Trophic status of Chlamydomonas reinhardtii influences the impact of iron deficiency on photosynthesis.

To investigate the impact of iron deficiency on bioenergetic pathways in Chlamydomonas, we compared growth rates, iron content, and photosynthetic parameters systematically in acetate versus CO(2)-grown cells. Acetate-grown cells have, predictably (2-fold) greater abundance of respiration components but also, counter-intuitively, more chlorophyll on a per cell basis. We found that phototrophic cells are less impacted by iron deficiency and this correlates with their higher iron content on a per cell basis, suggesting a greater capacity/ability for iron assimilation in this metabolic state. Phototrophic cells maintain both photosynthetic and respiratory function and their associated Fe-containing proteins in conditions where heterotrophic cells lose photosynthetic capacity and have reduced oxygen evolution activity. Maintenance of NPQ capacity might contribute to protection of the photosynthetic apparatus in iron-limited phototrophic cells. Acetate-grown iron-limited cells maintain high growth rates by suppressing photosynthesis but increasing instead respiration. These cells are also able to maintain a reduced plastoquinone pool.

Chlorophyll-deficient mutants of Chlamydomonas reinhardtii that accumulate magnesium protoporphyrin IX.

Two Chlamydomonas reinhardtii mutants defective in CHLM encoding Mg-protoporphyrin IX methyltransferase (MgPMT) were identified. The mutants, one with a missense mutation (chlM-1) and a second mutant with a splicing defect (chlM-2), do not accumulate chlorophyll, are yellow in the dark and dim light, and their growth is inhibited at higher light intensities. They accumulate Mg-protoporphyrin IX (MgProto), the substrate of MgPMT and this may be the cause for their light sensitivity. In the dark, both mutants showed a drastic reduction in the amounts of core proteins of photosystems I and II and light-harvesting chlorophyll a/b-binding proteins. However, LHC mRNAs accumulated above wild-type levels. The accumulation of the transcripts of the LHC and other genes that were expressed at higher levels in the mutants during dark incubation was attenuated in the initial phase of light exposure. No regulatory effects of the constitutively 7- to 18-fold increased MgProto levels on gene expression were detected, supporting previous results in which MgProto and heme in Chlamydomonas were assigned roles as second messengers only in the transient activation of genes by light.

Functional genomics of eukaryotic photosynthesis using insertional mutagenesis of Chlamydomonas reinhardtii.

The unicellular green alga Chlamydomonas reinhardtii is a widely used model organism for studies of oxygenic photosynthesis in eukaryotes. Here we describe the development of a resource for functional genomics of photosynthesis using insertional mutagenesis of the Chlamydomonas nuclear genome. Chlamydomonas cells were transformed with either of two plasmids conferring zeocin resistance, and insertional mutants were selected in the dark on acetate-containing medium to recover light-sensitive and nonphotosynthetic mutants. The population of insertional mutants was subjected to a battery of primary and secondary phenotypic screens to identify photosynthesis-related mutants that were pigment deficient, light sensitive, nonphotosynthetic, or hypersensitive to reactive oxygen species. Approximately 9% of the insertional mutants exhibited 1 or more of these phenotypes. Molecular analysis showed that each mutant line contains an average of 1.4 insertions, and genetic analysis indicated that approximately 50% of the mutations are tagged by the transforming DNA. Flanking DNA was isolated from the mutants, and sequence data for the insertion sites in 50 mutants are presented and discussed.

White mutants of Chlamydomonas reinhardtii are defective in phytoene synthase.

Carotenoids play an integral and essential role in photosynthesis and photoprotection in plants and algae. A collection of Chlamydomonas reinhardtii mutants lacking carotenoids was characterized for pigment and tocopherol (vitamin E) composition, growth phenotypes under different light conditions, and the molecular basis of their mutant phenotype. The carotenoid-less mutants, or "white" mutants, were also deficient in chlorophylls but had approximately twice the tocopherol content of the wild type. White mutants grew in the dark but were unable to survive in the light, even under very low light conditions on acetate-containing medium. Genetic crosses and recombination tests revealed that all individual white mutants in the collection are alleles of a single gene, lts1, and the white phenotype was closely linked to a marker located in the phytoene synthase gene. DNA sequencing of the phytoene synthase gene from each of the mutants revealed nonsense, missense, frameshift, and splice site mutations. Transformation with a wild-type copy of the phytoene synthase gene was able to complement the lts1-210 mutation. Together, these results show that all the white mutants examined in this work are affected in the phytoene synthase gene.