, negative, and empty (buffer) controls. Data are mean values ± SD; P values were >0.05 (Student’s t test). AtLURE1.2s, unmutated AtLURE1.2 peptide. (C) Dissected siliques of WT, hendecuple-1, hendecuple-2, and XIUQIU1-rescued hendecuple-1 plants. Asterisks indicate the aborted ovules. Scale bars, 1 mm. (D) Statistical analysis of seed set in WT, hendecuple-1, hendecuple-2, and XIUQIU1-rescued hendecuple-1 plants. Three repeats of silique examination were conducted, and seven to nine siliques were examined each time. Data are mean values ± SD. ***P < 0.01 (Student’s t test). (E) Aniline blue staining showing pollen tube behavior at WT and aborted ovules in an unsuccessfully targeted hendecuple-1 mutant at 48 HAP. The arrowhead indicates the tip of a pass-by pollen tube. Scale bars, 50 μm.