Truncated Hemoglobins 1 and 2 Are Implicated in the Modulation of Phosphorus Deficiency-Induced Nitric Oxide Levels in Chlamydomonas.

Truncated hemoglobins (trHbs) form a widely distributed family of proteins found in archaea, bacteria, and eukaryotes. Accumulating evidence suggests that trHbs may be implicated in functions other than oxygen delivery, but these roles are largely unknown. Characterization of the conditions that affect trHb expression and investigation of their regulatory mechanisms will provide a framework for elucidating the functions of these globins. Here, the transcription of Chlamydomonas trHb genes (THB1-12) under conditions of phosphorus (P) deprivation was analyzed. Three THB genes, THB1, THB2, and THB12 were expressed at the highest level. For the first time, we demonstrate the synthesis of nitric oxide (NO) under P-limiting conditions and the production of NO by cells via a nitrate reductase-independent pathway. To clarify the functions of THB1 and THB2, we generated and analyzed strains in which these THBs were strongly under-expressed by using an artificial microRNA approach. Similar to THB1 knockdown, the depletion of THB2 led to a decrease in cell size and chlorophyll levels. We provide evidence that the knockdown of THB1 or THB2 enhanced NO production under P deprivation. Overall, these results demonstrate that THB1 and THB2 are likely to contribute, at least in part, to acclimation responses in P-deprived Chlamydomonas.

Regulation of alternative oxidase 1 in Chlamydomonas reinhardtii during sulfur starvation.

The mitochondrial respiratory chain in plants, some protists and many fungi consists of the ATP-coupling cyanide-sensitive cytochrome pathway and the cyanide-resistant alternative respiratory pathway. The alternative pathway is mediated by alternative oxidase (AOX). Although AOX has been proposed to play essential roles in nutrient stress tolerance of plants and protists, the effects of sulfur (S) deprivation, on AOX are largely unknown. The unicellular green alga Chlamydomonas reinhardtii reacts to S limitation conditions with the induced expression of many genes. In this work, we demonstrated that exposure of C. reinhardtii to S deprivation results in the up-regulation of AOX1 expression and an increased AOX1 protein. Furthermore, S-deprived C. reinhardtii cells display the enhanced AOX1 capacity. Moreover, nitrate assimilation regulatory protein (NIT2) is involved in the control of the AOX1 gene expression in the absence of S. Together, the results clearly indicate that AOX1 relates to S limitation stress responses and is regulated in a NIT2-dependent manner, probably together with yet-unknown regulatory factor(s).

Regulation of sulfur deprivation-induced expression of the ferredoxin-encoding FDX5 gene Chlamydomonas reinhardtii in aerobic conditions.

The unicellular green alga Chlamydomonas reinhardtii reacts to sulfur (S) starvation with the increased expression of numerous genes. One gene which is induced in illuminated anaerobic S-deprived cells is the ferredoxin-5 gene (FDX5). To test FDX5 transcriptional regulation in aerobic cultures, we used a real-time PCR analysis and an artificial microRNA approach. We demonstrated that FDX5 gene is controlled by S deprivation independently of anoxia-treatment. The Ser/Thr kinase SNRK2.1 is necessary for expression of FDX5 during deprivation to S. Copper response regulator 1 (CRR1) is not involved in FDX5 up-regulation in S-deficient cells under aerobic conditions. Furthermore, expression of FDX5 is negatively regulated by nitric oxide (NO). Moreover, truncated hemoglobin 1 (THB1) underexpression resulted in the decrease in FDX5 transcript abundance in S-deficient cells under aerobic conditions. Together, our results imply that the FDX5 gene is controlled by NO in THB1-dependent pathway under conditions of depleted S supply.

Nitric oxide upregulates expression of alternative oxidase 1 in Chlamydomonas reinhardtii.

The mitochondrial respiratory chain in plants, many fungi and some protists consists of the ATP-coupling cyanide-sensitive cytochrome pathway and the cyanide-resistant alternative respiratory pathway. The alternative pathway is mediated by alternative oxidase (AOX). In unicellular algae, AOXs are monomeric fungi-type proteins. Studies performed in the model plant Chlamydomonas reinhardtii showed that a range of stress factors lead to induction of its AOX1. However, signaling molecules that trigger upregulation of AOX1 have not yet been identified. Here, we were able to discriminate between two alternative oxidases of the alga. In this work, we demonstrated that exposure of C. reinhardtii to nitric oxide (NO) resulted in the up-regulation of AOX1 expression and an increased AOX1 protein. Furthermore, NO-treated C. reinhardtii cells displayed the enhanced AOX1 capacity. We also clearly demonstrated that AOX1 can function in C. reinhardtii when the cytochrome oxidase became inhibited by NO. Although the pathway(s) that leads to increased AOX1 levels and activity upon NO treatment is yet unknown, it is now clear that NO serves as the signal to trigger this regulatory process in C. reinhardtii.

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

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.