Two aspartic proteases, BnaAP36s and BnaAP39s, regulate pollen tube guidance in Brassica napus

Abstract

The pollen tube (PTs) guidance to micropyle growth is the key to successful double fertilization. However, the regulatory mechanism leading to pollen tube orientation is still unclear in Brassica napus. In this study, two aspartate proteases, BnaAP36s and BnaAP39s, were identi ed in B. napus. BnaAP36s and BnaAP39s were localized on the plasma membrane. The homologs of BnaAP36 and BnaAP39 were highly expressed in ower organs, which were induced by pollination. Then, the mutants of sextuple and double mutants of BnaAP36 and BnaAP39 were generated by using CRISPR/Cas9 technology, respectively. Compared to WT, the seed set of cr-bnaap36 and cr-bnaap39 mutants was reduced by approximately 50% and 60%, respectively. Like WT, the pollen grain of cr-bnaap36 and cr-bnaap39 could germinate and the relative PTs could elongate. The reciprocal crosses results showed that the reduction of seed set was due to the defects of stigma. Interestingly, 36% and 33% cr-bnaap36 and cr-bnaap39 PTs failed to grow towards the micropyle, indicating BnaAP36s and BnaAP39s were essential for PTs guided growth. Further, Alexander's staining showed that 10% pollen abortion was observed in BnaAP39 mutants, but not in BnaAP36 mutants, suggesting BnaAP39s might also affect the microspore development. These results suggested that BnaAP36s and BnaAP39s play critical roles in PTs guidance growth in B. napus.

Dissection of quantitative trait nucleotides and candidate genes associated with agronomic and yield-related traits under drought stress in rapeseed varieties: integration of genome-wide association study and transcriptomic analysis

“…In B . napus , BnaAP36s and BnaAP39s genes play a critical role in pollen tube growth ( Wang et al., 2023 ). Chr13:3058428 ( P =1.36 × 10 −8 ) explained 42.99% of the phenotypic variance located within the position of the panicle architecture-related gene LAX PANICLE 2 ( LAX2 ) ( Figures 3M, N ).…”

“…Chr13:2852351 (P = 2.11 × 10 − 5 ) and Chr13:2878346 (P =1.36 × 10 −8 ) were located ~10 kbps downstream and upstream of aspartic proteinase oryzasin-1, respectively. In B. napus, BnaAP36s and BnaAP39s genes play a critical role in pollen tube growth (Wang et al, 2023). Chr13:3058428 (P =1.36 × 10 −8 ) explained 42.99% of the phenotypic variance located within the position of the panicle architecture-related gene LAX PANICLE 2 (LAX2) (Figures 3M, N).…”