Defective APETALA2 Genes Lead to Sepal Modification in Brassica Crops

Abstract

Many vegetable and oilseed crops belong to Brassica species. The seed production of these crops is hampered often by abnormal floral organs, especially under the conditions of abiotic conditions. However, the molecular reasons for these abnormal floral organs remains poorly understood. Here, we report a novel pistil-like flower mutant of B. rapa. In the flower of this mutant, the four sepals are modified to one merged carpel that look like a ring in the sepal positions, enveloping some abnormal stamens and a pistil, and resulting in poor seed production. This novel mutant is named sepal-carpel modification (scm). DNA sequencing showed that the BrAP2a gene, the ortholog of Arabidopsis APETALA2 (AP2) that specifies sepal identity, losses the function of in scm mutant due to a 119-bp repeated sequence insertion that resulted in an early transcription termination. BrAP2b, the paralog of BrAP2a featured two single-nucleotide substitutions that cause a single amino acid substitution in the highly conserved acidic serine-rich transcriptional activation domain. Each of the two BrAP2 genes rescues the sepal defective phenotype of the ap2-5 mutant of Arabidopsis. Furthermore, the knockout mutation of the corresponding BnAP2 genes of oilseed rape (B. napus) by CRISPR/Cas9-mediated genome editing system resulted in scm-like phenotype. These results suggest that BrAP2 gene plays a key role in sepal modification. Our finding provides an insight into molecular mechanism underlying morphological modification of floral organs and is useful for genetic manipulation of flower modification and improvement of seed production of Brassica crops.

Keywords: APETALA2 (AP2); Brassica; carpeloid sepal; flower development; oilseed rape; organ modification.

FIGURE 1

Characteristics the sepal carpeloid mutants of Brassica. (A–C): inflorescence; (D,E1,E2,F1–F3): flower; (G,H): floral organs; (I–K): SEM photos of flowers; (L1,L2,M1,M2,N1,N2): SEM photos of floral organs; (A,D,G,I,L1,L2): wild type Brassica (WT). (L1,L2) Are sepal (left) and stamen (right); (B,E1,E2,H,J,M1,M2): the typical sepal carpeloid mutant (scm). M1 is a sepal carpeloid organ, and M2 is an incomplete stamen to carpel organ with one ovule; (C,F1–F3,K,N1,N2): the incomplete sepal carpeloid mutant (scm2). Arrows indicate sepal carpel-like transition in F2 and petal stamen-like transition in F3. N1 is an incomplete petal to stamen organ, and N2 is a stamen of scm2. Bars in E1 and E2 are 5 mm; bars in I–K,L1,M1 are 1 mm; bars in L2,M2,N1,N2 are 500 μm; and bars in the others are 10 mm.

FIGURE 2

Expression of ABCE classic genes in the scm mutant and wild-type Brassica. WTr, wild-type B. rapa; WTn, wild-type B. napus; scm, sepal carpeloid mutant of Brassica.

FIGURE 3

Mutations of the two AP2 orthologs in the scm of B. rapa. (A) Schematic of the two AP2 gene mutations in scm. The BrAP2a cDNA from scm (SCM-a) had a 119-bp sequence repeat mutation, which was derived from the transcription of a 291-bp repeat in genomic DNA that consisted of nucleotides 648–938 repeated after nucleotide 938. BrAP2b cDNA of scm (SCM-b) had an A to C single nucleotide mutation that transformed the 18th glutamic acid into aspartic acid (E18D). (B) A schematic of the location of the designed primers. The relative locations of the AP2 primers in the scm and wild-type B. rapa (WTr) sequences are shown (upper part). PCR amplification of genome DNA and cDNA of WTr and the scm using primer pairs of AP2-F/R, AP2-F/R2, and AP2-F2/R (lower part).

FIGURE 4

Wild type and an ap2 weak mutant of Arabidopsis transformed with AP2 genes from B. rapa. (A) Wild-type Arabidopsis (Col-0) transformed with AtAP2 (control) and two AP2 genes of B. rapa. Their over-expressed lines p35S:AtAP2/Col, p35S::BrAP2a/Col, and p35S::BrAP2b/Col had normal flowers similar to the wild-type flower. (B) The AP2 mutant of Arabidopsis (ap2-5), which has a mild sepal-to-carpel and petal-to-stamen phenotype, transformed with AtAP2 gene and two B. rapa AP2 genes. All three transgenic plants, p35S:AtAP2/ap2-5, p35S::BrAP2a/ap2-5, and p35S::BrAP2a/ap2-5, rescued the ap2-5 mutant defect and developed normal sepals and petals. (C) The knockdown lines, p35S::AtAP2/ap2-5 KD, p35S::BrAP2a/ap2-5 KD, and p35S::BrAP2b/ap2-5 KD, from three transgenic group of p35S:AtAP2/ap2-5, p35S::BrAP2a/ap2-5, and p35S::BrAP2a/ap2-5. (D) semi-quantitative RT-PCR and Western blot assays to validate the transgenic lines. Bars, 500 μm.

FIGURE 5

ap2 quadruple mutant in B. napus generated by CRISPR/Cas9 genome editing system. (A) The designed target sequence used for CRISPR/Cas9. The PAM sequence is indicated in red. (B) a1–a3: wild-type Brassica (WT); b1–b3: a strong ap2 quadruple mutant; c1–c4: a weak ap2 quadruple mutant. a1, b1, c1: inflorescence; a2, b2, c2: flower; a3, b3, c3: floral organs; c4: enlargement of the floral organ shown in the red box in c3. The ap2 quadruple mutant of B. napus, which has a similar sepal-to-carpel and petal-to-stamen phenotype as the scm mutant of Brassica. (C) Sequencing results of the target sites in the strong ap2 mutant. Bars are 1 mm in c4 and 10 mm in all the others panels.