Splice Variants of the Castor WRI1 Gene Upregulate Fatty Acid and Oil Biosynthesis When Expressed in Tobacco Leaves

Abstract

The plant-specific WRINKLED1 (WRI1) is a member of the AP2/EREBP class of transcription factors that positively regulate oil biosynthesis in plant tissues. Limited information is available for the role of WRI1 in oil biosynthesis in castor bean (Ricinus connunis L.), an important industrial oil crop. Here, we report the identification of two alternatively spliced transcripts of RcWRI1, designated as RcWRI1-A and RcWRI1-B. The open reading frames of RcWRI1-A (1341 bp) and RcWRI1-B (1332 bp) differ by a stretch of 9 bp, such that the predicted RcWRI1-B lacks the three amino acid residues "VYL" that are present in RcWRI1-A. The RcWRI1-A transcript is present in flowers, leaves, pericarps and developing seeds, while the RcWRI1-B mRNA is only detectable in developing seeds. When the two isoforms were individually introduced into an Arabidopsiswri1-1 loss-of-function mutant, total fatty acid content was almost restored to the wild-type level, and the percentage of the wrinkled seeds was largely reduced in the transgenic lines relative to the wri1-1 mutant line. Transient expression of each RcWRI1 splice isoform in N. benthamiana leaves upregulated the expression of the WRI1 target genes, and consequently increased the oil content by 4.3-4.9 fold when compared with the controls, and RcWRI1-B appeared to be more active than RcWRI1-A. Both RcWRI1-A and RcWRI1-B can be used as a key transcriptional regulator to enhance fatty acid and oil biosynthesis in leafy biomass.

Keywords: RcWRI1; alternative splice form; castor (Ricinus communis L.); fatty acid and oil biosynthesis; tobacco (Nicotiana benthamiana L.).

Figure 1

Sequence alignment of two RcWRI1 and AtWRI1 protein isoforms. The alignment was performed by the ClustalW program. Most of the difference between protein sequences of RcWRI1s and AtWRI1 occurs at the C-terminal half of the protein. “VYL” motif in AtWRI1 and RcWRI1-A is absent in RcWRI1-B. The high and low consensus amino acid residues are denoted by black and gray colors, respectively. Two AP2 domains are marked by black boxes. The sequence “VYL” present in the first AP2 domain are marked by red box.

Figure 2

Phylogenic tree of RcWRI1s and WRI1 homologous from other plant species. Phylogenetic tree was generated using MEGA6 by the neighbor-joining method. Percentage values on each branch represent the corresponding bootstrap probability. Protein sequences used for phylogenic analysis included BnWRI1 (ADO16346), ZmWRI1-A (ACG32367.1), ZmWRI1-B(AIB05036.1), CeWRI1 (SRX1079431), PtWRI1 (XP_002311921.2), GmWRI1 (XP_006596986.1), BdWRI1 (Brai4g43877), AtWRI1 (AAP80382), AtWRI2 (ABG25074), AtWRI3 (NP_001320852.1), AtWRI4 (ABK32182.1), RcWRI2 (BAM75179.1) and EgWRI1 (AHX71676.1). The two RcWRI1s protein are highlighted by the grey arrowheads.

Figure 3

Expression of RcWRI1-A and RcWRI1-B in major castor bean organs. Expression profiles were determined by qRT-PCR using total RNA from leaves, flowers, pericarps, and developing seeds. RcActin was used as an internal control. Each value is the mean ± SD of six biological replicates. Letters a, b, c, d, e indicate significant differences at the level of p < 0.05 according to the Tukey’s test.

Figure 4

RcWRI1-A and RcWRI1-B complemented the Arabidopsis wrinkled seed phenotype. The seed phenotype was examined by a stereo microscope. (A) Wild type; (B) wri1-1 mutant; (C) wri1-1 expressing RcWRI1-A; (D) wri1-1 expressing RcWRI1-B.

Figure 5

Total fatty acid content in seeds of Arabidopsis wildtype, wri1-1 mutant, and the mutants expressing either RcWRI1-A or RcWRI1-B. Total fatty acids were extracted from the seed and transmethylated, followed by gas chromatography analysis. Fatty acid content was expressed as the percentage of seed dry weight. Each value is the mean ± SD of six biological replicates.

Figure 6

Expression profiles of WRI1 target genes in tobacco leaves expressing either RcWRI1-A or RcWRI1-B. Total RNA was isolated from the leaves expressing each of RcWRI1s, and used for qRT-PCR analysis. The primers were designed based on the coding sequences of PKp-β1, PKp-α, ACP1, PDH-E1α, BCCP2, and KAS1 of N. benthamiana. EV indicates empty-vector control. NbActin was used as an internal control. Each value is the mean ± SD of six biological replicates. Letters a, b, c indicate a significant difference at the level of p < 0.05 according to the Tukey’s test.

Figure 7

Total fatty acid content in N. benthamiana leaves expressing each of RcWRI1s. Fatty acids were extracted from the leaf samples and then transmethylated, followed by gas chromatography analysis. Fatty acid content was expressed as the percentage of leaf dry weight. EV indicates empty-vector control. Each value is the mean ± SE of twelve biological replicates. Letters a, b, c indicate a significant difference at the level p < 0.05 according to the Tukey’s test.

Figure 8

Fatty acid profiles in N. benthamiana leaves expressing each of RcWRI1s. Fatty acids were extracted from the leaf samples and then transmethylated, followed by gas chromatography analysis. Fatty acid content was expressed as the percentage of leaf dry weight. EV indicates empty-vector control. Each value is the mean ± SE of twelve biological replicates. Letters a, b indicate significant differences at the level p < 0.05 according to the Tukey’s test.

Figure 9

Schematic representation of RcWRI1-A, and RcWRI1-B expression constructs used for transient expression in Nicotiana benthamiana leaves and overexpression in the Arabidopsis wri1-1 mutant. (A) The RcWRI1-A expression cassette in pCAMBIA1303. (B) The RcWRI1-B expression cassette in pCAMBIA1303. (C) The RcWRI1-A expression cassette in pJC-Gly-DsRED. (D) The RcWRI1-B expression cassette in pJC-Gly-DsRED. 35S: CaMV 35S promoter. HPT: Hygromycin resistance gene. 35S polyA: 35S poly (A) signal sequence. Nos polyA: Nopaline synthase poly (A) signal sequence. Gly: Gly-promoter. BAR: Herbicide resistance bar gene. Gly term: Gly terminator. NOS term: Nopaline synthase terminator. GFP: Green fluorescent protein. RB: Right border. LR: Left border. DSRed: Discosoma red fluorescent protein.

Figure 10

Tobacco leaf image showing the regions infiltrated by the Agrobacterium. The left-half part of the leaf was infiltrated with Agrobacterium containing the RcWRI1 expression vector, while the right-half part of the leaf was infected by Agrobacterium containing an empty vector. The solid black circle shows the region infiltrated by the target gene, and dotted black circle marks the region infected by the empty-vector control.