Reliable reference genes and abiotic stress marker genes in Klebsormidium nitens.

Microalgae have recently emerged as a key research topic, especially as biological models. Among them, the green alga Klebsormidium nitens, thanks to its particular adaptation to environmental stresses, represents an interesting photosynthetic eukaryote for studying the transition stages leading to the colonization of terrestrial life. The tolerance to different stresses is manifested by changes in gene expression, which can be monitored by quantifying the amounts of transcripts by RT-qPCR. The identification of optimal reference genes for experiment normalization was therefore necessary. In this study, using four statistical algorithms followed by the RankAggreg package, we determined the best reference gene pairs suitable for normalizing RT-qPCR data in K. nitens in response to three abiotic stresses: high salinity, PEG-induced dehydration and heat shock. Based on these reference genes, we were able to identify marker genes in response to the three abiotic stresses in K. nitens.

The chaperone-like protein Cdc48 regulates ubiquitin-proteasome system in plants.

The degradation of misfolded proteins is mainly mediated by the ubiquitin-proteasome system (UPS). UPS can be assisted by the protein Cdc48 but the relationship between UPS and Cdc48 in plants has been poorly investigated. Here, we analysed the regulation of UPS by Cdc48 in tobacco thanks to two independent cell lines overexpressing Cdc48 constitutively and plant leaves overexpressing Cdc48 transiently. In the cell lines, the accumulation of ubiquitinated proteins was affected both quantitatively and qualitatively and the number of proteasomal subunits was modified, while proteolytic activities were unchanged. Similarly, the over-expression of Cdc48 in planta impacted the accumulation of ubiquitinated proteins. A similar process occurred in leaves overexpressing transiently Rpn3, a proteasome subunit. Cdc48 being involved in plant immunity, its regulation of UPS was also investigated in response to cryptogein, an elicitor of immune responses. In the cell lines stably overexpressing Cdc48 and in leaves transiently overexpressing Cdc48 and/or Rpn3, cryptogein triggered a premature cell death while no increase of the proteasomal activity occurred. Overall, this study highlights a role for Cdc48 in ubiquitin homeostasis and confirms its involvement, as well as that of Rpn3, in the processes underlying the hypersensitive response.

Nitric oxide production and signalling in algae.

Nitric oxide (NO) was the first identified gaseous messenger and is now well established as a major ubiquitous signalling molecule. The rapid development of our understanding of NO biology in embryophytes came with the partial characterization of the pathways underlying its production and with the decrypting of signalling networks mediating its effects. Notably, the identification of proteins regulated by NO through nitrosation greatly enhanced our perception of NO functions. In comparison, the role of NO in algae has been less investigated. Yet, studies in Chlamydomonas reinhardtii have produced key insights into NO production through the identification of NO-forming nitrite reductase and of S-nitrosated proteins. More intriguingly, in contrast to embryophytes, a few algal species possess a conserved nitric oxide synthase, the main enzyme catalysing NO synthesis in metazoans. This latter finding paves the way for a deeper characterization of novel members of the NO synthase family. Nevertheless, the typical NO-cyclic GMP signalling module transducing NO effects in metazoans is not conserved in algae, nor in embryophytes, highlighting a divergent acquisition of NO signalling between the green and the animal lineages.

Identification of Partner Proteins of the Algae Klebsormidium nitens NO Synthases: Toward a Better Understanding of NO Signaling in Eukaryotic Photosynthetic Organisms.

In animals, NO is synthesized from L-arginine by three isoforms of nitric oxide synthase (NOS) enzyme. NO production and effects have also been reported in plants but the identification of its sources, especially the enzymatic ones, remains one of the critical issues in the field. NOS-like activities have been reported, although there are no homologs of mammalian NOS in the land plant genomes sequenced so far. However, several NOS homologs have been found in algal genomes and transcriptomes. A first study has characterized a functional NOS in the chlorophyte Ostreococcus tauri and the presence of NOS homologs was later confirmed in a dozen algae. These results raise the questions of the significance of the presence of NOS and their molecular diversity in algae. We hypothesize that comparisons among protein structures of the two KnNOS, together with the identification of their interacting partner proteins, might allow a better understanding of the molecular diversification and functioning of NOS in different physiological contexts and, more generally, new insights into NO signaling in photosynthetic organisms. We recently identified two NOS homologs sequences in the genome of the streptophyte Klebsormidium nitens, a model alga in the study of plant adaptation to terrestrial life. The first sequence, named KnNOS1, contains canonical NOS signatures while the second, named KnNOS2, presents a large C-ter extension including a globin domain. In order to identify putative candidates for KnNOSs partner proteins, we draw the protein-protein interaction networks of the three human NOS using the BioGRID database and hypothesized on the biological role of K. nitens orthologs. Some of these conserved partners are known to be involved in mammalian NOSs regulation and functioning. In parallel, our methodological strategy for the identification of partner proteins of KnNOS1 and KnNOS2 by in vitro pull-down assay is presented.