Sunflower WRINKLED1 Plays a Key Role in Transcriptional Regulation of Oil Biosynthesis

Abstract

Sunflower (Helianthus annuus) is one of the most important oilseed crops worldwide. However, the transcriptional regulation underlying oil accumulation in sunflower is not fully understood. WRINKLED1 (WRI1) is an essential transcription factor governing oil accumulation in plant cells. Here, we identify and characterize a sunflower ortholog of WRI1 (HaWRI1), which is highly expressed in developing seeds. Transient production of HaWRI1 stimulated substantial oil accumulation in Nicotiana benthamiana leaves. Dual-luciferase reporter assay, electrophoretic mobility shift assay, fatty acid quantification, and gene expression analysis demonstrate that HaWRI1 acts as a pivotal transcription factor controlling the expression of genes involved in late glycolysis and fatty acid biosynthesis. HaWRI1 directly binds to the cis-element, AW-box, in the promoter of biotin carboxyl carrier protein isoform 2 (BCCP2). In addition, we characterize an 80 amino-acid C-terminal domain of HaWRI1 that is crucial for transactivation. Moreover, seed-specific overexpression of HaWRI1 in Arabidopsis plants leads to enhanced seed oil content as well as upregulation of the genes involved in fatty acid biosynthesis. Taken together, our work demonstrates that HaWRI1 plays a pivotal role in the transcriptional control of seed oil accumulation, providing a potential target for bioengineering sunflower oil yield improvement.

Keywords: WRI1; gene expression; plant oil biosynthesis; sunflower; transactivation; transcription factor.

Figure 1

Phylogenetic analysis of AtWRI1 homologs from H. annuus. The phylogenetic tree was created using protein sequences of AtWRI1 and all double AP2 domain-containing proteins. The tree was constructed by MEGA X using the neighbor-joining method. The number of bootstrap replicates is 1000. Putative WRI1 ortholog identified from H. annuus was highlighted by a gray box.

Figure 2

Expression analysis of HaWRI1 and WRI1 target genes in a variety of H. annuus tissues by quantitative real-time PCR (qRT-PCR). (A) HaWRI1 expression in different tissues of H. annuus plants as indicated. Results are shown as means ± SE (n = 3). (B) HaWRI1 expression at various stages during seed development [12–27 days after flowering (DAF) as indicated]. Results are shown as means ± SE (n = 4–5). (C) Expression analysis of selected genes known as WRI1 targets in developing seeds of H. annuus. Expression level of HaBCCP2, HaPKP-β1, HaKASI, and HaACP1, at various stages during seed development (12–27 DAF as indicated) was quantified by qRT-PCR. Results are shown as means ± SE (n = 4–5).

Figure 3

Functional analysis of HaWRI1. (A) Oil droplets in N. benthamiana leaves transiently expressing HaWRI1. Empty vector (EV) is used as a control. Confocal fluorescence images of N. benthamiana leaf mesophyll tissues displaying oil droplets stained with BODIPY (as indicated by the white arrows) were shown. Scale bar is 20 μM. (B) TAG content in N. benthamiana leaves transiently producing HaWRI1. Results are shown as means ± SE (n = 4). “**” indicates a significant difference (p < 0.01, Student’s t-test) between HaWRI1 and EV. (C) Schematic diagram of constructs used in the dual luciferase assay in N. benthamiana leaves through transient expression. (D) Transactivation activity of HaWRI1 on the promoters of BCCP2 and PKP-β1 (proBCCP2 and proPKP-β1) in N. benthamiana leaves. Results are shown as means ± SE (n = 6). “**” indicates a significant difference (p < 0.01, Student’s t-test) between HaWRI1 and control (reporter without addition of HaWRI1). (E) Binding of the HaWRI1 AP2 domain (amino acids 52–234) to probe containing AW-box in proBCCP2 using EMSA. (F) Schematic diagram of HaWRI1. Acidic amino acid residues in the C-terminal HaWRI1 are highlighted in red color. (G) Transactivation assay of HaWRI1 in yeast cells. HaWRI1 full-length and a series of HaWRI1 truncated variants were subcloned to vector fused with yeast GAL4 DNA binding domain (BD). Transactivation activity was measured via β–galactosidase assay of liquid cultures. Results are shown as means ± SE (n = 3).

Figure 4

Functional analysis of transgenic Arabidopsis overexpressing HaWRI1 (HaWRI1-OE). (A) Fatty acid content of seeds of Arabidopsis HaWRI1-OE lines. Results are shown as means ± SE (n = 3–4). (B) Mature dry seeds of WT and HaWRI1-OE lines. Scale bar is 200 μM. (C) Quantitative analysis of seed mass WT and HaWRI1-OE lines. Seed mass was attained by measuring 100 seeds per sample, and results are shown as means ± SE (n = 4–5). (D) Quantitative analysis of seed size of WT and HaWRI1-OE lines. Seed size is denoted by seed area, perimeter, length, and width with box plots displaying medians (lines), interquartile ranges (boxes) and 1.5× interquartile ranges (whiskers) of WT and HaWRI1-OE lines (n = 100). “*” and “**” indicate significant differences (p < 0.05 and p < 0.01, respectively, Student’s t-test) between WT and HaWRI1-OE lines. (E) Pooled siliques (10 DAF) from WT and HaWRI1-OE lines were used for the assay. Transcript levels of various WRI1 target genes (BCCP2, PKP-β1, ACP1, KASI, ENR1, ENO1, PDH-E1β, LPD1, BCCP1) were measured by qRT-PCR. Results are shown as means ± SE (n = 3). “*” and “**” indicate significant differences (p < 0.05 and p < 0.01, respectively, Student’s t-test) between WT and HaWRI1-OE lines in terms of expression of WRI1 target genes.