Abstract
The number of pollen grains varies within and between species. However, little is known about the molecular basis of this quantitative trait, in contrast with the many studies available on cell differentiation in the stamen. Recently, the first gene responsible for pollen number variation, REDUCED POLLEN NUMBER1 (RDP1), was isolated by genome-wide association studies of Arabidopsis thaliana and exhibited the signature of natural selection. This gene encodes a homolog of yeast Mrt4 (mRNA turnover4), which is an assembly factor of the large ribosomal subunit. However, no further data were available to link ribosome function to pollen development. Here, we characterized the RDP1 gene using the standard A. thaliana accession Col-0. The frameshift mutant, rdp1-3 generated by CRISPR/Cas9 revealed the pleiotropic effect of RDP1 in flowering, thus demonstrating that this gene is required for a broad range of processes other than pollen development. We found that the natural Col-0 allele conferred a reduced pollen number against the Bor-4 allele, as assessed using the quantitative complementation test, which is more sensitive than transgenic experiments. Together with a historical recombination event in Col-0, which was identified by sequence alignment, these results suggest that the coding sequence of RDP1 is the candidate region responsible for the natural phenotypic variation. To elucidate the biological processes in which RDP1 is involved, we conducted a transcriptome analysis. We found that genes responsible for ribosomal large subunit assembly/biogenesis were enriched among the differentially regulated genes, which supported the hypothesis that ribosome biogenesis is disturbed in the rdp1-3 mutant. Among the pollen-development genes, three key genes encoding basic helix-loop-helix (bHLH) transcription factors (ABORTED MICROSPORES (AMS), bHLH010, and bHLH089), as well as direct downstream genes of AMS, were downregulated in the rdp1-3 mutant. In summary, our results suggest a specialized function of ribosomes in pollen development through RDP1, which harbors natural variants under selection.
Highlights
Pollen grain number in seed plants is a key reproductive trait that has been studied extensively for decades, from agricultural and evolutionary viewpoints
The CRISPR/Cas9-generated frameshift rdp1-3 mutant in the Col-0 background produced about half the number of pollen grains compared with the wild-type counterpart (Tsuchimatsu et al, 2020)
We found that the bolting and flowering times were significantly delayed (Wilcoxon ranksum test; P = 4.33e–05 for bolting time, P = 1.08e–05 for flowering time, Figures 1C,D and Supplementary Video 1). These results support the notion that REDUCED POLLEN NUMBER1 (RDP1) functions as an mRNA turnover 4 (Mrt4) homolog in A. thaliana, in accordance with the slower growth observed in the Mrt4 null mutant in yeast (Rodriguez-Mateos et al, 2009a)
Summary
Pollen grain number in seed plants is a key reproductive trait that has been studied extensively for decades, from agricultural and evolutionary viewpoints. The Uod-1 accession had a long-haplotype variant at the RDP1 region, which conferred a lower number of pollen grains and exhibited a signature of selective sweep. The Bor-4 accession had an alternative variant that conferred a greater number of pollen grains. Both variants did not contain a gene disruptive mutation. Causal evidence of the allelic effects of RDP1 was obtained using Uod-1 and Bor-4 via the quantitative complementation test ( known as reciprocal hemizygosity test) (Tsuchimatsu et al, 2020), which compares allelic effects under the same copy number and positions of genes (Stern, 1998, 2014; Turner, 2014). Further quantitative complementation testing using an accession that experienced historical recombination events (such as Col-0) is another experiment that can narrow down the specific region responsible for the allelic effect of RDP1
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