Summary
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 Brassica napus. In this study, two aspartate proteases, BnaAP36s and BnaAP39s, were identified in B. napus. BnaAP36s and BnaAP39s were localized to the plasma membrane. The homologues of BnaAP36 and BnaAP39 were highly expressed in flower organs, especially in the anther. Sextuple and double mutants of BnaAP36s and BnaAP39s were then generated using CRISPR/Cas9 technology. Compared to WT, the seed-set of cr-bnaap36 and cr-bnaap39 mutants was reduced by 50% and 60%, respectively. The reduction in seed-set was also found when cr-bnaap36 and cr-bnaap39 were used as the female parent in a reciprocal cross assay. Like WT, cr-bnaap36 and cr-bnaap39 pollen were able to germinate and the relative PTs were able to elongate in style. Approximately 36% and 33% of cr-bnaap36 and cr-bnaap39 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 cr-bnaap39 pollen grains were aborted, but not cr-bnaap36, suggesting that BnaAP39s may also affect microspore development. These results suggest that BnaAP36s and BnaAP39s play a critical role in the growth of micropyle-directed PTs in B. napus.
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Acknowledgements
The authors extend their appreciation to the support from National Key Laboratory of Crop Genetic Improvement and Hubei Hongshan Laboratory, Huazhong Agricultural University.
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This study was supported by the NSFC (No. 32172070) to Chaozhi Ma, and NSFC (No. 32072105) to Cheng Dai.
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C.D., and L.W. designed the research. L.W., and X.L. performed the experiments. B.Y., T.F., and C.M. provided lab support. S.D., and L.W. analyzed the data. C.D., and L.W. wrote the manuscript. All authors read and approved the manuscript.
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Supplemental Figures and Tables. Supplemental Figure S1. Phylogenetic analysis of amino acid sequences of BnaAP36 and BnaAP39 in different plant species. Supplemental Figure S2. Multiple alignments of AP36 from A. thaliana, and B. napus. Supplemental Figure S3. Multiple alignments of AP39 from A. thaliana, and B. napus. Supplemental Figure S4. The tissue expression pattern of BnaAP36 and BnaAP39 homologs. Supplemental Figure S5. Prediction of potential C-terminal GPI-modification sites. Supplemental Table S1. Gene ID of BnaAP36 and BnaAP39 homologs. Supplemental Table S2. Segregation of self-progeny BnaAP36 mutants. Supplemental Table S3. Segregation of self-progeny BnaAP39 mutants. Supplemental Table S4. The list of primers used for making constructs. Supplemental Table S5. The list of primers used for Hi-TOM sequencing. Supplemental Table S6. The list of primers used for RT-qPCR. (DOCX 3575 kb)
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Wang, L., Liang, X., Dou, S. et al. Two aspartic proteases, BnaAP36s and BnaAP39s, regulate pollen tube guidance in Brassica napus. Mol Breeding 43, 27 (2023). https://doi.org/10.1007/s11032-023-01377-1
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DOI: https://doi.org/10.1007/s11032-023-01377-1