Long-distance transport of siRNAs with functional roles in pollen development

nature plants Article https://doi.org/10.1038/s41477-026-02219-6 Long-distance transport of siRNAs with functional roles in pollen development Received: 11 April 2025 Accepted: 4 January 2026 Jiali Zhu1, Juan Santos-González 2, Zhenxing Wang 2, Tinja Strothans 1, Thales Henrique Cherubino Ribeiro 3,4, Ai Zhang 2, Charles W. Melnyk 2, Blake C. Meyers 3,4 & Claudia Köhler 1,2 Published online: 28 January 2026 Check for updates Small interfering RNAs (siRNAs) play a crucial role in plant reproduction, yet their mobility and function remain incompletely understood. We report that a large proportion of siRNAs found in pollen of Capsella rubella relies on mobile siRNAs from maternal sporophytic tissues, highlighting the importance of non-cell-autonomous siRNAs in male gametophyte development. Unlike tapetal siRNAs, which guide DNA methylation and require CLASSY3 and DNA-dependent RNA polymerase IV (Pol IV) activity in the tapetum, we found that Pol IV-dependent mobile siRNAs (PMsiRNAs) mainly function post-transcriptionally and do not guide DNA methylation. Nevertheless, PMsiRNAs share key features with tapetal siRNAs, including Pol IV dependency, clustering and a size range of 21–24 nucleotides. Using a grafting approach, we show that sporophytic Pol IV-dependent siRNAs act as non-cell-autonomous mobile signals that trigger PMsiRNA formation through post-transcriptional gene silencing. This process parallels reproductive phased siRNA biogenesis, which is widespread across angiosperms but has been considered absent in Brassicaceae. Loss of PMsiRNAs causes pollen arrest, underscoring their essential role. Together, these findings highlight siRNAs as long-distance communication signals from maternal sporophytic tissues to the male gametophyte with critical functions in developmental regulation. In plants, the systemic movement of small RNAs (sRNAs), including siRNAs and microRNAs (miRNAs), represents a mechanism for intercellular communication and regulation1. These molecules can traverse long distances and influence gene expression and development in distal tissues2,3. Despite extensive evidence for this systemic transport, the biological impacts of endogenous mobile sRNAs remain poorly characterized, with functional roles attributed to only a limited subset of these molecules. Transgene-mediated RNA silencing studies have provided important insights into the systemic nature of sRNA mobility. Transgene-induced post-transcriptional gene silencing (PTGS) induced at one site in the plant can systemically move through vascular tissues to silence homologous sequences in distant cells4,5. The results of grafting studies strongly implicate siRNAs as mobile signals of transgene-triggered gene silencing2,6. Systemic silencing of transgenes was shown to involve two RNA silencing pathways. In the PTGS pathway, the primary siRNA derived from a specific transcript triggers the production of secondary siRNAs resembling phased secondary siRNAs (phasiRNAs). This pathway produces 21- and 22-nucleotide siRNAs and involves ARGONAUTE 1 (AGO1), SUPPRESSOR OF GENE SILENCING 3 (SGS3), RNA-DEPENDENT RNA POLYMERASE 6 (RDR6), DICER-LIKE 2 (DCL2) and DICER-LIKE 4 (DCL4)7. The second pathway involves the Department of Plant Reproductive Biology and Epigenetics, Max Planck Institute of Molecular Plant Physiology, Potsdam, Germany. 2Department of Plant Biology, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Centre for Plant Biology, Uppsala, Sweden. 3The Genome Center, University of California, Davis, CA, USA. 4Donald Danforth Plant Science Center, St. Louis, MO, USA. e-mail: 1 Nature Plants | Volume 12 | February 2026 | 386–399 386 Article https://doi.org/10.1038/s41477-026-02219-6 80 60 40 0 l nrpd1 /wt r nrpd1s/nrpd1r m nrpd1 / wt s r w s t w s /w t t/ nr pd 1 p nr p nrpd1s/nrpd1r n nrpd1 /wt s r 25 r 1 *** *** NS *** 20 15 10 5 0 wt ♀ ♂ w s t/ w t /s t nr pd 1 s k d1 r /n pd nr rp n 1/ s w r d1 r nrpd1s/nrpd1r j wts/ nrpd1r r i wts/nrpd1r r pd s Number of viable seeds per silique wts/ nrpd1r r 10 r h *** 20 r g f *** *** NS t wts/wtr wts/wtr 30 1 /s w wts/ wtr o e pd d c wtr nr nrpd1r w s w s t /w nr t /n t pd rp 1 /s d1 n nr rpd pd 1 1 /s w t nrpd1r Number of viable seeds per silique wtr r 20 r sRNAs? 100 r nrpd1s nr pd 1 /s w t wts *** *** *** NS r nrpd1s wts Percentage of mature pollen b a Fig. 1 | Grafting nrpd1 scion to wt rootstock restores pollen development and seed set in Capsella rubella. a, Scheme of the experimental design for hypocotyl grafting. b, Percentage of mature pollen dissected from anthers at stage 12–13 from wts/wtr, wts/nrpd1r, nrpd1s/nrpd1r and nrpd1s/wtr. Three biological replicates derived from independent plants were analysed. Technical replicates in each biological replicate are denoted by triangles, squares and circles. Statistical significance was assessed using a beta-binomial generalized linear model, followed by Tukey-adjusted pairwise comparisons, two-sided. The asterisks mark statistically significant differences (***P < 0.001). The exact P values are provided in the source data. c,d,f,g,i,j,l,m, Alexander staining testing the viability of mature pollen after grafting wts/wtr (c,d), wts/nrpd1r (f,g), nrpd1s/nrpd1r (i,j) and nrpd1s/ wtr (l,m). Scale bars, 100 µm. Arrows indicate aborted pollen. The experiment was repeated twice. e,h,k,n, Seed number per silique in wts/wtr (e), wts/nrpd1r (h), nrpd1s/nrpd1r (k) and nrpd1s/wtr (n). Scale bars, 1 mm. o, Seed number per silique of indicated genotypes. Numbers are based on four biological replicates from individual plants per genotype. Nine or ten siliques were analysed for each plant. Siliques from each replicate are denoted by triangles, squares, circles and crosses. The asterisks mark statistically significant differences (***P < 0.05), Kruskal– Wallis test followed by pairwise Mann–Whitney U-tests with Holm–Bonferroni correction for multiple comparisons. The exact P value is provided in the source data. p, Seed number per silique derived from crossing wt plants with pollen of grafted plants of the indicated genotypes. Seed numbers based on two biological replicates from individual plants per genotype (***P < 0.05), Kruskal–Wallis test followed by pairwise Mann–Whitney U-tests with Holm–Bonferroni correction for multiple comparisons. The exact P value is provided in the source data. In b, o and p, centre lines show the medians; box limits indicate the 25th and 75th percentiles; and whiskers extend 1.5× the interquartile range from the 25th and 75th percentiles. ♀, female; ♂, male; NS, not significant. nuclear components Pol IV and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2), DICER-LIKE 3 (DCL3) and ARGONAUTE 4 (AGO4)8–10. This pathway generates 23- and 24-nucleotide siRNAs. The latter directs RNA-dependent DNA methylation (RdDM) in the transcriptional gene silencing pathway11. Grafting exp (...truncated)