Cysteine-rich peptides promote interspecific genetic isolation in Arabidopsis

INTRODUCTION Reproductive isolation is the inability of a species to breed with a related species and thus is key to the formation and evolution of a new plant species. Within the genus Arabidopsis , pistils of A. thaliana can be fertilized by pollen of related species, such as A. lyrata , but conspecific (self) pollen is preferred to maintain reproductive isolation. The molecular mechanisms by which the plant’s own pollen tubes outperform heterospecific (alien) pollen tubes is largely unknown. In A. thaliana , maternal cysteine-rich peptides AtLURE1.1 to -1.5, secreted from the ovule, were reported to function through the male receptor PRK6 to mediate pollen tube attraction. RATIONALE Although AtLURE1s have been identified as species-specific pollen tube attractants in A. thaliana , down-regulation of AtLURE1 genes and knockout of their receptor PRK6 did not disturb fertilization and seed set, indicating that the biological functions of AtLURE1s are not fully understood. We initially aimed to fully knock out genes for AtLURE1.1 to -1.5, as well as additional related AtLURE1 genes, to understand their contribution to fertilization success and reproductive isolation. RESULTS In addition to the five reported AtLURE1 genes, we identified two further A. thaliana –specific AtLURE1 genes and generated a loss-of-function atlure1 null septuple mutant by knocking out the whole gene family. Although atlure1 null mutants, resembling prk6 receptor mutants, exhibited normal fertility, pollen tubes in atlure1 null pistils, like prk6 pollen tubes, displayed delayed emergence at the septum. These data collectively suggest that AtLURE1-PRK6 signaling accelerates pollen tubes to penetrate the septum and to grow toward ovules. We further tested the function of AtLURE1-PRK6 signaling in reproductive isolation by depositing alien A. lyrata pollen onto A. thaliana pistils and showed that A. lyrata pollen tubes emerged much more slowly out of the septum. However, the emergence of A. lyrata pollen tubes in A. thaliana pistils was not further impaired in atlure1 null pistils, suggesting that AtLURE1s promote reproductive isolation by accelerating their own pollen tubes, which thus outperform alien tubes. This hypothesis requires the existence of more general, genus- and/or family-specific ovular guidance molecules. Therefore, we next investigated the functions of four AtLURE1-related Brassicaceae-conserved cysteine-rich peptides named XIUQIU1 to -4. We found that XIUQIU peptides attract pollen tubes in a non–species-specific manner and independently of the PRK6 receptor. This finding suggests that evolutionarily ancient XIUQIUs function as general pollen tube attractants in the Brassicaceae. Finally, after combining atlure1 null with xiuqiu loss-of-function mutations, fertility in A. thaliana was reduced, supporting the biological importance of these cysteine-rich peptides in plant reproduction. CONCLUSION In A. thaliana, species-specific female AtLURE1 peptides and their male receptor PRK6 promote and maintain reproductive isolation by accelerating conspecific pollen tube growth to penetrate the septum. The AtLURE1-related cysteine-rich XIUQIU peptides are evolutionarily ancient and conserved attractants in the Brassicaceae and attract pollen tubes in a non–species-specific manner. XIUQIUs contribute to reproductive success and heterospecific fertilization. Their activity may lead to new genome combinations and thus to the formation of new species.