Methylcrotonyl-CoA Carboxylase Regulates Triacylglycerol Accumulation in the Model Diatom Phaeodactylum tricornutum.

Feng Ge,Chris Bowler,Weichao Huang,Jin Xu,Juan Yang,Qian Xiong,Chunye Zhang,Zhuo Chen,Hanhua Hu

doi:10.1105/tpc.114.124982

Abstract

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.

Highlights

Diatoms are a diverse group of eukaryotic, unicellular, photosynthetic microalgae believed to be responsible for ;40% of the total carbon fixation in the oceans and 20% of the primary production on Earth (Falkowski et al, 1998; Field et al, 1998)
Relative mRNA levels of MCC2 (A), MCC2 protein levels (B), growth (C), accumulation of TAGs detected by Nile Red assay and gas chromatography (D), nitrate utilization (E), and total lipid content (F) of wild-type and two RNA interference (RNAi) silenced lines grown in f/2 (NaNO3 concentration was reduced to 500 mM) enriched artificial seawater medium. mRNA levels, immunoblotting, and lipid content analyses were performed at day 8
The formation and accumulation of TAGs may be important for carbon and energy storage in response to environmental stress (Hu et al, 2008), but despite intense interest, the mechanisms of TAG accumulation are largely uncharacterized to date

Summary

Introduction

Diatoms are a diverse group of eukaryotic, unicellular, photosynthetic microalgae believed to be responsible for ;40% of the total carbon fixation in the oceans and 20% of the primary production on Earth (Falkowski et al, 1998; Field et al, 1998). P. tricornutum can grow rapidly to high cell densities, has a short biomass doubling time, is constituted of at least 20% lipids by dry cell weight under normal culture conditions (Chisti, 2007), and accumulates even higher levels of lipids under nutrient depletion (Valenzuela et al, 2013). This organism has attracted increasing attention for biofuel production. Together with the fact that P. tricornutum can be genetically transformed (Siaut et al, 2007; De Riso et al, 2009), these resources provide the possibilities to perform comparative genomic and proteomic studies and to understand more about the molecular mechanisms involved in diatom lipid accumulation under different growth conditions

Methods

Results

Conclusion