Truncated hemoglobin 1 is a new player in Chlamydomonas reinhardtii acclimation to sulfur deprivation

Abstract

Truncated hemoglobins constitute a large family, present in bacteria, in archaea and in eukaryotes. However, a majority of physiological functions of these proteins remains to be elucidated. Identification and characterization of a novel role of truncated hemoglobins in the model alga provides a framework for a more complete understanding of their biological functions. Here, we use quantitative RT-PCR to show that three truncated hemoglobins of Chlamydomonas reinhardtii, THB1, THB2 and THB12, are induced under conditions of depleted sulfur (S) supply. THB1 underexpression results in the decrease in cell size, as well in levels of proteins, chlorophylls and mRNA of several S-responsive genes under S starvation. We provide evidence that knock-down of THB1 enhances NO production under S deprivation. In S-deprived cells, a subset of S limitation-responsive genes is controlled by NO in THB1-dependent pathway. Moreover, we demonstrate that deficiency for S represses the nitrate reduction and that THB1 is involved in this control. Thus, our data support the idea that in S-deprived cells THB1 plays a dual role in NO detoxification and in coordinating sulfate limitation with nitrate assimilation. This study uncovers a new function for the Chlamydomonas reinhardtii THB1 in the control of proper response to S deprivation.

Fig 1. Effects of sulfur deprivation on the relative transcript abundance for genes encoding truncated hemoglobins THB1-12 in Chlamydomonas reinhardtii.

Vegetative cells of cw15-325 were treated as described in Materials and methods. Levels of gene transcripts are given as times of relative abundance with respect to the housekeeping control gene (RACK1) that has a value of 1. Data are the means±SE from three biological and two technical replicates obtained by real-time RT-PCR. ND, transcripts are not detected. See Supporting experimental procedures (Figures A, B and C and Tables A and B in S1 File) for more information.

Fig 2. Effects of sulfur deprivation on THB1 transcript accumulation under different light conditions.

Light-grown Chlamydomonas reinhardtii cw15-325 cells were transferred to TAP-S medium in the light (A) or in the dark (B) for 0.5h, 1h, 2h, 4h or 6h. Levels of gene transcripts are given as times of relative abundance with respect to the housekeeping control gene (RACK1) that has a value of 1. Data are the means±SE from three biological and two technical replicates obtained by real-time RT-PCR. See Supporting experimental procedures (Figures D, E and F and Tables C and D in S1 File) for more information.

Fig 3. Characterization of Chlamydomonas reinhardtii THB1 knock-down strains.

(A) Real time reverse transcription PCR analysis of THB1 transcript levels, comparing parental strain cw15-325 to amiRNA-THB1 strains. Relative expression levels were normalized with the gene expression of RACK1 and calculated using ΔC_T; all measurements were done in triplicate. Additional underlying data can be found in S1 Fig. (B) Comparative chlorophyll contents and protein contents of parental strain cw15-325 and amiRNA-THB1 strains. Vegetative cells were grown in TAP medium. Additional underlying data can be found in S2 Fig. Insert shows test tubes with the same cell density of cultures (2 10⁶ cells/ml) in TAP. (C) Expression of selected S limitation-responsive genes in cw15-325 and amiRNA-THB1 strains subjected to S-depleted conditions. Relative expression levels were normalized with the gene expression of RACK1 and calculated using ΔCt; all measurements were done in triplicate. Cells were treated as described in legend to Fig 2A. See Supporting experimental procedures (Figures G and H and Tables E and F in S1 File) for more information.

Fig 4. Nitric oxide–dependent expression of selected S limitation-responsive genes in Chlamydomonas reinhardtii.

Cells grown in TAP were washed in S-free-medium and incubated for 30 min or 1 h in the absence or presence of 50 μM DEA-NONOate. The effect of 100 μM cPTIO was analyzed when added simultaneously with DEA NONOate at time 0. The value 1 was assigned to the expression level of internal standard RACK1 gene in each condition. Data are the means±SE from three biological and two technical replicates obtained by real-time RT-PCR. See Supporting experimental procedures (Figures I, J and K and Tables G and H in S1 File) for more information.

Fig 5. NO visualization in Chlamydomonas reinhardtii by confocal microscopy.

(A) Images of cell populations grown in TAP (TAP) or incubated in S-free medium (-S) for 15 min. S-deprived cells were treated with 50 μM (-S+50 μM NO) or with 100 μM (-S+100 μM NO) DEA NONOate. Imaging was also performed on the same starved culture supplemented with 100 μM cPTIO and 100 μM DEA NONOate (-S+100 μM NO+cPTIO). The left-hand panels show DAF-FM fluorescence (green color) while the right-hand panels show Chl autofluorescence (red color). Green and red fluorescence images were processed as indicated in Materials and Methods. Scale bar equals 100 μm. Additional underlying data can be found in S3 Fig. (B) Representative cell images. Cw15-325 and amiRNA-THB1-11 cells were treated as described in legend to (A). Scale bar equals 10 μm.

Fig 6. NO production in Chlamydomonas reinhardtii following the removal of S from the medium.

(A) Vegetative cells of cw15-325 strain were grown in TAP medium and transferred to TAP-S medium in the light for 15 min. Cells were treated with 100 μM DEA NONOate. The effect of 300 μM cPTIO was analyzed when added simultaneously with DEA NONOate at time 0. Fluorescence intensity due to intracellular NO was determined using DAF-FM DA and was expressed as arbitrary units per chlorophyll cells 10⁻⁶. Cell autofluorescence was subtracted from the total fluorescence obtained. Data are the means±SE from three technical replicates of a representative experiment. (B) Fluorescence increase was measured and expressed in amiRNA-THB1 cells following the removal of S from the medium as described in (A).

Fig 7. Effects of sulfur depletion on nitrate-induced activity of NR in wild-type and THB1 knock-down strains.

NR was quantified in the cells incubated in TAP containing 8 mM of NH₄⁺ (ammonium), 4 mM of NO₃^- with S (nitrate) or without S (nitrate-S).