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.
The MEX1 locus of Chlamydomonas reinhardtii was identified in a genetic screen as a factor that affects starch metabolism. Mutation of MEX1 causes a slow-down in the mobilization of storage polysaccharide. Cosegregation and functional complementation analyses were used to assess the involvement of the Mex1 protein in starch degradation. Heterologous expression experiments performed in Escherichia coli and Arabidopsis thaliana allowed us to test the capacity of the algal protein in maltose export. In contrast to the A. thaliana mex1 mutant, the mutation in C. reinhardtii does not lead to maltose accumulation and growth impairment. Although localized in the plastid envelope, the algal protein does not transport maltose efficiently across the envelope, but partly complements the higher plant mutant. Both Mex orthologs restore the growth of the E. coli ptsG mutant strain on glucose-containing medium, revealing the capacity of these proteins to transport this hexose. These findings suggest that Mex1 is essential for starch mobilization in both Chlamydomonas and Arabidopsis, and that this protein family may support several functions and not only be restricted to maltose export across the plastidial envelope.
Chlamydomonas reinhardtii represents an ideal model microbial system to decipher starch metabolism. In this green algae, in cells growing in photosynthetic conditions, starch mainly accumulates as a sheath surrounding the pyrenoid while in cells subjected to a nutrient starvation, numerous starch granules are filling up the plastid stroma. The mechanisms underlying and regulating this switch from photosynthetic to storage starch metabolisms are not known. In this work, we have isolated a Chlamydomonas mutant strain containing a deletion in chromosome 2 which displays abnormal starch granule distribution. Under nitrogen starvation, this strain contains an additional starch granules population. These granules are twice as big as the wild-type granules and display characteristics of photosynthetic starch. Genetic and functional complementation analyses allowed us to identify the gene responsible for this original phenotype which was called BSG1 for “Bimodal Starch Granule”. Possible roles of BSG1 in starch metabolism modifications during the transition from photosynthetic to starved growth conditions are discussed.
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