LEAFY COTYLEDON1 is a key regulator of fatty acid biosynthesis in Arabidopsis

Abstract

In plants, fatty acids are de novo synthesized predominantly in plastids from acetyl-coenzyme A. Although fatty acid biosynthesis has been biochemically well studied, little is known about the regulatory mechanisms of the pathway. Here, we show that overexpression of the Arabidopsis (Arabidopsis thaliana) LEAFY COTYLEDON1 (LEC1) gene causes globally increased expression of fatty acid biosynthetic genes, which are involved in key reactions of condensation, chain elongation, and desaturation of fatty acid biosynthesis. In the plastidial fatty acid synthetic pathway, over 58% of known enzyme-coding genes are up-regulated in LEC1-overexpressing transgenic plants, including those encoding three subunits of acetyl-coenzyme A carboxylase, a key enzyme controlling the fatty acid biosynthesis flux. Moreover, genes involved in glycolysis and lipid accumulation are also up-regulated. Consistent with these results, levels of major fatty acid species and lipids were substantially increased in the transgenic plants. Genetic analysis indicates that the LEC1 function is partially dependent on ABSCISIC ACID INSENSITIVE3, FUSCA3, and WRINKLED1 in the regulation of fatty acid biosynthesis. Moreover, a similar phenotype was observed in transgenic Arabidopsis plants overexpressing two LEC1-like genes of Brassica napus. These results suggest that LEC1 and LEC1-like genes act as key regulators to coordinate the expression of fatty acid biosynthetic genes, thereby representing promising targets for genetic improvement of oil production plants.

Figure 1.

Characterization of pER8-LEC1 transgenic plants. A, Ten-day-old pER8-LEC1 plants germinated and grown on MS medium in the absence (left) or presence (right) of different concentrations of estradiol as indicated at top. Bars = 2 mm. B, Northern-blot analysis of LEC1 expression in transgenic plants. Total RNA was prepared from 10-d-old pER8-LEC1 transgenic plants treated with different concentrations of estradiol as indicated at top for 16 h. Each lane contained 20 μg of RNA. C, Oil bodies (white dots in the bottom panels; stained with Nile Red) in vegetative tissues of pER8-LEC1 transgenic seedlings germinated and grown in the presence of 10 μm estradiol for 5 d. D, Fat Red 7B staining of 10-d-old pER8-LEC1 transgenic plants germinated and grown on MS medium in the absence (left) or presence (right) of different concentrations of estradiol as indicated at top. Bars = 2 mm. E, The accumulation of eicosenoic acid (C20:1; a fatty acid maker for the formation of TAG) in pER8-LEC1 transgenic seedlings germinated and grown in the presence of 10 μm estradiol for 10 d. Four independent transgenic lines (line numbers are given below the graph) were analyzed. Data obtained from line 23 are presented hereafter. See Table I for more details. FW, Fresh weight.

Figure 2.

Up-regulated differentially expressed genes in the fatty acid biosynthesis pathway in LEC1-OXi plants. A, Diagram of the fatty acid biosynthetic pathway with major steps in the plastid and the cytosol (endoplasmic reticulum). Up-regulated differentially expressed genes in LEC1-OXi plants are indicated by the Arabidopsis Genome Initiative codes. See Supplemental Table S3 for a complete list of the up-regulated differentially expressed genes of the pathway and their full names. B, Expression of representative fatty acid synthetic genes analyzed by quantitative real-time RT-PCR using total RNA prepared from wild-type and LEC1-OXi seedlings germinated and grown in the presence of 10 μm estradiol for 4 d. The relative expression level of each gene was normalized with that of ACTIN7. Error bars indicate sd values of three independent experiments. C, Expression of representative fatty acid synthetic genes analyzed by RT-PCR. The PCR was cycled 26 times for all samples. RNA was prepared from seedlings germinated and grown in the presence of 10 μm estradiol for 4 d. D, Expression of the β-subunit (AtCg00500) gene analyzed by northern blot. See Figure 1B for technical details.

Figure 3.

ABI3 and FUS3 are required for LEC1 function in fatty acid biosynthesis. A, RT-PCR analysis of the expression of LEC1, L1L, ABI3, FUS3, and LEC2 in wild-type and LEC1-OXi plants germinated and grown in the presence of 10 μm estradiol for 4 d. See Figure 2C legend for other technical details. B, Fatty acid contents in 10-d-old seedlings germinated and grown in the presence of 10 μm estradiol. Data presented are mean values of three independent experiments. FW, Fresh weight. Error bars denote sd. C, Quantitative RT-PCR analysis of the expression of representative fatty acid synthetic genes in 4-d-old seedlings germinated and grown in the presence of 10 μm estradiol. See Figure 2B legend for other technical details.

Figure 4.

LEC1-regulated fatty acid biosynthesis is partially dependent on WRI1. A, Quantitative RT-PCR analysis of WRI1 expression in wild-type (Col-0) and LEC1-OXi plants germinated and grown in the presence of 10 μm estradiol for 4 d. Results are mean values of three independent experiments. See Figure 2B legend for technical details. Error bars denote sd. B, Ten-day-old seedlings germinated and grown in the absence (MS) or presence of 0.1 μm estradiol. Bars = 2 mm. C, Fatty acid levels in Col-0, wri1, LEC1-OXi, and LEC1-OXi wri1 seedlings germinated and grown in the presence of 0.1 μm estradiol for 10 d. Results are mean values obtained from three independent experiments. FW, Fresh weight. Error bars indicate sd. [See online article for color version of this figure.]

Figure 5.

Functional characterization of BnLEC1 and BnL1L1. A, Ten-day-old seedlings germinated and grown in the absence (MS) or presence of 10 μm estradiol. Bars = 2 mm. B, RT-PCR analysis of the expression of representative fatty acid synthetic genes in BnL1L-OXi seedlings germinated and grown in the presence of 10 μm estradiol for 4 d (transgenic line 12). C, Fatty acid levels in pER10-BnL1L transgenic seedlings germinated and grown in the presence of 10 μm estradiol for 10 d. Three independent transgenic lines were tested. Results are mean values obtained from two independent experiments. Error bars indicate sd. Note that the levels of C20:0, C20:1, and C22:1 were under the detection limit in wild-type seedlings (Col-0; dark blue bars). FW, Fresh weight. D, Fatty acid levels in pER10-BnLEC1 transgenic seedlings analyzed as described for C. [See online article for color version of this figure.]