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

doi:10.21203/rs.3.rs-2220984/v1

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

https://doi.org/10.21203/rs.3.rs-2220984/v1

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published 05 Apr, 2023

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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 identified in B. napus. BnaAP36s and BnaAP39s were localized on the plasma membrane. The homologs of BnaAP36 and BnaAP39 were highly expressed in flower 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.

Brassica napus

BnaAP36s

BnaAP39s

CRISPR/Cas9

pollen tube growth

seed setting rate

In angiosperms, the successful double fertilization plays a crucial role in plant reproduction. Mature pollen grains land on the stigma, and the compatible interaction between pollen and papilla cells triggers the pollen grains hydration and germination (Palanivelu and Tsukamoto, 2012). The germinated pollen tubes (PTs) grow toward to ovule guiding by the internal signals, and then deliver the two sperm cells into the embryo sac, which fuse with egg cells and central cells to complete double fertilization (Swanson et al., 2004, Dresselhaus et al., 2016, Berger et al., 2008). Unlike animal sperms, plant sperm cells cannot move autonomously and rely on PTs transport to successfully reach the ovule. Therefore, PTs growth and guidance are key to complete double fertilization (Weterings & Russell, 2004, Márton et al., 2005, Zhong & Qu, 2019), which is controlled by a complicated signaling cascade involving male and female gametes (Lausser & Dresselhaus, 2010, Higashiyama & Takeuchi, 2015).

Plant male and female gametes secret a variety of signaling substances to regulate the PTs growth and guidance. In Arabidopsis, the LURE1.2 small peptide is secreted by ovule, which could be recognized by its pollen-specific receptor-like kinase 6 (PRK6), and guides ovule specifically attract its own pollen tubes, thus promoting reproductive isolation among species (Zhong et al., 2019, Zhang et al., 2017b, Wang et al., 2016). Lacking receptor like kinase Feronia (FER), ANJEA (ANJ), and HERCULES RECEPTOR KINASE 1 (HERK1) in Arabidopsis female gametes result in unstopped PTs elongation, even entering the micropylar and reaching the synergid cells (Escobar-Restrepo et al., 2007, Galindo-Trigo et al., 2020). Further studies indicate that the PTs expressed rapid alkalinization factor (RALF) peptides are the ligands of FER, ANJ, and HERK1(Zhong et al., 2022). Mutation of these peptides leads to the simultaneous penetration of multiple pollen tubes out of the septum (Zhong et al., 2022). After fertilization, the EGG CELL-SPECIFIC1 (ECS1) and ECS2 peptides are secreted by the egg cells, which could degrade the LURE1, and avoid the entry of multiple sperm cells into the egg cell (Yu et al., 2021). Few studies also report the PTs growth and guidance in crops. For instance, the maize polypeptide ZmEA1 is involved in the signal transduction process of PTs attraction, and down-regulation of ZmEA1 gene leads to ovule sterility (Márton et al., 2005). However, the mechanism of PTs growth and guidance is very complex, and many regulatory pathways remain to be discovered.

Aspartate proteases (APs) are a class of important proteolytic enzymes, which contain two Asp residues within the conserved Asp-Thr/Ser-Gly motifs at the active site (Simões & Faro, 2004, Takahashi et al., 2008). The acidic pH condition could active APs, and the activates of APs are specifically inhibited by pepstatin A (Simões & Faro, 2004, Takahashi et al., 2008). Aspartate proteases exist widely in many plants and play an indispensable role in their growth and development. CND41 is a DNA-binding aspartic protease, which is expressed in the tobacco chloroplast, and plays an important role in leaf senescence (Takahashi et al., 2008). Overexpressing CND41 promotes leaf senescence (Takahashi et al., 2008). An atypical aspartic protease (ASPR1) modulates lateral root development in Arabidopsis (Soares et al., 2019). APs also involve in plant hormone response. Overexpression of aspartate protease constitutive disease resistance (CDR1) in Arabidopsis and rice can enhance salicylic acid-mediated disease resistance (Xia et al., 2004, Prasad et al., 2009). The Arabidopsis AtASPG1 (ASPARTIC PROTEASE IN GUARD CELL 1) positively regulates ABA-mediated drought resistance, and overexpression of AtASPG1 can enhance plant drought resistance (Yao et al., 2012). Subsequent studies suggest that AtASPG1 is also involved in the GA signaling mediated storage protein degradation (Shen et al., 2018). In addition, aspartate proteases also play an important role in female and male gamete formation. For example, rice aspartate proteases, OsAP25, OsAP37 and OsAP65, are essential for tapetum degradation and pollen tube formation during pollen grain development (Huang et al., 2013, Niu et al., 2013). The Arabidopsis aspartic proteases encoding genes, AtA36 and AtA39, are highly expressed in pollen and pollen tubes (Gao et al., 2017b). AtA36 and AtA39 loss of function leads to programmed apoptosis of microspores, degeneration of female gametophytes, and impaired pollen tube guidance (Gao et al., 2017b). These findings imply functional specialization of plant APs, and suggest a strict regulation of APs activity in different biological processes.

Brassica napus is an important oil crop, which is originated from a spontaneous hybridization between Brassica rapa and Brassica oleracea (Hu et al., 2022). We previously identified 154 APs genes in B. napus (Huang et al., 2022). Here, we identified two plasma membrane localized aspartate proteases, BnaAP36s and BnaAP39s, in B. napus. The homologs of BnaAP36 and BnaAP39 were mainly expressed in flower organs, and significantly up-regulated after pollination. Then, the sextuple and double mutants of BnaAP36 and BnaAP39 were generated by CRISPR/Cas9 genome editing technology, respectively. The sextuple and double mutants of BnaAP36 and BnaAP39 displayed lower seed setting, and PTs failed to grow towards the micropyle. Our results indicate that BnaAP36s and BnaAP39s emerge as the first AP with an anticipated role in the regulation of PTs guided growth in B. napus, which will elucidate the fundamental role of APs on pollen grain survival and egg-sperm communications in plants.

BnaAP36s and BnaAP39s are highly expressed in flower organs in B. napus

We previously identified 154 APs genes in B. napus (Huang et al., 2022). Among them, BnaAP36 and BnaAP39 homologs, were significantly up-regulated after pollination (Fig. 1A). There were six and two homologs of BnaAP36 (BnaA08G0069800WE, BnaC08G0065400WE, BnaA04G0084200WE, BnaC04G0369400WE, BnaA05G0142500WE, and BnaC06G0142300WE) and BnaAP39 (BnaA06G0191600WE and BnaC03G0512100WE) in B. napus genome, respectively (Figure S1). We named these BnaAP36 and BnaAP39 homologs, BnaA08.AP36, BnaC08.AP36, BnaA04.AP36, BnaA05.AP36, BnaC06.AP36, BnaA06.AP39 and BnaC03.AP39, respectively. The protein sequences of these homologs of BnaAP36 and BnaAP39 shared high identity with the Arabidopsis AP36 and AP39, from 81.19% to 85.59%, and 83.01% to 83.05%, respectively (Figure S2 and S3). Both BnaAP36s and BnaAP39s contained signal peptide in N-terminal, and aspartic protease domain in C terminal (Figure S2 and S3). Like other eukaryotic aspartic proteases, both BnaAP36s and BnaAP39s contain two catalytic Asp residues within two conserved active sites with the sequence Asp-Thr/Ser-Gly-Ser/Thr (Figure S2 and S3), suggesting these proteins could be functional as aspartate proteases.

Subsequently, the tissue expression data of BnaAP36s and BnaAP39s was obtained from BnTIR (http://yanglab.hzau.edu.cn/BnTIR)(Liu et al., 2021). The homologs of BnaAP36 were found to be highly expressed in 4 mm flower buds (Figure S4A-F). Interestingly, only BnaA08.AP36, BnaC08.AP36, BnaA04.AP36 and BnaC04.AP36 were highly expressed in anther (Figure S4A-D). Compared to BnaAP36s, BnaA06.AP39 and BnaC03.AP39 were expressed in anther and 4 mm flower bud (Figure S4G-H). Then, the expression of BnaAP36s and BnaAP39s was verified by RT-qPCR, and the results showed that BnaAP36 homologs was highly expressed in open flowers (Figure 1C). Notably, BnaA08.AP36, BnaA04.AP36, and BnaC04.AP36 were also expressed in other tissues, including root, stem, leaf, flower bud, stigma and seed (Figure 1C). Like RNA-seq results, BnaA06.AP39 and BnaC03.AP39 were highly expressed in open flower (Figure 1D). These two genes were also expressed in stigma (Figure 1C-D). To gain the accurate tissue expression pattern of BnaAP36s and BnaAP39s, BnaC08.AP36 and BnaA06.AP39 were chosen, and the constructs which contained GUS reporter gene driven by ~2kb promoter of BnaC08.AP36 and BnaA06.AP39 were generated, and transformed into Arabidopsis, individually. No GUS signal was observed in vegetative tissue, such as root and leaf (Figure 1E (i) and 1F (i)). The BnaC08.AP36pro::GUS activity was observed in the stigma and stamen (Figure 1E (ii-v)). Like BnaC08.AP36, the BnaA06.AP39pro::GUS signal was also reported in the stigma and stamen (Figure 1F (ii-v)).

Sequence analysis indicated that BnaAP36s and BnaAP39s were GPI-anchored proteins (http://mendel.imp.ac.at/gpi/gpi_server.html) (Figure S5). To determine the subcellular localization of BnaAP36s and BnaAP39s, we transiently expressed the BnaC08.AP36-GFP and BnaA06.AP39-GFP fusion genes in tobacco epidermal cells. The fusion proteins were predominantly localized to the plasma membrane (PM) (Figure 1G), which is colocalized with the PM marker CBL-RFP (Yang et al., 2021). These results indicate that BnaAP36s and BnaAP39s belong to membrane-anchored aspartic proteases.

Generation the functionally deficient mutants of BnaAP36s and BnaAP39s in B. napus

To study the function of BnaAP36s and BnaAP39s in B. napus, we then generated mutant lines of the BnaAP36s and BnaAP39s by CRISPR/Cas9-mediated genome editing toolbox. Four single-guide RNAs (sgRNAs) were designed to target the homologs of BnaAP36 (Figure 2A). Among them, sgRNA1 targeted the first exon of BnaA08.AP36, BnaC08.AP36, BnaA05.AP36 and BnaC06.AP36; sgRNA2 targeted the first exon of BnaA05.AP36 and BnaC06.AP36; sgRNA3 targeted the sixth exon of BnaA08.AP36, BnaC08.AP36, BnaA04.AP36, BnaC04.AP36, and BnaA05.AP36; and sgRNA4 targeted the seventh exon of all six BnaAP36 homologs (Figure 2A). Two sgRNAs, sgRNA5 and sgRNA6, were designed to target the two homologs of BnaAP39 at the first and second exons, respectively (Figure 2C). Then, these two constructs were transformed into B. napus Westar (WT) individually. Ten and nine mutants of BnaAP36s and BnaAP39s were isolated in the T₀ generation (Table 1 and 2). Short deletions or insertions at the sgRNA target sites were validated by Hi-TOM sequencing (Figure 2, Table 1 and 2). The editing efficiency was quite different among the sgRNAs. For example, no mutation site was identified in sgRNA1, sgRNA2, and sgRNA3 at the BnaAP36 targeted sites, while sgRNA4 was simultaneously generated mutations at six homologs of BnaAP36 (Figure 2B, Table 1). In T₀ generation, two lines cr-bnaap36-3 and cr-bnaap36-6 were homozygous sextuple mutants, while the other cr-bnaap36 lines and all cr-bnaap39 lines were heterozygous mutants (Table 1 and 2). Then, the two sextuple homozygous mutants, cr-bnaap36-3, cr-bnaap36-6, and two homozygous mutants cr-bnaap39-4 and cr-bnaap39-9, were isolated by Hi-TOM sequencing in T₁ generation (Figure 2C and 2D; Table 1 and 2), and these four mutants were chosen for phenotypic characterization.

Disruption of BnaAP36s or BnaAP39s genes result in lower seed setting rate

Then, the two sextuple homozygous mutants, cr-bnaap36-3, cr-bnaap36-6, and two homozygous mutants cr-bnaap39-4 and cr-bnaap39-9, were grown in the field for phenotype observation. Like wild-type and transgenic negative plants (without editing), cr-bnaap36-3, cr-bnaap36-6, cr-bnaap39-4 and cr-bnaap39-9 mutants exhibit normal development during vegetative stage, and the flower organs didn’t show any defect in BnaAP36 and BnaAP39 mutant lines (data not shown). Then, the silique lengths of cr-bnaap36 were about 3.8-4.9 cm, which was shorter than wild-type (5.5 cm) and transgenic negative plants (5.6 cm)(Figure 3A and 3B). The seed set was also reduced in cr-bnaap36, about 11-18 seeds per silique in cr-bnaap36, which was significantly less than wild-type (28 seeds/ silique) and transgenic negative plants (27 seeds/ silique) (Figure 3C). To further investigate the function of male or female gametophytes in responsible for the seed set reduction, the silique length and seed set were calculated in which cr-bnaap36 plants were used as the male or female parent in crosses with wild-type plants. When using cr-bnaap36 as the male parent, the silique length and the seed set were no difference between cr-bnaap36 × wild-type and wild-type × wild-type (Figure 3A – 3C). When taking cr-bnaap36 as the female parent, the silique length and the seed set were reduced (silique length: 3.9-5.2cm; and seed set: 12-19 seeds/ silique) (Figure 3A – 3C), similar to cr-bnaap36 self-crossing. Like cr-bnaap36, the silique lengths were 3.8-4.0 cm in cr-bnaap39, which was shorter than wild-type (5.5cm) and transgenic negative plants (5.6 cm)(Figure 3D and 3E). The seed set was also reduced in cr-bnaap39, about 11-14 seeds per silique in cr-bnaap39, which was significantly less than wild-type and transgenic negative plants (Figure 3F), and the seed set reduction was due to the female gametophytes (Figure 3D – 3F).

Next, the viability of mature pollen grains in the BnaAP36 and BnaAP39 mutants was examined by Alexander staining. Compared with wild-type plants, no obvious differences were detected in the mature pollen grains of the cr-bnaap36 mutants (Figure 4A and 4B). Compared with 98.6% of pollen grains in the wild type, approximately 9% of pollen grains from the cr-bnaap39-4 (8.4%) or cr-bnaap39-9 (9.1%) mutant could not be stained by Alexander, and the pollen grain was smaller with abnormal cytoplasm (Figure 4A and 4B). These results suggested that a portion of pollen grains may be dead or unviable in the cr-bnaap39 mutant.

PTs in BnaAP36s and BnaAP39s mutants display abnormal micropylar guidance

Very low inactive pollen ratio in the BnaAP36s and BnaAP39s mutants (Figure 4) could not explain why the lower seed-set markedly higher than that of wild-type plants (Figure 3). Therefore, we observed pollen tube growth after self- and cross-pollination using aniline blue assay. The pollen grains of wild-type, cr-bnaap36-3, cr-bnaap36-6, cr-bnaap39-4 and cr-bnaap39-9 could germinate normally, and a large number of PTs could elongate and cross the stigma after 24 hours (Figure 5A-5B), suggesting the reducing the seed-set does not due to the failure of pollen - stigma recognition and PTs elongation.

Further, we tracked the path of PTs growth toward to the micropyles after self-pollination. Compared with 88% entered (Figure 5C (i) and 5D) in the wild type, less PTs grew normally and entered the micropyles directly (cr-bnaap36: 64%-65%, cr-bnaap39: 66%-67%; Figure 5D). About 35%, and 33% of cr-bnaap36 and cr-bnaap39 pollen tubes showed abnormal guidance, and pollen tubes were growing on the ovule surface or twisting around the funicular surface, ultimately missing the micropyles (Figure 5C (ii-iii) and 5D). Then, the path of WT PTs was tracked after pollinating the cr-bnaap36 and cr-bnaap39 stigma. After 24 h, about 34%, and 36% WT PTs were failed to enter the micropyles directly (Figure 5D). Therefore, the micropylar guidance is hampered in the pollen tube of cr-bnaap36 and cr-bnaap39.

The communication between female tissues and growing PTs is required for successful the double fertilization. Many components involve in regulating the PTs growth toward to the micropyles (Müller et al., 2016). Here, we identified two aspartate proteases BnaAP36s and BnaAP39s, in B. napus. Loss of function of BnaAP36s and BnaAP39s resulted in lower seed setting rate by mediating the guided PTs growth toward the micropyles, indicating the aspartate proteases plays an important role in this process.

Aspartate proteases are the important hydrolytic enzyme, playing the important role in plant reproduction process (Ge et al., 2005). For example, rice aspartate proteases OsAP25, OsAP37 and OsAP65 are essential for tapetum degradation and pollen tube formation during pollen grain development (Huang et al., 2013). Further studies confirm that ETERNAL TAPETUM 1 (EAT1) promotes the PCD of pollen tapetum by regulating the expression of OsAP25 and OsAP37(Huang et al., 2013, Niu et al., 2013) Microspore PCD was also observed in Arabidopsis A36 A39 double mutants (Gao et al., 2017b). In our study, we show that BnaAP36s and BnaAP39s, which both are mainly expressed in flower organs, function as an micropyle guidance of the PTs growth; while BnaAP39 was also essential for sustaining mature pollen grain survival (Fig. 4A and Fig. 4B). In Arabidopsis, the BnaAP36 and BnaAP39 homologs, A36 and A39, have important roles in suppressing pollen grain program cell death (PCD) by modulating intracellular ROS levels (Gao et al., 2017a). Besides, the A36 and A39 are implicated in the micropyle guidance of the pollen tube (Gao et al., 2017b). These results indicate that the biological functions of AP36 and AP39 in regulating pollen grain development and pollen tube growth into micropores were conserved.

There is complex signal regulation between pollen tube and ovule recognition. For instance, the Arabidopsis synergid cells secrete pollen tube attraction signal LURE1, and is recognized by pollen-specific receptor-like kinases, LIP1 and LIP2 (Lost in Pollen Tube Guidance 1 and 2), which regulate pollen tube growth and guidance (Takeuchi & Higashiyama, 2016, Liu et al., 2013). We identified BnaAP36s and BnaAP39s as GPI-modified aspartate proteases (Fig. 1, and Supplemental Fig. 5). In BnaAP36s and BnaAP39s mutants, about 35% the pollen tubes fail to target the ovule. According to aniline blue assay, pollen tubes in BnaAP36s and BnaAP39s mutants showed significant defects in micropylar guidance (Fig. 5E), which are similar to the phenotype of GPI-anchored aspartate proteases a36 a39 double mutants, and other mutants of GPI-anchored proteins, such as COBL10 (Gao et al., 2017a). GPI-anchored proteins are localized in lipid rafts, which serve as the platforms for protein trafficking and cell signaling transduction(Varma & Mayor, 1998). One possibility is that BnaAP36s and BnaAP39s might have some relationship with LIP1/ LIP2- LURE1 cassette, which need to be investigated in the future.

Brassica napus (AACC) is an important allopolyploid crop derived from interspecific crosses between Brassica rapa and Brassica oleracea (Chalhoub et al., 2014). The Arabidopsis homologue genes in B. napus are multiple-copy that exhibit high sequence similarity. Six and two homologs of BnaAP36 and BnaAP39 were identified in B. napus genome, respectively, with conserved protein domains (Supplemental Figure S2 and S3). Although the expression levels were a little bit different among the BnaAP36 and BnaAP39 homologs, these homologs showed the similar expression patterns (Fig. 1C-1D and Supplemental Figure S4), indicating the homologs of BnaAP36 and BnaAP39 might function redundancy in B. napus. Indeed, the sextuple and double mutants of BnaAP36 and BnaAP39 displayed lower seed setting, and PTs failed to grow towards the micropyle (Fig. 5); while the single mutants of BnaAP36 and BnaAP39 didn’t show any defects in micropylar guidance (data not shown), suggesting the function redundance among different homologs. Notably, the reduction of seed setting rate in BnaAP36 and BnaAP39 were more severe than Arabidopsis A36 and A39 single gene mutants (Fig. 5) (Gao et al., 2017b), and the pollen abortion occurred in the BnaAP39 mutants, not in BnaAP39 mutants (Fig. 4), indicating that the gene functional differentiation might be affected by polyploidization process.

Plant materials and growth condition

All BnaAP36s and BnaAP39s mutants and wild-type (WT) seeds were soaked in water for 7 days and then placed in soil and grown in the transgenic plant field at Huazhong Agricultural University. Arabidopsis thaliana transgenic lines, and tobacco (Nicotiana benthamiana) were grown in greenhouse under a light intensity of 100 µmol m^− 2s^− 1 with a 16/8 h light/dark photoperiod at 22°C.

Plasmid construction

The genes were amplified from B. napus DNA library by PCR using gene-specific primers. For the GUS staining assay, the ~ 2kb promoters of BnaC08.AP36 and BnaA06.AP39 were amplified by PCR and cloned into the vector pCAMBIA2300::GUS, respectively. For the subcellular localization assay, the CDS of BnaC08.AP36 and BnaA06.AP39 were amplified by PCR and then cloned into pRI101-GFP vector. To generate BnaAP36s and BnaAP39s mutants, the GENE-sgRNA plant expression vectors were constructed as previously described with minor modifications (Xing et al., 2014). Briefly, four and two sgRNAs were designed to target the homologs of BnaAP36 and BnaAP39 exon by CRISPR-P (http://cbi.hzau.edu.cn/cgi-bin/CRISPR), respectively (Lei et al., 2014). The sgRNAs cassettes were amplified by PCR using pCBC-DT₁T₂ as temple, and the PCR fragments were then inserted into pKSE401G by Golden Gate Assembly(Gao et al., 2013). All primers were listed in Table S1

Plant transformation and genotyping

The procedure of Agrobaterium-mediated B. napus transformation was carried out as previously described (Dai et al., 2020). To genotype the CRISPR/Cas9-mediated mutation, CTAB method was used to extract the genomic DNA (Molecular Cloning 4th edition). The targeted mutations were determined in transgenic plants using the Hi-TOM platform (Liu et al., 2019). Target-specific and barcoding PCR, that is two rounds of PCR, were performed to amplify the genomic region encompassing the specific targets of independent samples, and the resulting PCR products were mixed in equal amounts and purified for next-generation sequencing (the Illumina HiSeq platform at the Novogene Bioinformatics Institute, Beijing, China). The resulting sequencing data were then decoded by a corresponding online tool to track the mutations of the target sites (http://www.hi-tom.net/hi-tom/). The target-specific primer sets are listed in Table S2.

RNA isolation and reverse-transcription quantitative PCR (RT-qPCR)

The B. napus roots, stems, leaves, buds, open flowers, stigmas and seeds were collected and immediately frozen by liquid nitrogen. Total RNA was extracted using an RNAprep pure plant Kit (TIANGEN Cat. #DP441) following the manufacturer’s instructions. The cDNA was synthesis using a Transcript RT kit (TaKaRa, Japan, Cat. #RR047A). For qRT-PCR, a total volume of 10 µl reaction mixtures was used containing 5 µl of 2× SYBR Green Master Mix (BioRad, USA), 4.6 µl of 100× diluted cDNA, 0.2 µl of each primer. Amplification was performed using a CFX384 Real-Time System (TOYOBO, JAPAN). The amplification program was performed as previous describe (Wu et al., 2020). The expression level of each gene was calculated using the 2^−ΔΔCT method (Livak & Schmittgen, 2001). The primers were designed to target the conserved region of paralogues of FA biosynthesis relative genes. All analyses were repeated at least three times. The primers used for qRT-PCR were listed in Supplemental Table S3.

Measurement of seeds per silique

The stigmas were emasculated one day before anthesis to avoid pollen contamination. Then, the emasculated stigmas were sufficiently pollinated by WT’s or mutant’s pollen. After pollination, the stigmas were covered by paper bags and kept for 10 days. Then the bags were removed, and mature siliques were opened to calculate the number of seeds and silique length. The experiment was performed independently three times. Each time, at least five single plants of each line were selected, and ten stigmas of each plant were randomly pollinated. After pollination, siliques (n ≥ 50) were measured from each line, and the number of seeds was calculated for each seed pot.

Pollen viability analysis

Pollen grains from fully opened flower was used pollen viability analysis. Pollen viability was determined as the percentage of stainable pollen grains in Alexander. More than 1000 pollen grains from each plant were counted.

Aniline blue assay

The aniline blue staining assay was performed according to the established procedure with minor modifications (Sankaranarayanan et al., 2015). The anther was removed the day before flowering. The emasculated stigmas were pollinated with the corresponding pollen on the day of flowering. After 16 h, the pollinated pistils were put into the fixer buffer (3:1 ethanol, glacial acetic acid) for 2 h, and then the samples were incubated in 1 M NaOH at 65°C water for 1 h. Next, the pistils were washed three times with distilled water and stained with the basic aniline blue staining (0.1% aniline blue staining in 0.1 M K₃PO₄) for 3 h. The stained pistils were placed on the glass slide in distilled water to detect the pollen germination and pollen tube growth using the blue channel (UV 340–380 nm) of a fluorescence microscope (SP8; Leica, Wetzlar, Germany).

Phytology tree and sequence alignment analysis

Amino acid sequences of BnaAP36 and BnaAP39 homologs were downloaded from BnIR (http://yanglab.hzau.edu.cn/BnIR) and Brassicaceae Database (http://brassicadb.cn). The software MEGA (version 7.0) was used to perform the sequence alignment and clustering analysis through the nearest neighbor method with 1000 bootstrap replicates. The visualization of the sequence alignment results was performed with GeneDoc (version 2.7). GPI-anchor sequence was predicted by big-PI Pre-dictor (http://mendel.imp.ac.at/gpi/gpi_server.html;(Eisenhaber et al., 1998)).

Subcellular localization assay

The protein subcellular localization analysis in N. benthamiana was performed as previously described (Yang et al., 2021). In brief, in N. benthamiana, Agrobacterium tumefaciens (strain GV3101) cells containing the desired constructs were co-infiltrated into 4- to 5-week-old N. benthamiana leaves with infiltration buffer (50 mM MES, pH = 5.6, 2 mM Na₃PO₄ and 1 mM acetosyringone). The final concentrations of the bacteria were adjusted to an OD₆₀₀ = 0.6 for each construct. Infected leaves were analyzed at 48 h after infiltration. GFP fluorescence (Excitation /Emission wavelength: 488 nm / 509 nm), and RFP fluorescence (Excitation /Emission wavelength: 559 nm / 583 nm) was observed under a fluorescence microscope (SPX8, Leica).

Histochemical GUS assay

The ~ 2kb promoters of BnaC08.AP36 and BnaA06.AP39 were amplified by PCR and cloned into the vector pCAMBIA2300::GUS, respectively. Then transformed into Arabidopsis (Col-0). The positive transgenic plants at seedling and flowering stages were incubated at 37°C overnight in X-Gluc solution and then photographed using a microscope as previously described (Liu et al., 2020). At least three biological replicates were performed.

Statistical analyses

Statistical analysis was performed to identify significant between genotypes, using Student’s t-test, at P values < 0.05 or < 0. 01.

Funding Support

This study was supported by the NSFC (No. 32172070) to Chaozhi Ma, and NSFC (No. 32072105) to Cheng Dai.

Author contributions

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.

Ethics declarations

Ethics approval

All authors approved the submission.

Consent to participate

N/A

Consent for publication

Yes.

Conflict of interest

The authors declare no conflicts of interest.

Data availability

Not applicable.

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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Table 1. Genotypic analysis of BnaAP36s mutants in T₀ and T₁ generations (The red labeled ID indicated the two individual lines in this study)

Plant ID

Generation

genotype at targets of BnaA08.AP36

genotype at targets of BnaC08.AP36

genotype at targets of BnaA04.AP36

sgRNA1

sgRNA2

sgRNA3

sgRNA4

sgRNA1

sgRNA2

sgRNA3

sgRNA4

sgRNA1

sgRNA2

sgRNA3

sgRNA4

cr-bnaap36-L1

Homo (id)

Homo (i)

cr-bnaap36-L2

cr-bnaap36-L3

Homo (id)

cr-bnaap36-L3-1

Homo (id)

cr-bnaap36-L3-2

Homo (id)

cr-bnaap36-L3-3

Homo (id)

cr-bnaap36-L3-4

Homo (id)

Homo (i)

Homo (id)

cr-bnaap36-L3-5

Homo (i)

Homo (id)

cr-bnaap36-L6

Homo (id)

Homo (i)

cr-bnaap36-L6-1

Homo (id)

Homo (i)

cr-bnaap36-L6-2

Homo (id)

Homo (i)

cr-bnaap36-L6-3

Homo (id)

Homo (i)

cr-bnaap36-L6-4

Homo (id)

Homo (i)

cr-bnaap36-L6-5

Homo (id)

Homo (i)

cr-bnaap36-L7

Hetero

cr-bnaap36-L8

cr-bnaap36-L9

cr-bnaap36-L10

Homo (i)

Hetero

cr-bnaap36-L11

Hetero

cr-bnaap36-L12

Hetero

Plant ID

Generation

genotype at targets of BnaC04.AP36

genotype at targets of BnaA05.AP36

genotype at targets of BnaC06.AP36

sgRNA1

sgRNA2

sgRNA3

sgRNA4

sgRNA1

sgRNA2

sgRNA3

sgRNA4

sgRNA1

sgRNA2

sgRNA3

sgRNA4

cr-bnaap36-L1

Homo (id)

Homo (i)

cr-bnaap36-L2

Hetero

cr-bnaap36-L3

Homo (id)

cr-bnaap36-L3-1

Homo (id)

cr-bnaap36-L3-2

Homo (id)

cr-bnaap36-L3-3

Homo (id)

cr-bnaap36-L3-4

Homo (id)

cr-bnaap36-L3-5

Homo (id)

cr-bnaap36-L6

Homo (id)

Homo (i)

cr-bnaap36-L6-1

Homo (i)

cr-bnaap36-L6-2

Homo (i)

cr-bnaap36-L6-3

Homo (i)

cr-bnaap36-L6-4

Homo (i)

cr-bnaap36-L6-5

Homo (i)

cr-bnaap36-L7

Hetero

cr-bnaap36-L8

Hetero

cr-bnaap36-L9

Hetero

cr-bnaap36-L10

Homo (i)

Homo (id)

Homo (d)

cr-bnaap36-L11

Hetero

Homo (id)

Hetero

cr-bnaap36-L12

Hetero

Hetero, heterozygous; Homo, homozygous; wt, no mutations were identified; #d, #of bp deleted from a target site; #i, # of bp inserted at target site; #s, #of bp substitute at target site.

Table 2. Genotypic analysis of BnaAP39s mutants in T₀ and T₁ generations. (The red labeled ID indicated the two individual lines in this study)

Plant ID	Generation	genotype at targets of BnaA06.AP39		genotype at targets of BnaC03.AP39
Plant ID	Generation	sgRNA5	sgRNA6	sgRNA5	sgRNA6
cr-bnaap39-L1	T0	WT	Hetero	WT	Hetero
cr-bnaap39-L2	T0	WT	WT	WT	Hetero
cr-bnaap39-L3	T0	Hetero	WT	Hetero	Hetero
cr-bnaap39-L4	T0	Hetero	Hetero	Hetero	Hetero
cr-bnaap39-L4-1	T1	Hetero	Homo (i)	Hetero	Homo (i)
cr-bnaap39-L4-2	T1	Hetero	Homo (i)	Hetero	Homo (i)
cr-bnaap39-L4-3	T1	Hetero	Homo (i)	Hetero	Homo (i)
cr-bnaap39-L4-4	T1	Hetero	Homo (i)	Hetero	Homo (i)
cr-bnaap39-L4-5	T1	Hetero	Homo (i)	Hetero	Homo (i)
cr-bnaap39-L5	T0	WT	WT	Hetero	Hetero
cr-bnaap39-L6	T0	Hetero	Hetero	Hetero	Hetero
cr-bnaap39-L7	T0	Hetero	Hetero	Hetero	Hetero
cr-bnaap39-L7-1	T1	Hetero	Homo (i)	Hetero	Hetero
cr-bnaap39-L7-2	T1	WT	WT	WT	Hetero
cr-bnaap39-L7-3	T1	Hetero	Hetero	Hetero	Hetero
cr-bnaap39-L7-4	T1	Hetero	Hetero	Hetero	Hetero
cr-bnaap39-L7-5	T1	Hetero	WT	Hetero	Hetero
cr-bnaap39-L8	T0	Hetero	Hetero	Hetero	Hetero
cr-bnaap39-L8-1	T1	WT	WT	WT	Hetero
cr-bnaap39-L8-2	T1	WT	WT	WT	WT
cr-bnaap39-L8-3	T1	Hetero	Hetero	Hetero	Hetero
cr-bnaap39-L8-4	T1	Hetero	WT	Hetero	Hetero
cr-bnaap39-L8-5	T1	WT	WT	WT	Hetero
cr-bnaap39-L9	T0	Hetero	WT	Hetero	Hetero
cr-bnaap39-L9-1	T1	Homo (id)	WT	Homo (id)	Homo (i)
cr-bnaap39-L9-2	T1	Homo (id)	WT	Homo (id)	Homo (i)
cr-bnaap39-L9-3	T1	Homo (id)	WT	Homo (id)	Homo (i)
cr-bnaap39-L9-4	T1	Homo (id)	WT	Homo (id)	Homo (i)
cr-bnaap39-L9-5	T1	Homo (id)	WT	Homo (id)	Homo (i)

Hetero, heterozygous; Homo, homozygous; wt, no mutations were identified; #d, #of bp deleted from a target site; #i, # of bp inserted at target site; #s, #of bp substitute at target site.

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

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Introduction

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Discussion

Material And Methods

Plant materials and growth condition

Plasmid construction

Plant transformation and genotyping

RNA isolation and reverse-transcription quantitative PCR (RT-qPCR)

Measurement of seeds per silique

Pollen viability analysis

Aniline blue assay

Phytology tree and sequence alignment analysis

Subcellular localization assay

Histochemical GUS assay

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Tables

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