BnaMPK3s promote organ size by interacting with BnaARF2s in Brassica napus

The larger organs have potential economic value in Brassica napus improvement and bioenergy production. Mitogen-activated protein kinase (MAPK) cascades are highly conserved signalling modules, playing pivotal roles in multiple processes related to plant growth and development (Xu and Zhang, 2015). We identified two homologues of AtMPK3 in B. napus, BnaA06.MPK3 (BnaA06g18440D) and BnaC03.MPK3 (Bna03g55440D), respectively (Figure S1a,b,e). These two BnaMPK3s were highly expressed in vegetative tissues and increasingly expressed in seeds during development (Figure S1c, d). We found that the seed size of Arabidopsismpk3mutants was significantly smaller than wild type (Figure S2). To examine the roles of BnaMPK3s in the seed size control, the BnaMPK3s double mutants (bnampk3, L4 and L5) were generated by CRISPR/Cas9 toolkit in T0 generation and further confirmed in T1 generation (Figure S3; Table S1). The cotyledon size and fresh weight of T1 bnampk3 plants were less than WT (Figure S4a–d). The silique width, 1000 seed weight (TSW) and the yield per plant were decreased, while other yield related agronomic traits (e.g. branch number) were not affected (Figure 1a, Figure S4e), suggesting MPK3s play conserved functions in promoting organ size. MAPKs regulate diverse biological processes through interacting with different proteins (Guan et al., 2014). Thus, we used BnaC03.MPK3 as bait to screen the B. napus cDNA library, and identified BnaC03.ARF2 (506–1536 bp) that could interact with BnaC03.MPK3. There were four homologues of ARF2 in B. napus, which were widely expressed in different tissues (Figure S5a,b). All BnaARF2s were down-regulated during the seed development (Figure S5c). Then, the interaction between MPK3 and ARF2 were confirmed by using B. napus and Arabidopsis MPK3 and ARF2 homologues (Figure 1b; Figures S6 and S7). Further analysis revealed that BnaMPK3s interacted with BnaARF2s via Auxin Response domain (Figure 1c–e; Figure S8). The seed size was enlarged in Arabidopsis atarf2 mutants (Figure S9), consistent with previous report (Schruff et al., 2006). Two BnaARF2 quadruple mutants (bnaarf2, L35 and L37, T2 generation, Table S2) were isolated from our previous study (Tang et al., 2018), which also had enlarged organs (Figure S10a–d). Specifically, silique length and width, 1000 seed weight, and yield per plant of bnaarf2 were all greatly increased (Figure 1f,g, Figure S10e). On the contrary, overexpressing BnaC03.ARF2 reduced the organ size, especially the TSW and yield per plant were lower than WT (Figure 1f,g, Figure S11). These results suggest that BnaARF2s negatively regulate organ size and seed weight. Furthermore, the seed size of atmpk3atarf2 double mutant was similar to that of atarf2 (Figure S12), indicating that ARF2 is epistatic to MPK3 in regulating seed size. To identify the downstream genes of MPK3-ARF2 module, RNA-seq analysis was performed using 2-week-old seedlings of WT, bnampk3 and 35 S::BnaC03.ARF2. Compared with WT, a total of 4760 and 4688 differential expressed genes (DEGs) were identified in bnampk3 and 35 S::BnaC03.ARF2, respectively (Figure 1h; Table S3). Among them, 1555 genes were coregulated by BnaMPK3 and BnaARF2 (Figure 1i; Table S4). The coup-regulated genes were mainly enriched in the GO terms of seed development, cell size, MAPK cascade and auxin response network (Figure 1j; Table S5). The homologue genes involved in seed size regulation, such as BnaA07.CKX2 and BnaA06.ABA2 (IKU pathway genes), BnaA08.SK41 (Auxin signalling related gene), BnaA05.BG1 (Auxin transporter related gene) and BnaA05.IQD26 (BR related gene), were confirmed to have consistent expression changes in WT, bnampk3, bnaarf2 and 35 S::BnaC03.ARF2 seedlings and developing seeds (Figure S13), suggesting that these genes could be co-regulated by BnaMPK3 and BnaARF2. Furthermore, the dual-luciferase assay showed that expression of pBnaA07.CKX2-LUC was inhibited by BnaARF2s but derepressed by co-infiltration of BnaARF2s and BnaMPK3s (Figure 1k, Figure S14). These results suggest that the BnaARF2s transcriptional activities are regulated by BnaMPK3s. To validate the function of BnaMPK3 and BnaARF2 homologues in other B. napus accessions, the candidate gene association analysis of 1000 seed weight (TSW) was performed using 505 B. napus natural population (Tang et al., 2021). Only seven TSW associated SNPs (promoter: 6 SNPs; fifth exon: 1 SNP) were identified in BnaA06.ARF2, but not in BnaMPK3s and other BnaARF2s (Figure 1l; Figure S15; Table S6). The SNP in the fifth exon only caused a synonymous mutation (Table S6). Then, these SNPs were grouped into three distinct haplotypes, H1, H2 and H3 (reference sequence). TSW of the H1 and H2 haplotypes was significantly higher than the H3 haplotype (Figure 1m). Interestingly, H1 and H2 were enriched in semi-winter type, whereas H3 was predominant in spring type of B. napus (Figure 1n), and TSW of spring B. napus was lower than semi-winter type (Figure S16). In natural population, the expression of BnaA06.ARF2 in H1 and

The larger organs have potential economic value in Brassica napus improvement and bioenergy production. Mitogen-activated protein kinase (MAPK) cascades are highly conserved signalling modules, playing pivotal roles in multiple processes related to plant growth and development (Xu and Zhang, 2015). We identified two homologues of AtMPK3 in B. napus, BnaA06.MPK3 (BnaA06g18440D) and BnaC03.MPK3 (Bna03g55440D), respectively ( Figure S1a,b,e). These two BnaMPK3s were highly expressed in vegetative tissues and increasingly expressed in seeds during development (Figure S1c,d). We found that the seed size of Arabidopsis mpk3 mutants was significantly smaller than wild type ( Figure S2). To examine the roles of BnaMPK3s in the seed size control, the BnaMPK3s double mutants (bnampk3 CR , L4 and L5) were generated by CRISPR/Cas9 toolkit in T 0 generation and further confirmed in T 1 generation ( Figure S3; Table S1). The cotyledon size and fresh weight of T 1 bnampk3 CR plants were less than WT ( Figure S4a-d). The silique width, 1000 seed weight (TSW) and the yield per plant were decreased, while other yield related agronomic traits (e.g. branch number) were not affected (Figure 1a, Figure S4e), suggesting MPK3s play conserved functions in promoting organ size.
MAPKs regulate diverse biological processes through interacting with different proteins (Guan et al., 2014). Thus, we used BnaC03.MPK3 as bait to screen the B. napus cDNA library, and identified BnaC03.ARF2 (506-1536 bp) that could interact with BnaC03.MPK3. There were four homologues of ARF2 in B. napus, which were widely expressed in different tissues ( Figure S5a,b). All BnaARF2s were down-regulated during the seed development ( Figure S5c). Then, the interaction between MPK3 and ARF2 were confirmed by using B. napus and Arabidopsis MPK3 and ARF2 homologues (Figure 1b; Figures S6 and S7). Further analysis revealed that BnaMPK3s interacted with BnaARF2s via Auxin Response domain (Figure 1c-e; Figure S8).
Specifically, silique length and width, 1000 seed weight, and yield per plant of bnaarf2 CR were all greatly increased (Figure 1f,g, Figure S10e). On the contrary, overexpressing BnaC03.ARF2 reduced the organ size, especially the TSW and yield per plant were lower than WT (Figure 1f,g, Figure S11). These results suggest that BnaARF2s negatively regulate organ size and seed weight. Furthermore, the seed size of atmpk3 CR atarf2 CR double mutant was similar to that of atarf2 CR ( Figure S12), indicating that ARF2 is epistatic to MPK3 in regulating seed size.
To identify the downstream genes of MPK3-ARF2 module, RNA-seq analysis was performed using 2-week-old seedlings of WT, bnampk3 CR and 35 S::BnaC03.ARF2. Compared with WT, a total of 4760 and 4688 differential expressed genes (DEGs) were identified in bnampk3 CR and 35 S::BnaC03.ARF2, respectively ( Figure 1h; Table S3). Among them, 1555 genes were coregulated by BnaMPK3 and BnaARF2 (Figure 1i; Table S4). The coup-regulated genes were mainly enriched in the GO terms of seed development, cell size, MAPK cascade and auxin response network ( Figure 1j; Table S5). The homologue genes involved in seed size regulation, such as BnaA07.CKX2 and BnaA06.ABA2 (IKU pathway genes), BnaA08.SK41 (Auxin signalling related gene), BnaA05.BG1 (Auxin transporter related gene) and BnaA05.IQD26 (BR related gene), were confirmed to have consistent expression changes in WT, bnampk3 CR , bnaarf2 CR and 35 S::BnaC03.ARF2 seedlings and developing seeds (Figure S13), suggesting that these genes could be co-regulated by BnaMPK3 and BnaARF2. Furthermore, the dual-luciferase assay showed that expression of pBnaA07.CKX2-LUC was inhibited by BnaARF2s but derepressed by co-infiltration of BnaARF2s and BnaMPK3s (Figure 1k, Figure S14). These results suggest that the BnaARF2s transcriptional activities are regulated by BnaMPK3s.
To validate the function of BnaMPK3 and BnaARF2 homologues in other B. napus accessions, the candidate gene association analysis of 1000 seed weight (TSW) was performed using 505 B. napus natural population (Tang et al., 2021). Only seven TSW associated SNPs (promoter: 6 SNPs; fifth exon: 1 SNP) were identified in BnaA06.ARF2, but not in BnaMPK3s and other BnaARF2s (Figure 1l; Figure S15; Table S6). The SNP in the fifth exon only caused a synonymous mutation (Table S6). Then, these SNPs were grouped into three distinct haplotypes, H1, H2 and H3 (reference sequence). TSW of the H1 and H2 haplotypes was significantly higher than the H3 haplotype ( Figure 1m). Interestingly, H1 and H2 were enriched in semi-winter type, whereas H3 was predominant in spring type of B. napus (Figure 1n), and TSW of spring B. napus was lower than semi-winter type ( Figure S16). In natural population, the expression of BnaA06.ARF2 in H1 and H2 haplotypes were lower than that of H3 haplotype at 20-DAF seeds ( Figure 1o). Moreover, the relative LUC activities driven by H1 and H2 haplotype promoters were lower than that driven by H3 haplotype (Figure 1p), suggesting that the varied expression levels of BnaA06.ARF2 is closely associated with the SNPs in its promoter.
In this study, several lines of evidence showed that the BnaMPK3s-BnaARF2s module is critical for the regulation of organ size and yield in B. napus. The natural mutations in the promoter of BnaA06.ARF2 may contribute to the TSW of B. napus. These findings provide valuable target genes for genome editing and molecular maker development that can be applied in B. napus breeding.

Acknowledgements
We would like to thank Prof. Chunying Kang from Huazhong Agricultural University for revising the manuscript. This research was supported by NSFC (No. 32072105) to Cheng Dai. Data analysis was performed on the bioinformatics computing platform of the National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University.

Conflicts of interest
The authors declare no conflicts of interest.

Supporting information
Additional supporting information may be found online in the Supporting Information section at the end of the article.

Figure S1
Characterization of MPK3 homologues in B. napus. Figure S2 Knockout of AtMPK3 decreased seed size. Figure S3 The genotype of bnampk3 CR in T 1 generation. Figure S4 The agronomic traits of bnampk3 CR and WT. Figure S5 Characterization of ARF2 homologues in B. napus. Figure S6 BnaMPK3s interacted with BnaARF2s in Y2H assay. Figure S7 AtMPK3 interacted with AtARF2. Figure S8 BnaMPK3s interacted with the Auxin Response domain of BnaARF2s. Figure S9 atarf2 CR increased the seed size. Figure S10 The agronomic traits of bnaarf2 CR and WT. Figure S11 The cotyledon and leaf size of 35 S::BnaC03.ARF2 and WT. Figure S12 ARF2 is epistatic to MPK3 in regulating seed size. Figure S13 The expression levels of selected DEGs in different materials. Figure S14 The constructs used for the dual-luciferase assay. Figure S15 Association analysis the BnaA06.MPK3, BnaARF2s. Figure S16 The TSW of spring and semi-winter cultivars.