Abstract
PGRL1 RNA and protein levels are increased in iron-deficient Chlamydomonas reinhardtii cells. In an RNAi strain, which accumulates lower PGRL1 levels in both iron-replete and -starved conditions, the photosynthetic electron transfer rate is decreased, respiratory capacity in iron-sufficient conditions is increased, and the efficiency of cyclic electron transfer under iron-deprivation is diminished. Pgrl1-kd cells exhibit iron deficiency symptoms at higher iron concentrations than wild-type cells, although the cells are not more depleted in cellular iron relative to wild-type cells as measured by mass spectrometry. Thiol-trapping experiments indicate iron-dependent and redox-induced conformational changes in PGRL1 that may provide a link between iron metabolism and the partitioning of photosynthetic electron transfer between linear and cyclic flow. We propose, therefore, that PGRL1 in C. reinhardtii may possess a dual function in the chloroplast; that is, iron sensing and modulation of electron transfer.
Highlights
PGRL1 RNA and protein levels are increased in iron-deficient Chlamydomonas reinhardtii cells
The Cellular PGRL1 Content Is Controlled by Iron Availability in C. reinhardtii—PGRL1 protein and RNA abundance is increased under iron-deficient conditions in wild-type C. rein
We found that Cox2b levels did not decline in both wild-type and mutant cells under iron deficiency, consistent with the notion that the mitochondrial respiratory chain is more resistant to iron starvation than is the photosynthetic electron transport chain in acetate-grown C. reinhardtii (16)
Summary
Strains and Culture Conditions—Cells were grown in the light (60 E mϪ2sϪ1) in Tris acetate-phosphate (TAP) medium (27) at 25 °C and shaken at 120 rpm. For generation of the inverted repeat PGRL1 cassette, a 694-bp fragment corresponding to the coding region of PGRL1 was amplified by using cDNA isolated from iron-deficient cells as template and the primers 42-FORS-P (5Ј-aactgcaggttccttgcggcgatgac-3Ј, the underline indicates the PstI site) and 42-REV-E (5Ј-cggaattccagcttctcctgcttgacct-3Ј, the underline indicates the EcoRI site), adding the respective restriction sites. Nuclear Transformation and Selection—Biolistic transformation of the RNAi construct into a cell wall containing the wild-type strain and the subsequent screening was carried out as described in Naumann et al (32). Samples were incubated in solution B (described above) in the presence of ferrozine at room temperature for 20 min and were allowed to react with AMS without trichloroacetic acid precipitation. Samples were set at a density of 2 ϫ 106 cells/ml in TAP and were dark-adapted for 15 min before each measurement. Illumination was provided by light red LEDs centered on 650 nm
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