biostudies-literatureWang LNSFC27https://www.ebi.ac.uk/biostudies/studies/S-EPMC10248713biostudies-literatureUnknown43(4)Pollen tube (PT) growth towards the micropyle is critical for successful double fertilization. However, the mechanism of micropyle-directed PT growth is still unclear in <i>Brassica napus</i>. In this study, two aspartate proteases, BnaAP36s and BnaAP39s, were identified in <i>B. napus</i>. BnaAP36s and BnaAP39s were localized to the plasma membrane. The homologues of <i>BnaAP36</i> and <i>BnaAP39</i> were highly expressed in flower organs, especially in the anther. Sextuple and double mutants of <i>BnaAP36s</i> and <i>BnaAP39s</i> were then generated using CRISPR/Cas9 technology. Compared to WT, the seed-set of <i>cr-bnaap36</i> and <i>cr-bnaap39</i> mutants was reduced by 50% and 60%, respectively. The reduction in seed-set was also found when <i>cr-bnaap36</i> and <i>cr-bnaap39</i> were used as the female parent in a reciprocal cross assay. Like WT, <i>cr-bnaap36</i> and <i>cr-bnaap39</i> pollen were able to germinate and the relative PTs were able to elongate in style. Approximately 36% and 33% of <i>cr-bnaap36</i> and <i>cr-bnaap39</i> PTs, respectively, failed to grow towards the micropyle, indicating that BnaAP36s and BnaAP39s are essential for micropyle-directed PT growth. Furthermore, Alexander's staining showed that 10% of <i>cr-bnaap39</i> pollen grains were aborted, but not <i>cr-bnaap36</i>, suggesting that <i>BnaAP39s</i> may also affect microspore development. These results suggest that BnaAP36s and BnaAP39s play a critical role in the growth of micropyle-directed PTs in <i>B. napus</i>.<h4>Supplementary information</h4>The online version contains supplementary material available at 10.1007/s11032-023-01377-1.Molecular breeding : new strategies in plant improvementTwo aspartic proteases, BnaAP36s and BnaAP39s, regulate pollen tube guidance in <i>Brassica napus</i>.PMC102487133207210532172070Fu TMa CDai CLiang XYi BWang LDou SfalseTwo aspartic proteases, BnaAP36s and BnaAP39s, regulate pollen tube guidance in <i>Brassica napus</i>.Pollen tube (PT) growth towards the micropyle is critical for successful double fertilization. However, the mechanism of micropyle-directed PT growth is still unclear in <i>Brassica napus</i>. In this study, two aspartate proteases, BnaAP36s and BnaAP39s, were identified in <i>B. napus</i>. BnaAP36s and BnaAP39s were localized to the plasma membrane. The homologues of <i>BnaAP36</i> and <i>BnaAP39</i> were highly expressed in flower organs, especially in the anther. Sextuple and double mutants of <i>BnaAP36s</i> and <i>BnaAP39s</i> were then generated using CRISPR/Cas9 technology. Compared to WT, the seed-set of <i>cr-bnaap36</i> and <i>cr-bnaap39</i> mutants was reduced by 50% and 60%, respectively. The reduction in seed-set was also found when <i>cr-bnaap36</i> and <i>cr-bnaap39</i> were used as the female parent in a reciprocal cross assay. Like WT, <i>cr-bnaap36</i> and <i>cr-bnaap39</i> pollen were able to germinate and the relative PTs were able to elongate in style. Approximately 36% and 33% of <i>cr-bnaap36</i> and <i>cr-bnaap39</i> PTs, respectively, failed to grow towards the micropyle, indicating that BnaAP36s and BnaAP39s are essential for micropyle-directed PT growth. Furthermore, Alexander's staining showed that 10% of <i>cr-bnaap39</i> pollen grains were aborted, but not <i>cr-bnaap36</i>, suggesting that <i>BnaAP39s</i> may also affect microspore development. These results suggest that BnaAP36s and BnaAP39s play a critical role in the growth of micropyle-directed PTs in <i>B. napus</i>.<h4>Supplementary information</h4>The online version contains supplementary material available at 10.1007/s11032-023-01377-1.2023-01-01T00:00:00Z2023 Apr2025-04-18T20:14:00.098Z2025-04-07T08:13:26.314ZS-EPMC102487133731352910.1007/s11032-023-01377-1