Defective pollen tube tip growth induces neo-polyploid infertility

Genome duplication (generating polyploids) is an engine of novelty in eukaryotic evolution and a promising crop improvement tool. Yet newly formed polyploids often have low fertility. Here we report that a severe fertility-compromising defect in pollen tube tip growth arises in new polyploids of Arabidopsis arenosa. Pollen tubes of newly polyploid A. arenosa grow slowly, have aberrant anatomy and disrupted physiology, often burst prematurely, and have altered gene expression. These phenotypes recover in evolved polyploids. We also show that gametophytic (pollen tube) genotypes of two tip-growth genes under selection in natural tetraploid A. arenosa are strongly associated with pollen tube performance in the tetraploid. Our work establishes pollen tube tip growth as an important fertility challenge for neo-polyploid plants and provides insights into a naturally evolved multigenic solution. Editor’s summary Plants often throw out offspring with different chromosome numbers (ploidy). This phenomenon can create bigger flowers and fruits or provide environmental resilience, and it has become a target in plant breeding for crop improvement. Westermann et al. investigated why de novo polyploids show reduced fertility compared with established descendant polyploid plants. Fertility loss was originally thought to result primarily from issues relating to segregation of multiple chromosomes during meiosis, but this is not the only obstacle. In a survey of genes under selection, the authors identified two: AGC1, which is important for pollen tube growth, and ACA8, a calcium transporter, both of which are known to affect pollen tube structure. Experiments verified the role that adaptations in the expression of these genes play in correcting defective pollen tube growth after ploidy generation. —Caroline Ash and Ankit Walia Mechanistic follow-up to a genome scan shows selection targeted pollen tube tip growth to rectify neopolyploid fertility defects. INTRODUCTION Polyploidy arises from whole-genome duplication. It is implicated in evolution and adaptation and thus is of fundamental interest in evolutionary biology. Adaptation of polyploid lineages to their genome-duplicated state provides an interesting case study for evolutionary cell biology: In newly formed polyploids, a diploid-optimized genome is suddenly in a novel cellular context to which it is not adapted, and this could necessitate evolutionary retuning of cellular functions. There is also increasing interest in using polyploidy as a crop improvement tool, because polyploids are often resistant to climate-relevant stresses—most prominently, drought. However, for understanding their evolution or using them in agriculture, the fact that most new polyploids have very low fertility remains a key stumbling block. Meiotic chromosome segregation is a well-known challenge for new polyploids, but we have recently shown that improving neo-polyploid meiotic stability does not rescue fertility on its own. This suggests, as G. L. Stebbins already proposed 75 years ago, that there is something else that affects polyploid fertility—what that might be has, to date, remained mysterious. RATIONALE We used a “reverse genomics of adaptation” approach to identify what additional challenges to fertility might be important in newly formed polyploids. We did this by focusing on a list of genes under selection that we had previously identified from a genome scan in a natural autotetraploid lineage of Arabidopsis arenosa. Among these genes, we found two with strong signals of positive selection that are known, from mutant studies in Arabidopsis thaliana, to play a bona fide role in pollen tube tip growth. Given that pollen tube tip growth is essential for fertility across sexually reproducing seed plants, we aimed to test whether it might indeed present a challenge for neo-polyploids and whether the novel alleles of the two candidate genes that led us to this hypothesis might play a role in solving it. RESULTS We show that pollen tube tip growth is severely challenged in neo-polyploid A. arenosa and A. thaliana. The pollen tubes from experimentally induced neo-polyploids grow slowly, with extensive abnormalities, including stalling, branching, bulging, premature bursting, disrupted calcium gradients, defective actin filament formation, and ectopic cell wall softening. In established tetraploids, which exist in A. arenosa but not A. thaliana, the physiology and morphology of pollen tubes have returned to normal. The defects seen in pollen tubes of neo-tetraploids are coupled with perturbations in gametophytic gene expression, including expression of several genes that could be involved in generating the observed phenotypes; many, but not all, of these return to the ancestral expression state in the evolved tetraploids. We tested genetically whether genotypes of the two strongly ploidy-differentiated genes we identified, which encode a kinase AGC1.5 and a calcium-exporting adenosine triphosphatase ACA8, correlate with pollen tube performance in tetraploids. We find that diploid pollen tubes homozygous for tetraploid alleles at both loci strongly outcompete pollen tubes carrying diploid alleles in vivo when used to fertilize tetraploid females. In segregating lines, homozygotes for tetraploid alleles at AGC1.5 and ACA8 loci produce pollen tubes with markedly improved growth compared with plants homozygous for diploid alleles, both in terms of growth rate and prevention of premature bursting. The two loci thus additively associate with improved growth of pollen tubes after whole-genome duplication and may represent good candidates for improving fertility of neo-polyploids in other species. CONCLUSION Our work shows that pollen tube tip growth is defective in newly formed A. arenosa and A. thaliana polyploids, providing a previously unexplored component of neo-polyploid sterility and a potential explanation for Stebbins’s nonmeiotic sterility factor. Our transcriptome work provides insights into what problems arise in neo-polyploid pollen tubes and shows that established tetraploids did not simply return pollen tube gene expression to the ancestral state during their evolution to overcome the neo-polyploid challenge. Notably, we identify two candidate genes and show genetically that derived tetraploid alleles associate with improved pollen tube tip growth. We also show that it is the gametophyte, not the sporophyte, genotype that conditions these effects. The two genes we identify are promising candidates, motivating further studies to confirm their mechanistic roles, find additional players, and, importantly, test whether they can be transferred to other species to improve neo-polyploid fertility. Abnormal pollen tubes from a neo-tetraploid A. arenosa plant. Microscope image showing pollen grains and two defective pollen tubes from a neo-tetraploid A. arenosa plant growing in vitro. These defects show that neo-tetraploids suffer a defect that disrupts the normal polar growth of pollen tubes. One grain has a pollen tube that burst immediately after germination. Several grains have just started germinating. Tubes are stained with aniline blue, which marks callose, and the bright bands show callose plugs.