Ge Feng, Huang Weichao, Chen Zhuo, Zhang Chunye, Xiong Qian, Bowler Chris, Yang Juan, Xu Jin, Hu Hanhua
Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China.
Diatom Biology Group, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China.
Plant Cell. 2014 Apr;26(4):1681-1697. doi: 10.1105/tpc.114.124982. Epub 2014 Apr 25.
The model marine diatom Phaeodactylum tricornutum can accumulate high levels of triacylglycerols (TAGs) under nitrogen depletion and has attracted increasing attention as a potential system for biofuel production. However, the molecular mechanisms involved in TAG accumulation in diatoms are largely unknown. Here, we employed a label-free quantitative proteomics approach to estimate differences in protein abundance before and after TAG accumulation. We identified a total of 1193 proteins, 258 of which were significantly altered during TAG accumulation. Data analysis revealed major changes in proteins involved in branched-chain amino acid (BCAA) catabolic processes, glycolysis, and lipid metabolic processes. Subsequent quantitative RT-PCR and protein gel blot analysis confirmed that four genes associated with BCAA degradation were significantly upregulated at both the mRNA and protein levels during TAG accumulation. The most significantly upregulated gene, encoding the β-subunit of methylcrotonyl-CoA carboxylase (MCC2), was selected for further functional studies. Inhibition of MCC2 expression by RNA interference disturbed the flux of carbon (mainly in the form of leucine) toward BCAA degradation, resulting in decreased TAG accumulation. MCC2 inhibition also gave rise to incomplete utilization of nitrogen, thus lowering biomass during the stationary growth phase. These findings help elucidate the molecular and metabolic mechanisms leading to increased lipid production in diatoms.
典型海洋硅藻三角褐指藻在氮缺乏条件下能够积累高水平的三酰甘油（TAGs），作为一种潜在的生物燃料生产系统，它已引起越来越多的关注。然而，硅藻中TAG积累所涉及的分子机制在很大程度上尚不清楚。在此，我们采用无标记定量蛋白质组学方法来评估TAG积累前后蛋白质丰度的差异。我们总共鉴定出1193种蛋白质，其中258种在TAG积累过程中发生了显著变化。数据分析揭示了参与支链氨基酸（BCAA）分解代谢过程、糖酵解和脂质代谢过程的蛋白质发生了主要变化。随后的定量逆转录-聚合酶链反应（RT-PCR）和蛋白质凝胶印迹分析证实，与BCAA降解相关的四个基因在TAG积累过程中mRNA和蛋白质水平均显著上调。选择上调最显著的基因，即编码甲基巴豆酰辅酶A羧化酶（MCC2）β亚基的基因进行进一步的功能研究。通过RNA干扰抑制MCC2的表达扰乱了碳（主要以亮氨酸形式）向BCAA降解的通量，导致TAG积累减少。MCC2抑制还导致氮的不完全利用，从而降低了稳定生长期的生物量。这些发现有助于阐明导致硅藻脂质产量增加的分子和代谢机制。
