A Forward Genetic Approach in Chlamydomonas reinhardtii as a Strategy for Exploring Starch Catabolism

Abstract

A screen was recently developed to study the mobilization of starch in the unicellular green alga Chlamydomonas reinhardtii. This screen relies on starch synthesis accumulation during nitrogen starvation followed by the supply of nitrogen and the switch to darkness. Hence multiple regulatory networks including those of nutrient starvation, cell cycle control and light to dark transitions are likely to impact the recovery of mutant candidates. In this paper we monitor the specificity of this mutant screen by characterizing the nature of the genes disrupted in the selected mutants. We show that one third of the mutants consisted of strains mutated in genes previously reported to be of paramount importance in starch catabolism such as those encoding β-amylases, the maltose export protein, and branching enzyme I. The other mutants were defective for previously uncharacterized functions some of which are likely to define novel proteins affecting starch mobilization in green algae.

Time-resolved transcriptome analysis and lipid pathway reconstruction of the oleaginous green microalga Monoraphidium neglectum reveal a model for triacylglycerol and lipid hyperaccumulation

“…The starch, lipid, and central carbon metabolism of M. neglectum was reconstructed based on pathways described for A. thaliana [ 87 , 140 , 141 ] and C. reinhardtii [ 8 , 25 , 32 , 142 , 143 ]. Several filters were applied to ensure a high-quality pathway reconstruction for subsequent analysis of transcriptional regulation.…”

“…Today, the most detailed time course experiments were performed for the model green alga C. reinhardtii [ 12 – 14 , 22 ], where the nutrient starvation phase was investigated in great detail at several time points and in different mutants, such as the starchless strain sta6 [ 14 ]. However, in those works, the reverse phase of nutrient resupply after the starvation phase, triggering degradation of TAGs [ 24 ] and other storage compounds such as starch [ 25 ], has not been investigated so far. Furthermore, C. reinhardtii is not considered as the optimal choice for large-scale biofuel production [ 26 ].…”

A Review of Starch Biosynthesis in Relation to the Building Block-Backbone Model

“…Evidence supporting a role for SBEI in storage starch formation comes from maize mutants which show reduced germination (and therefore degradation of storage starch by α-amylases) when SBEI is absent [ 217 ]. Interestingly loss of SBEI in the green alga Chlamydomonas reinhardtii shows reduced transient starch degradation, suggesting that when it is expressed SBEI creates amylopectin structures more amenable to amylolysis [ 218 ]. Not all plants express SBEI, and plants such as Arabidopsis and Canola ( Brassica napus L.) possess only SBEII class enzymes.…”

The Chlamydomonas genome project: a decade on

“…These include: rapid doubling time (~8–12h), well-defined media and growth requirements, the ability to synchronize cultures with periodic light exposure, the capacity for classical genetic crosses to characterize mutant strains and efficient long-term cryopreservation [2]. The Chlamydomonas molecular and genetic toolbox has grown over the years: irradiated or chemically mutagenized lines have been identified with classical genetic screens [3–5], and RNAi-based knock-downs [6, 7]; zinc-finger nuclease-based mutagenesis [8] and efficient protocols for gene-specific mutant screens [9] are now available. A growing collection of laboratory-generated and environmentally-isolated strains is available at the Chlamydomonas resource center (http://chlamy.org/).…”