The characteristic of BnaFAX sequences and the expression pattern of BnaFAXs in different tissues
In Arabidopsis, there are 7 FAX members, FAX1-7. The FAX sequences of Arabidopsis were used to do an alignment in the database of B. napus. The results show that there are multiple copies of each FAX in B. napus, including 6 copies of FAX1, 2 copies of FAX2, 4, 5 and 7, and 3 copies of FAX3 and 6 (Fig. 1A). Analysis of the motifs of all FAXs, data show that the motif number of different FAX is different, and the motif number of most FAX subfamily is the same. Among them, FAX1 had 7 motifs except BnaC04. FAX1, and FAX2 have 6 motifs, FAX3 and FAX4 have 4 motifs, FAX5 and FAX6 (except BnaA03. FAX6) have 5 motifs, and FAX7 has only 3 motifs (Fig. 1A). The ZS11 transcriptome database (http://yanglab.hzau.edu.cn/BnIR) was used to analyze the expression of different copies of FAX1. The results show that the BnaA09. FAX1 has the highest expression level in all tissues, compared to other copies, followed by BnaC08.FAX1, the rest 4 homologous genes have lower or no expression (Fig. 1B). Furthermore, the expression of BnaA09. FAX1 and BnaC08. FAX1 is higher in leaf, sepal, petal and silique than that of other tissues (Fig. 1B). The data also show that most of the other FAX subfamilies (except FAX6) have high expression in different tissues except in anther (Fig. S1). Interestingly, almost all FAXs have no expression in anther (Fig. 1B, Fig. S1).
Generation Of Transgenic Plants And The Subcellular Localization Of Bnaa09.fax1
To verify the function of FAX1 in B. napus, the transgenic plants were obtained using the transformation mediated by Agrobacterium tumefaciens GV3101. Six overexpression lines, OE1-OE6, were confirmed by PCR. Subsequently, the qRT-PCR and western blot were employed to test the gene and protein expression, respectively. The results show that BnaA09.FAX1 has been expressed successfully in B. napus (Fig. 2A and B). Additionally, the plants containing Cas9 were sequenced to confirm the mutations. The results show that two types of homozygous mutants (C2 and C3) are obtained (Fig. 2C). Line C2 has 1 base pair (bp) insertion in BnaA09.FAX1 and 1 bp deletion in BnaC08.FAX1, while C3 has 8 bp deletion in BnaA09.FAX1 and 1 bp insertion in BnaC08.FAX1 (Fig. 2B).
To detect the subcellular localization of BnaA09.FAX1, the recombinant vector of pMDC83-BnaA09.FAX1-GFP was constructed and injected into the epidermal cells of tobacco leaves. The imaging showed that the green fluorescent signal from fused protein could overlap with the auto-fluorescent signal of chloroplast (Fig. 2D), suggesting BnaA09.FAX1 was located on the chloroplast membrane.
Overexpression of BnaA09.FAX1 increases plant biomass and FA content in B. napus leaves
To verify the effect of BnaA09.FAX1 on plant growth, we compared the plant growth among OE, mutants, and WT. The 3-week-old plants grown in liquid media were compared. The results display that OE1-OE3 plants are larger than WT, while the mutants have no obvious phenotypical change (Fig. 3A). Compared to WT, the plant fresh weight and root length of OE lines are increased by 0.7–1.7 g and 1.63–1.79 cm, respectively, while there are no changes in mutant lines (Fig. 3B). The size of the same-age leaves of OE is also significantly larger than that of WT, but no changes are observed in the mutant lines (Fig. 3C and D). The FA was also extracted from the same-age leaves of each sample. Compared to WT, the content of total FA in OE lines is significantly increased by 0.6–0.9%, but no difference in mutant lines (Fig. 3D). Together, the results reveal that the BnaA09.FAX1 can improve plant growth and FA accumulation in leaves.
BnaA09.FAX1 changes plant height in B. napus
To investigate the effect of FAX1 on agronomic traits of B. napus, OE lines, mutants and WT plants were planted under natural conditions with well-watered. When plants were grown to full flowering (120 days after sowing), the OE lines are significantly higher than WT, while there is no difference between WT and mutants (Fig. 4A). After plants were harvested, the agronomic traits including plant height, inflorescence length, silique number of the main inflorescence, number of effective branches, silique number per plant and silique length were measured. The results show that OE lines are 7.8–12.2 cm taller than WT, while the mutants have no difference from WT (Fig. 4B). There are no significantly different changes of other agronomic traits among OE, mutants and WT plants (Fig. 4B). These results indicate that FAX1 can increase plant height, but has no effect on other agronomic traits.
BnaA09.FAX1 affects seed oil content and FA composition in B. napus
To determine the effect of BnaA09.FAX1 on seed oil and protein content. The mature seeds from naturally grown plants were scanned by a near-infrared instrument. The results show that the oil content of OE1 and OE3 is increased by 1.7 and 1.4% compared to WT, respectively. However, there is no significant difference between WT and mutants (Fig. 5A). The thousand seed weight was also measured, and the data display that there is no difference in the three materials (Fig. 5B). The protein content of seeds has no difference among OE, mutants, and WT (Fig. 5C). In addition, the FA composition of mature seeds was analyzed by GC-FID. The results show that the content of C18:0 and C18:3 in OE is significantly decreased, while the C18:1 and C20:1 are significantly increased, compared to WT. However, the content of C18:3 in the mutants is decreased significantly, but C20:0 is increased significantly (Fig. 5D). These data imply that BnaA09.FAX1 has a role in seed oil accumulation and also affects FA composition.
BnaA09.FAX1 affects the lipid content of B. napus seeds
To explore whether BnaA09.FAX1 changes the lipid content of B. napus seeds, we extracted and analyzed the lipids of WT, OE, and mutants. Compared to WT, the content of neutral lipids including TAG and diacylglycerol (DAG) in OE is increased by 25.2% and 70.4%, respectively, but no significant change is observed in the mutant (Fig. 6A and C). Compared to WT, the species of TAG-54:1 and − 56:0 are decreased significantly and TAG-52:3, -52:5 and − 54:6 are increased significantly in OE seeds. TAG-50:1, -50:2, -52:3 and − 60:1 are increased significantly and TAG-52:6, -54:8 and − 54:8 are decreased significantly in the mutant (Fig. 6B). The species of DAG-32:0, -34:0, -34:1, -36:1 and − 36:2 are decreased and DAG-34:6, -36:3 and − 36:4 are increased in OE3. While the DAG-34:5 and − 36:3 ware increased and DAG-36:2 and − 36:3 are decreased in the mutant (Fig. 6D).
The content of phospholipids is also changed. The data show the content of phosphatidylcholines (PC) is increased by 26.8% in OE, but no difference is observed in the mutant, compared to WT (Fig. 6E). The species of PC-34:4, -34:2 and − 36:3 ~ -36:5 are increased significantly in OE seeds, and PC-36:2 is decreased significantly. The species of PC-32:0, 34:2 and 34:1 are increased significantly and PC-36:6 and 36:5 are decreased significantly in the mutants (Fig. 6F). The content of phosphatidylethanolamine (PE) and phosphatidylserine (PS) has no changes among OE, mutants and WT, although some species of PE and PS are changed significantly (Fig. S2A-D). Together, the results indicate that overexpression of BnaA09g.FAX1 causes change the content and species of lipids in B. napus seeds.