System Analysis of an Arabidopsis Mutant Altered in de Novo Fatty Acid Synthesis Reveals Diverse Changes in Seed Composition and Metabolism
System Analysis of an Arabidopsis Mutant Altered in de Novo Fatty Acid Synthesis Reveals Diverse Changes in Seed Composition and Metabolism
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DOI：
10.1104/pp.108.134882
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发表时间：
2009-05-01
期刊：
影响因子：
7.4
通讯作者：
Thelen, Jay J.
中科院分区：
文献类型：
--
作者：
Chen, Mingjie;Mooney, Brian P.;Thelen, Jay J.
胚特异性过表达生物素羧基载体蛋白2(BCCP2)抑制了拟南芥成熟种子中的叶绿体乙酰辅酶A羧基酶(ACCase)，导致种子的油、蛋白质和碳水化合物组成发生改变。为了表征该突变的基因和蛋白质调控结果，并行进行了全球微阵列、二维差示凝胶电泳法、iTRAQ和定量免疫印迹。这些分析表明：(1)BCCP2转基因过表达不影响其他三个ACCase亚基的表达；(2)四个亚基在蛋白质水平上下调25%-70%，但不在转录水平上下调；(3)关键的糖酵解和从头合成脂肪酸/脂肪合成酶被诱导；(4)多种贮藏蛋白上调，但不同来源的转录本上调；(5)生物素合成途径在转录水平和蛋白质水平上都上调。生物素产量似乎与内源BCCP水平密切相关，因为BCCP2的过度表达主要产生apo-BCCP2，并由此产生ACCase受损的低油表型。糖酵解、质体丙酮酸脱氢酶复合体、脂肪酸和脂肪合成活性的差异表达表明了多种复杂的调节反应，包括反馈以及在脂肪酸和脂肪生物合成酶的情况下无效的“前馈”诱导。贮藏蛋白的诱导表明，油脂和蛋白质的合成共享碳中间体(S)，减少丙二酰辅酶A流入脂肪酸，将碳转移到氨基酸和蛋白质的合成。
Embryo-specific overexpression of biotin carboxyl carrier protein 2 (BCCP2) inhibited plastid acetyl-coenzyme A carboxylase (ACCase), resulting in altered oil, protein, and carbohydrate composition in mature Arabidopsis (Arabidopsis thaliana) seed. To characterize gene and protein regulatory consequences of this mutation, global microarray, two-dimensional difference gel electrophoresis, iTRAQ, and quantitative immunoblotting were performed in parallel. These analyses revealed that (1) transgenic overexpression of BCCP2 did not affect the expression of three other ACCase subunits; (2) four subunits to plastid pyruvate dehydrogenase complex were 25% to 70% down-regulated at protein but not transcript levels; (3) key glycolysis and de novo fatty acid/lipid synthesis enzymes were induced; (4) multiple storage proteins, but not cognate transcripts, were up-regulated; and (5) the biotin synthesis pathway was up-regulated at both transcript and protein levels. Biotin production appears closely matched to endogenous BCCP levels, since overexpression of BCCP2 produced mostly apo-BCCP2 and the resulting ACCase-compromised, low-oil phenotype. Differential expression of glycolysis, plastid pyruvate dehydrogenase complex, fatty acid, and lipid synthesis activities indicate multiple, complex regulatory responses including feedback as well as futile "feed-forward" elicitation in the case of fatty acid and lipid biosynthetic enzymes. Induction of storage proteins reveals that oil and protein synthesis share carbon intermediate(s) and that reducing malonyl-coenzyme A flow into fatty acids diverts carbon into amino acid and protein synthesis.