Expression of Camelina WRINKLED1 Isoforms Rescue the Seed Phenotype of the Arabidopsis wri1 Mutant and Increase the Triacylglycerol Content in Tobacco Leaves

Abstract

Triacylglycerol (TAG) is an energy-rich reserve in plant seeds that is composed of glycerol esters with three fatty acids. Since TAG can be used as a feedstock for the production of biofuels and bio-chemicals, producing TAGs in vegetative tissue is an alternative way of meeting the increasing demand for its usage. The WRINKLED1 (WRI1) gene is a well-established key transcriptional regulator involved in the upregulation of fatty acid biosynthesis in developing seeds. WRI1s from Arabidopsis and several other crops have been previously employed for increasing TAGs in seed and vegetative tissues. In the present study, we first identified three functional CsWRI1 genes (CsWRI1A. B, and C) from the Camelina oil crop and tested their ability to induce TAG synthesis in leaves. The amino acid sequences of CsWRI1s exhibited more than 90% identity with those of Arabidopsis WRI1. The transcript levels of the three CsWRI1 genes showed higher expression levels in developing seeds than in vegetative and floral tissues. When the CsWRI1A. B, or C was introduced into Arabidopsis wri1-3 loss-of-function mutant, the fatty acid content was restored to near wild-type levels and percentages of the wrinkled seeds were remarkably reduced in the transgenic lines relative to wri1-3 mutant line. In addition, the fluorescent signals of the enhanced yellow fluorescent protein (eYFP) fused to the CsWRI1 genes were observed in the nuclei of Nicotiana benthamiana leaf epidermal cells. Nile red staining indicated that the transient expression of CsWRI1A. B, or C caused an enhanced accumulation of oil bodies in N. benthamiana leaves. The levels of TAGs was higher by approximately 2.5- to 4.0-fold in N. benthamiana fresh leaves expressing CsWRI1 genes than in the control leaves. These results suggest that the three Camelina WRI1s can be used as key transcriptional regulators to increase fatty acids in biomass.

Keywords: Camelina sativa; WRINKLED1; fatty acid; leaves; oil; triacylglycerol.

FIGURE 1

Alignment of the deduced amino acid sequences of WRI1 isoforms from C. sativa and A. thaliana. Non-conserved and conservatively changed amino acid residues are shaded in black and gray, respectively. Bright green and orange boxes indicate conserved AP2/EREBP DNA binding motifs and the ‘VYL’ motif for transcriptional activation of WRI1, respectively.

FIGURE 2

Phylogenetic tree of WRI1 from higher plants including C. sativa. Phylogenetic tree was generated using MEGA6.06 by the maximum likelihood method. Bootstrap value percentages of 500 replicates are shown at the branching points. AlWRI1, Arabidopsis lyrata (EFH52510.1); AsWRI1, Avena sativa (SRX1079426); BnWRI1, Brassica napus (ABD16282.1); CeWRI1, Cyperus esculentus (SRX1079431); CnWRI1, Cocos nucifera (JQ040545); EgWRI1, Elaeis guineensis; GhWRI1, Gossypium hirsutum (TC200263); JcWRI1, Jatropha curcas (AIA57945.1); OsWRI1, Oryza sativa (CAE00853.1); PtWRI1, Populus trichocarpa (SRX1079428); RcWRI1, Ricinus communis (AB774159.1, AB774160.1); StWRI1, Solanum tuberosum (SRX1079426); VvWRI1, Vitis vinifera (CBI32013.3); ZmWRI1, Zea mays (ACF83189.1, ACF80269.1).

FIGURE 3

Expression of three CsWRI1 isoforms in various C. sativa organs. (A) The primer specificity of three CsWRI1 isoforms. Plasmids harboring CsWRI1A. CsWRI1B, or CsWRI1C were used in PCR analysis as templates. PCR products were analyzed on 1% agarose gels. (B) Total RNAs were isolated from the roots (R) of 2-week-old stems (S) of 4-week-old, leaves (L) and buds (B) from 5-week-old, and open flowers (OF) and developing seeds (DS1, DS2, and DS3; 10, 20, and 30 days after flowering, respectively) of 6-week-old C. sativa plants.

FIGURE 4

Subcellular localization of CsWRI1s:eYFP proteins in N. benthamiana epidermis. (A) Schematic diagram of CsWRI1A:eYFP. CsWRI1B:eYFP, and CsWRI1C:eYFP constructs. 35S-P, cauliflower mosaic virus 35S promoter; LB, left border; RB, right border; rbs-T, the terminator of ribulose-1,5-bisphosphate carboxylase and oxygenase small subunit from pea (Pisum sativum). (B–J) Agrobacterium harboring the CsWRI1A:eYFP (upper row), CsWRI1B:eYFP (middle row) or CsWRI1C:eYFP (bottom row) construct was infiltrated into N. benthamiana leaves and then the fluorescent signals were visualized under laser confocal scanning microscopy. YFP signals (B,E,H) from the CsWRI1s:eYFP constructs. The nucleus (C,F,I) was visualized by staining with DAPI under the UV filter. Merged image between signals of YFP and the nucleus (D,G,J). EV, empty vector (pBA002). Bars = 10 μm.

FIGURE 5

Isolation of T-DNA inserted wri1-3 knockout mutant and complementation of CsWRI1A. CsWRI1B, and CsWRI1C in wri1-3 mutants. (A) Genomic organization of the WRI1 gene inserted with T-DNA in wri1-3. (B) SEM image of the seed morphology of WT and wri1-3 mutant seeds. Bar = 10 μm. (C) Schematic diagram of the binary vector constructs for the expression of CsWRI1s in Arabidopsis wri1-3 mutant. (D) Genomic DNA-PCR of CsWRI1s transgenes of WT, wri1-3 mutant, and complementation transgenic lines. w-EV represents transgenic Arabidopsis wri1-3 mutant introducing empty vector. The numbers indicate independent transgenic lines (T₁).

FIGURE 6

Fatty acid content and rescue of the wrinkled phenotype from seeds of WT, wri1-3, and transgenic plants. Fatty acids were extracted from dry seeds of WT, wri1-3, and transgenic plants (T₂), transmethylated, and analyzed using gas chromatography. Fatty acid content was expressed on the basis of seed dry weight (A) or seed number (B). Error bar indicates SE of three independent measurements. (^∗∗P < 0.01; ^∗P < 0.05; Student’s t-test). (C) The graph showing morphology of dry seeds from WT, wri1-3, and transgenic lines (T₂). w-EV represents transgenic Arabidopsis of wri1-3 mutant introducing empty vector. The numbers indicate independent transgenic lines.

FIGURE 7

Expression of CsWRI1s and WRI1 downstream targets in developing Arabidopsis seeds. Total RNA was isolated from developing seeds 6–8 days after flowering of wri1-3 mutant and transgenic plants (T₂). The isolated RNAs were subjected to RT-PCR analysis. The EIF4A1 gene was used to determine RNA quality and quantity. PI-PKβ1, At5g52920. BCCP2, At5g15530.

FIGURE 8

Transient expression of three CsWRI1 isoforms in N. benthamiana leaves. (A) Schematic diagram of the binary vector constructs for the transient expression of CsWRI1s in N. benthamiana leaves. (B) Oil body (OB) counts in N. benthaminana leaves expressing empty vector (pBA002), CsWRI1A. CsWRI1B, or CsWRI1C. Values are averages and SD of three individual images. Data were statistically analyzed using Student’s t-test (^∗P < 0.01). (C) Agrobacterium harboring CsWRI1A. CsWRI1B, or CsWRI1C was infiltrated in N. benthamiana leaves and then the leaf disks were stained with Nile red solution. The fluorescent signals were visualized by laser confocal scanning microscopy. The white arrows indicate OBs. Bars = 20 μm.

FIGURE 9

Fatty acid content present in triacylglycerols (A) and total lipids (B) of N. benthamiana leaves transiently overexpressing three CsWRI1 isoforms. Agrobacterium harboring empty vector (EV), CsWRI1A. CsWRI1B, or CsWRI1C was infiltrated to 5-week-old N. benthamiana leaves and the N. benthamiana plants were further incubated for 5 days. Total lipids were extracted from N. benthamiana leaf disks including the injection region and analyzed by gas chromatography or fractionated on thin layer chromatography. The eluted TAG fractions were transmethylated and the fatty acid methyl esters were analyzed by gas chromatography. Each value is the mean ± SE of three independent measurements. Data were statistically analyzed using Student’s t-test (^∗P < 0.01).