The Yin-Yang of the Green Fluorescent Protein: Impact on Saccharomyces cerevisiae stress resistance.

Although fluorescent proteins are widely used as biomarkers (Yin), no study focuses on their influence on the microbial stress response. Here, the Green Fluorescent Protein (GFP) was fused to two proteins of interest in Saccharomyces cerevisiae. Pab1p and Sur7p, respectively involved in stress granules structure and in Can1 membrane domains. These were chosen since questions remain regarding the understanding of the behavior of S. cerevisiae facing different heat kinetics or oxidative stresses. The main results showed that Pab1p-GFP fluorescent mutant displayed a higher resistance than that of the wild type under a heat shock. Moreover, fluorescent mutants exposed to oxidative stresses displayed changes in the cultivability compared to the wild type strain. In silico approaches showed that the presence of the GFP did not influence the structure and so the functionality of the tagged proteins meaning that changes in yeast resistance were certainly related to GFP ROS-scavenging ability (Yang).

Metal stresses modify soluble proteomes and toxin profiles in two Mediterranean strains of the distributed dinoflagellate Alexandrium pacificum.

HABs involving Alexandrium pacificum have been reported in metal-contaminated ecosystems, suggesting that this distributed species adapts to and/or can tolerate the effects of metals. Modifications in soluble proteomes and PST contents were characterized in two Mediterranean A. pacificum strains exposed to mono- or polymetallic stresses (zinc, lead, copper, cadmium). These strains were isolated from two anthropized locations: Santa Giusta Lagoon (Italy, SG C10-3) and the Tarragona seaport (Spain, TAR C5-4F). In both strains, metals primarily downregulated key photosynthesis proteins. Metals also upregulated other proteins involved in photosynthesis (PCP in both strains), the oxidative stress response (HSP 60, proteasome and SOD in SG C10-3; HSP 70 in TAR C5-4F), energy metabolism (AdK in TAR C5-4F), neoglucogenesis/glycolysis (GAPDH and PEP synthase in SG C10-3) and protein modification (PP in TAR C5-4F). These proteins, possibly involved in adaptive proteomic responses, may explain the development of these A. pacificum strains in metal-contaminated ecosystems. The two strains showed different proteomic responses to metals, with SG C10-3 upregulating more proteins, particularly PCP. Among the PSTs, regardless of the metal and the strain studied, C2 and GTX4 predominated, followed by GTX5. Under the polymetallic cocktail, (i) total PSTs, C2 and GTX4 reached the highest levels in SG C10-3 only, and (ii) total PSTs, C2, GTX5 and neoSTX were higher in SG C10-3 than in TAR C5-4F, whereas in SG C10-3 under copper stress, total PSTs, GTX5, GTX1 and C1 were higher than in the controls, revealing variability in PST biosynthesis between the two strains. Total PSTs, C2, GTX4 and GTX1 showed significant positive correlations with PCP, indicating that PST production may be positively related to photosynthesis. Our results showed that the A. pacificum strains adapt their proteomic and physiological responses to metals, which may contribute to their ecological success in highly anthropized areas.

Increased xerotolerance of Saccharomyces cerevisiae during an osmotic pressure ramp over several generations.

Although mechanisms involved in response of Saccharomyces cerevisiae to osmotic challenge are well described for low and sudden stresses, little is known about how cells respond to a gradual increase of the osmotic pressure (reduced water activity; aw ) over several generations as it could encounter during drying in nature or in food processes. Using glycerol as a stressor, we propagated S. cerevisiae through a ramp of the osmotic pressure (up to high molar concentrations to achieve testing-to-destruction) at the rate of 1.5 MPa day-1 from 1.38 to 58.5 MPa (0.990-0.635 aw ). Cultivability (measured at 1.38 MPa and at the harvest osmotic pressure) and glucose consumption compared with the corresponding sudden stress showed that yeasts were able to grow until about 10.5 MPa (0.926 aw ) and to survive until about 58.5 MPa, whereas glucose consumption occurred until 13.5 MPa (about 0.915 aw ). Nevertheless, the ramp conferred an advantage since yeasts harvested at 10.5 and 34.5 MPa (0.778 aw ) showed a greater cultivability than glycerol-shocked cells after a subsequent shock at 200 MPa (0.234 aw ) for 2 days. FTIR analysis revealed structural changes in wall and proteins in the range 1.38-10.5 MPa, which would be likely to be involved in the resistance at extreme osmotic pressure.

Automatic Counting of Intra-Cellular Ribonucleo-Protein Aggregates in Saccharomyces cerevisiae Using a Textural Approach.

In the context of microbiology, recent studies show the importance of ribonucleo-protein aggregates (RNPs) for the understanding of mechanisms involved in cell responses to specific environmental conditions. The assembly and disassembly of aggregates is a dynamic process, the characterization of the stage of their evolution can be performed by the evaluation of their number. The aim of this study is to propose a method to automatically determine the count of RNPs. We show that the determination of a precise count is an issue by itself and hence, we propose three textural approaches: a classical point of view using Haralick features, a frequency point of view with generalized Fourier descriptors, and a structural point of view with Zernike moment descriptors (ZMD). These parameters are then used as inputs for a supervised classification in order to determine the most relevant. An experiment using a specific Saccharomyces cerevisiae strain presenting a fusion between a protein found in RNPs (PAB1) and the green fluorescent protein was performed to benchmark this approach. The fluorescence was observed with two-photon fluorescence microscopy. Results show that the textural approach, by mixing ZMD with Haralick features, allows for the characterization of the number of RNPs.

Insights into B-type RR members as signaling partners acting downstream of HPt partners of HK1 in the osmotic stress response in Populus.

The B-type response regulators (B-type RRs), final elements of a signaling pathway called "histidine/aspartate phosphorelay system" in plants, are devoted to the regulation of response genes through a transcription factor activity. Signal transduction consists in the transfer of a phosphoryl group from a transmembrane histidine kinase (HK) which recognizes a given stimulus to nuclear RRs via cytosolic shuttle phosphotransfer proteins (HPts). In Arabidopsis, the receptors HK are to date the major characterized candidates to be responsible for initiation of osmotic stress responses. However, little information is available concerning the signaling partners acting downstream of HKs. In Populus, three HPts and five B-type RRs were previously identified as interacting partners of HK1, the Arabidopsis AHK1 homolog. Here, we report the isolation of RR18, a member of the B-type RR family, which shares high sequence similarities with ARR18 characterized to act in the osmosensing signaling pathway in Arabidopsis, from poplar cuttings subjected to osmotic stress conditions. By using yeast and in planta interaction assays, RR18 was further identified as acting downstream of HK1 and its three preferential HPt partners. Besides, our results are in favor of a possible involvement of both RR18 and RR13, the main expressed poplar B-type RR, in the osmotic signaling pathway. Nonetheless, different behaviors of these two B-type RRs in this pathway need to be noted, with one RR, RR13, acting in an early phase, mainly in roots of poplar cuttings, and the other one, RR18, acting in a late phase, mainly in leaves to supply an adequate response.

Characterization of histidine-aspartate kinase HK1 and identification of histidine phosphotransfer proteins as potential partners in a Populus multistep phosphorelay.

In poplar, we identified proteins homologous to yeast proteins involved in osmosensing multistep phosphorelay Sln1p-Ypd1p-Ssk1p. This finding led us to speculate that Populus cells could sense osmotic stress by a similar mechanism. This study focuses on first and second protagonists of this possible pathway: a histidine-aspartate kinase (HK1), putative osmosensor and histidine phosphotransfer proteins (HPt1 to 10), potential partners of this HK. Characterization of HK1 showed its ability to homodimerize in two-hybrid tests and to act as an osmosensor with a kinase activity in yeast, by functional complementation of sln1Δ sho1Δ strain. Moreover, in plant cells, plasma membrane localization of HK1 is shown. Further analysis on HPts allowed us to isolate seven new cDNAs, leading to a total of 10 different HPts identified in poplar. Interaction tests showed that almost all HPts can interact with HK1, but two of them exhibit stronger interactions, suggesting a preferential partnership in poplar. The importance of the phosphorylation status in these interactions has been investigated with two-hybrid tests carried out with mutated HK1 forms. Finally, in planta co-expression analysis of genes encoding these potential partners revealed that only three HPts are co-expressed with HK1 in different poplar organs. This result reinforces the hypothesis of a partnership between HK1 and these three preferential HPts in planta. Taken together, these results shed some light on proteins partnerships that could be involved in the osmosensing pathway in Populus.

Identification of five B-type response regulators as members of a multistep phosphorelay system interacting with histidine-containing phosphotransfer partners of Populus osmosensor.

BACKGROUND: In plants, the multistep phosphorelay signaling pathway mediates responses to environmental factors and plant hormones. This system is composed of three successive partners: hybrid Histidine-aspartate Kinases (HKs), Histidine-containing Phosphotransfer proteins (HPts), and Response Regulators (RRs). Among the third partners, B-type RR family members are the final output elements of the pathway; they act as transcription factors and clearly play a pivotal role in the early response to cytokinin in Arabidopsis. While interactions studies between partners belonging to the multistep phosphorelay system are mainly focused on protagonists involved in cytokinin or ethylene pathways, very few reports are available concerning partners of osmotic stress signaling pathway. RESULTS: In Populus, we identified eight B-type RR proteins, RR12-16, 19, 21 and 22 in the Dorskamp genotype. To assess HPt/B-type RR interactions and consequently determine potential third partners in the osmosensing multistep phosphorelay system, we performed global yeast two-hybrid (Y2H) assays in combination with Bimolecular Fluorescence Complementation (BiFC) assays in plant cells. We found that all B-type RRs are able to interact with HPt predominant partners (HPt2, 7 and 9) of HK1, which is putatively involved in the osmosensing pathway. However, different profiles of interaction are observed depending on the studied HPt. HPt/RR interactions displayed a nuclear localization, while the nuclear and cytosolic localization of HPt and nuclear localization of RR proteins were validated. Although the nuclear localization of HPt/RR interaction was expected, this work constitutes the first evidence of such an interaction in plants. Furthermore, the pertinence of this partnership is reinforced by highlighting a co-expression of B-type RR transcripts and the other partners (HK1 and HPts) belonging to a potential osmosensing pathway. CONCLUSION: Based on the interaction studies between identified B-type RR and HPt proteins, and the co-expression analysis of transcripts of these potential partners in poplar organs, our results favor the model that RR12, 13, 14, 16 and 19 are able to interact with the main partners of HK1, HPt2, 7 and 9, and this HPt/RR interaction occurs within the nucleus. On the whole, the five B-type RRs of interest could be third protagonists putatively involved in the osmosensing signaling pathway in Populus.