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

In plants, fatty acids are de novo synthesized predominantly in plastids fromacetyl-CoA. Although fatty acid biosynthesis has been biochemically well-studied, little isknown about the regulatory mechanisms of the pathway. Here, we show that overexpressionof the Arabidopsis (Arabidopsis thaliana) LEAFY COTYLEDON1 (LEC1) gene causesglobally increased expression of fatty acid biosynthetic genes, which are involved in keyreactions of condensation, chain elongation and desaturation of fatty acid biosynthesis. Inthe plastidial fatty acid synthetic pathway, over 58% of known enzyme-coding genes areupregulated in LEC1-overexpressing transgenic plants, including those encoding threesubunits of acetyl-CoA carboxylase, a key enzyme controlling the fatty acid biosynthesisflux. Moreover, genes involved in glycolysis and lipid accumulation are also upregulated.Consistent with these results, levels of major fatty acid species and lipids were substantiallyincreased in the transgenic plants. Genetic analysis indicates that the LEC1 function ispartially dependent on ABSCISIC ACID INSENSITIVE3, FUSCA3 and WRINKLED1 in theregulation of fatty acid biosynthesis. Moreover, a similar phenotype was observed intransgenic 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 theexpression of fatty acid biosynthetic genes, thereby representing a promising target forgenetic improvement of oil-production plants. Overall design: The pER8-LEC1 transgenic seedlings were germinated and grown in the presence of 10 µM estradiol for 4 days. The control sample was germinated and grown under the identical conditions without estradiol but containing 0.1% DMSO. Total RNA was prepared from fresh or frozen plant materials using the RNAeasy Plant Mini Kit (Qiagen China, Beijing). The first strand cDNA was synthesized, and then hybridized with the ATH1 oligonucleotide chips as described by the manufacture’s instructions (Affymetrix). The microarray hybridization data were collected and analyzed using related R (http://www.r-project.org/) packages provided by Bioconductor (http://www.biocoductor.org/). In brief, genes differentially expressed between wild type and mutant plants were selected by first removing “absent” genes which were never detected to be expressed in the experiments, then a two-side t-test was applied to remaining genes in order to test the expression difference between wide type and mutant plants. To avoid multiple testing problems, raw p-values were then adjusted into False Discovery Rate (FDR) using Benjamini and Hochberg approach. Finally, differentially expressed genes were defined as those FDR less than 0.2. Functional analysis of differentially expressed genes was carried out using the biological process category of Arabidopsis Gene Ontology. The hierarchical map of GO annotation was constructed according to the ontology tree provided by the Gene Ontology website (http://www.geneontology.com) as described previously (Zheng and Wang, 2008). Ontology categories that are significantly enriched among differentially expressed genes (hypergeometric test and FDR less than 0.1) were displayed as boxes in the map.

在植物中，脂肪酸主要在质体（plastids）中由乙酰辅酶A（acetyl-CoA）从头合成。尽管脂肪酸生物合成的生化过程已被充分解析，但该通路的调控机制仍有待深入阐明。本研究发现，拟南芥（Arabidopsis thaliana）LEAFY COTYLEDON1（LEC1）基因的过表达可全局上调脂肪酸生物合成相关基因的表达，这些基因参与脂肪酸生物合成的缩合、链延长及脱氢等关键反应。在质体脂肪酸合成通路中，已知的酶编码基因中有超过58%在LEC1过表达的转基因植株中被上调，其中包括控制脂肪酸合成通量的关键酶——乙酰辅酶A羧化酶的三个亚基编码基因。此外，糖酵解及脂质积累相关基因也呈现上调表达。与上述结果一致，转基因植株中主要脂肪酸种类及脂质的水平显著升高。遗传分析表明，在调控脂肪酸生物合成过程中，LEC1的功能部分依赖于脱落酸不敏感3（ABSCISIC ACID INSENSITIVE3）、FUSCA3及WRINKLED1。此外，在过表达甘蓝型油菜（Brassica napus）两个LEC1同源基因的转基因拟南芥植株中，也观察到了相似的表型。上述结果表明，LEC1及其同源基因可作为关键调控因子，协同调控脂肪酸生物合成相关基因的表达，因此有望成为油料作物遗传改良的潜在靶点。
整体实验设计：将pER8-LEC1转基因幼苗在含有10 μM雌二醇（estradiol）的培养基中萌发并生长4天；对照组幼苗在相同条件下培养，但不添加雌二醇，仅含有0.1%二甲基亚砜（DMSO）。使用RNAeasy植物总RNA提取试剂盒（Qiagen China, 北京）从新鲜或冷冻的植物材料中提取总RNA。反转录合成第一链cDNA后，按照制造商说明书（Affymetrix）使用ATH1寡核苷酸芯片进行杂交。利用Bioconductor（http://www.bioconductor.org）提供的R（http://www.r-project.org/）相关包对芯片杂交数据进行收集与分析。简言之，筛选差异表达基因的步骤如下：首先移除实验中未检测到表达的"absent"基因，随后对剩余基因进行双侧t检验，以比较野生型与突变体植株的表达差异。为避免多重检验问题，采用Benjamini-Hochberg法将原始p值校正为错误发现率（False Discovery Rate, FDR）。最终将FDR<0.2的基因定义为差异表达基因。采用拟南芥基因本体（Gene Ontology, GO）的生物过程分类对差异表达基因进行功能富集分析。按照Gene Ontology官网（http://www.geneontology.org）提供的本体树构建GO注释层级图，具体方法参照此前Zheng与Wang（2008年）的研究。将差异表达基因中显著富集的本体类别（超几何检验，FDR<0.1）以方框形式展示在层级图中。