Targeted mutagenesis of flavonoid biosynthesis pathway genes reveals functional divergence in seed coat colour, oil content and fatty acid composition in Brassica napus L

Abstract

Yellow-seed is widely accepted as a good-quality trait in Brassica crops. Previous studies have shown that the flavonoid biosynthesis pathway is essential for the development of seed colour, but its function in Brassica napus, an important oil crop, is poorly understood. To systematically explore the gene functions of the flavonoid biosynthesis pathway in rapeseed, several representative TRANSPARENT TESTA (TT) genes, including three structural genes (BnaTT7, BnaTT18, BnaTT10), two regulatory genes (BnaTT1, BnaTT2) and a transporter (BnaTT12), were selected for targeted mutation by CRISPR/Cas9 in the present study. Seed coat colour, lignin content, seed quality and yield-related traits were investigated in these Bnatt mutants together with Bnatt8 generated previously. These Bnatt mutants produced seeds with an elevated seed oil content and decreased pigment and lignin accumulation in the seed coat without any serious defects in the yield-related traits. In addition, the fatty acid (FA) composition was also altered to different degrees, i.e., decreased oleic acid and increased linoleic acid and α-linolenic acid, in all Bnatt mutants except Bnatt18. Furthermore, gene expression analysis revealed that most of BnaTT mutations resulted in the down-regulation of key genes related to flavonoid and lignin synthesis, and the up-regulation of key genes related to lipid synthesis and oil body formation, which may contribute to the phenotype. Collectively, our study generated valuable resources for breeding programs, and more importantly demonstrated the functional divergence and overlap of flavonoid biosynthesis pathway genes in seed coat colour, oil content and FA composition of rapeseed.

Keywords: Brassica napus; CRISPR/Cas9; Fatty acid composition; Flavonoid biosynthesis pathway; Oil content; Seed coat colour.

Figure 1

The gene structures and the tissue expression patterns of the BnaTT genes. (a) The analysis of the conserved domain. (b) The tissue expression patterns. The red inverted triangle in (a) is the sgRNA location.

Figure 2

Mature seeds phenotypic observations of Bnatts. (a) Mature seeds phenotypes of Bnatts. Scale = 1 cm. (b) The yellow‐seeded degree (YSD) of Bnatts. The colour of Bnatt1 to Bnatt18 was analysed on freshly harvested seeds, whereas Bnatt10‐old were analysed on Bnatt10 seeds after a period of storage. (c) Mature seeds embryo of Bnatts. Scale = 500 μm. (d) The reciprocal crosses F₁ seed of Bnatt10 with WT. Scale = 1 cm.

Figure 3

The observations of seed coat PAs staining and analysis of expression level of phenylpropanoid pathway genes in Bnatt mutants. (a) Vanillin (left) and DMACA (right) staining were performed on the seed coat of wild type and Bnatts at different developmental stages. DAF, days after flowering. (b) The expression level of genes involved in phenylpropyl and downstream flavonoids and lignin metabolism in Bnatts. PAL, phenylalanine ammonia‐lyase; C4H, cinnamic acid 4‐hydroxylase; 4CL, 4‐coumaric acid: CoA ligase; TT, Transparent testa; BAN, BANYULS; CCoAOMT, caffeoyl CoA 3‐O‐methyltransferase; COMT, caffeate/5‐hydroxyferulate 3‐O‐methyltransferase; Total RNA was extracted from 21, 28 and 31 DAF seeds. Relative expression level was calculated by comparison to Actin7's expression level. Values are means±SD (n = 3).

Figure 4

The Bnatt mutants changed the lignin content, seed coat thickness and seed coat content. (a) The seed coat structure, thickness and lignin content of WT and Bnatts at 28 DAF, 35 DAF, 42 DAF, 49 DAF were observed under microscope. The observation of seed coat thickness was performed on mature seeds. Scale = 100 μm. oi, outer integument; pl, palisade layer; ii, inner integument; en, endosperm; em, embryo. (b) Determination of lignin content in seed and seed coat in Bnatt mutants and WT. The data and error bars represent the mean ± SD; Student's t‐test was used for statistical analysis between the mutant and its corresponding WT (*P ≤ 0.05). (c) The thickness of seed coat at mature stage. n = 30. (d) The seed coat content at mature stage. Seed coat content = seed coat weight/seed weight, n = 100. In c and d, values are mean ± SD; different letters represent significant differences at P < 0.05, based on an ANOVA analysis with Fisher LSD test. (e) Correlation analysis between seed coat content, seed coat lignin content and seed coat thickness.

Figure 5

Seed oil content, protein content and the contents of fatty acid components in WT and Bnatts. (a) Oil content. (b) Protein content. (c–e) The contents of fatty acid components C18:1, C18:2, C18:3 in seed, seed coat and embryos. Values are mean ± SD; different letters represent significant differences at P < 0.05, based on an ANOVA analysis with Fisher LSD test.

Figure 6

Bnatt mutants promoted seed oil accumulation in mature seeds. (a) OBs in mature seeds were examined by scanning transmission electron microscopy. The magnification is 3000 times. (b) PBs in mature seeds were examined by scanning transmission electron microscopy. The magnification is 2000 times. (c) Number of OBs per 100 μm². (d) Size per large OB. (e) Size per small OB. (f) Number of PBs per 100 μm². (g) Size per PB. Image J software was used to calculate the number and size of OBs. OB, oil body; LOB, larger oil body; SOB, small oil body; PB, protein body. In c–g, values are mean ± SD; different letters represent significant differences at P < 0.05, based on an ANOVA analysis with Fisher LSD test. (h) The expression level of genes involved in seed oil metabolism. MCMT, malonyltransferase; ENR, enoyl‐ACP reductase; FATA, acyl‐ACP thioesterase A; GPAT9, glycerol‐3‐phosphate acyltransferase 9; LPCAT, lysophosphatidylcholine acyltransferase; DGAT2, DAG acyltransferase 2; CPT, CDP choline: DAG cholinephosphotransferase; LEC, Leafy cotyledon; FUS3, Fusca 3; WRI1, wrinkled1; FAD, fatty acid desaturase; PDCT, phosphatidylcholine: DAG choline phosphotransferase; FAE1, fatty acidelongation 1; CTS, peroxisomal ABC transporter 1; OBO1, oil body oleosin 1; OBO2, oil body oleosin 2; OBO3, oil body oleosin 3; CALO, caleosin; Total RNA was extracted from 21, 28 and 31 DAF seeds. Relative expression level was calculated by comparison to Actin7's expression level. Values are means ±SD (n = 3).

Figure 7

A proposed working model of Bnatt mutants in the regulation of seed coat colour, oil content and fatty acid composition. ER, endoplasmic reticulum; OB, oil body. Solid line indicates direct regulation, whereas dashed line indicates direct or indirect regulation. Arrows and T bars indicate promoting and inhibitory effects, respectively.