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SUMMARY: The successful interaction between pollen and stigma is a critical process for plant sexual reproduction, involving a series of intricate molecular and physiological events. After self‐compatible pollination, a significant reduction in reactive oxygen species (ROS) production has been observed in stigmas, which is essential for pollen grain rehydration and subsequent pollen tube growth. Several scavenging enzymes tightly regulate ROS homeostasis. However, the potential role of these ROS‐scavenging enzymes in the pollen‐stigma interaction in Brassica napus remains unclear. Here, we showed that the activity of ascorbate peroxidase (APX), an enzyme that plays a crucial role in the detoxification of hydrogen peroxide (H2O2), was modulated depending on the compatibility of pollination in B. napus. We then identified stigma‐expressed APX1s and generated pentuple mutants of APX1s using CRISPR/Cas9 technology. After compatible pollination, the BnaAPX1 pentuple mutants accumulated higher levels of H2O2 in the stigma, while the overexpression of BnaA09.APX1 resulted in lower levels of H2O2. Furthermore, the knockout of BnaAPX1 delayed the compatible response‐mediated pollen rehydration and germination, which was consistent with the effects of a specific APX inhibitor, ρ‐Aminophenol, on compatible pollination. In contrast, the overexpression of BnaA09.APX1 accelerated pollen rehydration and germination after both compatible and incompatible pollinations. However, delaying and promoting pollen rehydration and germination did not affect the seed set after compatible and incompatible pollination in APX1 pentuple mutants and overexpression lines, respectively. Our results demonstrate the fundamental role of BnaAPX1 in pollen rehydration and germination by regulating ROS homeostasis during the pollen‐stigma interaction in B. napus. Significance Statement: Our research found that self‐incompatibility in Brassica napus requires the accumulation of reactive oxygen species (ROS) levels in the stigma. We identified the importance of BnaAPX1 in regulating ROS homeostasis, which is critical for pollen rehydration and germination in B. napus. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Self-incompatibility (SI) in plants genetically prevents self-fertilization to promote outcrossing and genetic diversity. Its hybrids in Brassica have been widely cultivated due to the propagation of SI lines by spraying a salt solution. We demonstrated that suppression of Brassica napus SI from edible salt solution treatment was ascribed to sodium chloride and independent of S haplotypes, but it did not obviously change the expression of SI-related genes. Using the isobaric tags for relative and absolute quantitation (iTRAQ) technique, we identified 885 differentially accumulated proteins (DAPs) in Brassica napus stigmas of un-pollinated (UP), pollinated with compatible pollen (PC), pollinated with incompatible pollen (PI), and pollinated with incompatible pollen after edible salt solution treatment (NA). Of the 307 DAPs in NA/UP, 134 were unique and 94 were shared only with PC/UP. In PC and NA, some salt stress protein species, such as glyoxalase I, were induced, and these protein species were likely to participate in the self-compatibility (SC) pathway. Most of the identified protein species were related to metabolic pathways, biosynthesis of secondary metabolites, ribosome, and so on. A systematic analysis implied that salt treatment-overcoming SI in B.napus was likely conferred by at least five different physiological mechanisms: (i) the use of Ca2+ as signal molecule; (ii) loosening of the cell wall to allow pollen tube penetration; (iii) synthesis of compatibility factor protein species for pollen tube growth; (iv) depolymerization of microtubule networks to facilitate pollen tube movement; and (v) inhibition of protein degradation pathways to restrain the SI response. [ABSTRACT FROM AUTHOR]

Self-incompatibility (SI) is an important genetic mechanism exploited by numerous angiosperm species to prevent inbreeding. This mechanism has been widely used in the breeding of SI trilinear hybrids of Brassica napus. The SI responses in these hybrids can be overcome by using a salt (NaCl) solution, which is used for seed propagation in SI lines. However, the mechanism underlying the NaCl-induced breakdown of the SI response in B. napus remains unclear. Here, we investigated the role of two key proteins, BnaPLDα1 and BnaMPK6, in the breakdown of SI induced by NaCl. Pollen grain germination and seed set were reduced in BnaPLDα1 triple mutants following incompatible pollination with NaCl treatment. Conversely, SI responses were partially abolished by overexpression of BnaC05.PLDα1 without salt treatment. Furthermore, we observed that phosphatidic acid (PA) produced by BnaPLDα1 bound to B. napus BnaMPK6. The suppression and enhancement of the NaCl-induced breakdown of the SI response in B. napus were observed in BnaMPK6 quadruple mutants and BnaA05.MPK6 overexpression lines, respectively. Moreover, salt-induced stigmatic reactive oxygen species (ROS) accumulation had a minimal effect on the NaCl-induced breakdown of the SI response. In conclusion, our results demonstrate the essential role of the BnaPLDα1-PA-BnaMPK6 pathway in overcoming the SI response to salt treatment in SI B. napus. Additionally, our study provides new insights into the relationship between SI signaling and salt stress response. A new molecular mechanism underlying the breakdown of the NaCl-induced self-incompatibility (SI) response in B. napus has been discovered. It involves the induction of BnaPLDα1 expression by NaCl, followed by the activation of BnaMPK6 through the production of phosphatidic acid (PA) by BnaPLDα1. Ultimately, this pathway leads to the breakdown of SI. The involvement of the BnaPLDα1-PA-BnaMPK6 pathway in overcoming the SI response following NaCl treatment provides new insights into the relationship between SI signalling and the response to salt stress. • BnaPLD1 positively regulates salt-breaking the SI response. • PA produced by BnaPLD1 bound to BnaMPK6, and BnaMPK6 positively regulates salt-breaking the SI response. • BnaPLDα1-PA-BnaMPK6 pathway is essential for salt-breaking the SI response in B.napus. [ABSTRACT FROM AUTHOR]