Osmotic Stress Signaling Research Articles

Changes in plasma membrane aquaporin gene expression under osmotic stress and blue light in tomato

Divergent abiotic stresses induce osmotic stress on plant cells resulting in an imbalance in water homeostasis which is preserved by aquaporins. Since the plasma membrane aquaporins (PIPs) were shown to be involved in seed development and responses to abiotic stresses, we focused on determining the contribution of mannitol-induced osmotic stress, blue light (BL), and 7B-1 mutation to their gene expression in tomato (Solanum lycopersicum L.) seeds. To assess that, we used a quantitative RT-PCR to determine the expression profiles of genes encoding PIPs. Subsequently, a multiple linear regression analysis was used to evaluate the impact of studied stressors (mannitol and BL) and 7B-1 mutation on PIP gene expressions. We found that mannitol-induced osmotic stress and 7B-1 mutation (conferring the lower responsiveness to osmotic stress- and BL-induced inhibition of seed germination) decreased expression of PIP1;3, PIP2;3 and PIP1;2, PIP2;1 genes, respectively. This might be a way to retain water for radicle elongation and seed germination under the stress conditions. Interestingly, the expression of PIP1;3 gene was downregulated not only by osmotic stress, but also by BL. Altogether, our data indicate the existence of a link between osmotic stress and BL signalling and the involvement of the 7B-1 mutation in this crosstalk.

Metabolic Pathways Regulated by Chitosan Contributing to Drought Resistance in White Clover.

Increased endogenous chitosan (CTS) could be associated with improved drought resistance in white clover (Trifolium repens). Plants were pretreated with or without 1 mg/mL CTS and then were subjected to optimal or water-limited condition in controlled growth chambers for 6 days. Phenotypic and physiological results indicated that exogenous CTS significantly improved drought resistance of white clover. Metabolome results showed that exogenous CTS induced a significant increase in endogenous CTS content during dehydration accompanied by the maintenance of greater accumulation of sugars, sugar alcohols, amino acids, organic acids, and other metabolites (ascorbate, glutathione, flavonoids, putrescine, and spermidine). These compounds are associated with osmotic adjustment, antioxidant defense, stress signaling, and energy metabolism under stress condition. Similarly, transcriptome revealed that many genes in relation to amino acid and carbohydrate metabolism, energy production and conversion, and ascorbate-glutathione and flavonoid metabolism were significantly up-regulated by CTS in response to dehydration stress. CTS-induced drought resistance was associated with the accumulation of stress protective metabolites, the enhancement of ascorbate-glutathione and tricarboxylic acid cycle, and increases in the γ-aminobutyric acid shunt, polyamine synthesis, and flavonoids metabolism contributing to improved osmotic adjustment, antioxidant capacity, stress signaling, and energy production for stress defense, thereby maintaining metabolic homeostasis under dehydration stress.

Metabolic Effects of Acibenzolar-S-Methyl for Improving Heat or Drought Stress in Creeping Bentgrass.

Acibenzolar-S-methyl (ASM) is a synthetic functional analog of salicylic acid which can induce systemic acquired resistance in plants, but its effects on abiotic stress tolerance is not well known. The objectives of this study were to examine effects of acibenzolar-S-methyl on heat or drought tolerance in creeping bentgrass (Agrostis stolonifera) and to determine major ASM-responsive metabolites and proteins associated with enhanced abiotic stress tolerance. Creeping bentgrass plants (cv. ‘Penncross’) were foliarly sprayed with ASM and were exposed to non-stress (20/15°C day/night), heat stress (35/30°C), or drought conditions (by withholding irrigation) in controlled-environment growth chambers. Exogenous ASM treatment resulted in improved heat or drought tolerance, as demonstrated by higher overall turf quality, relative water content, and chlorophyll content compared to the untreated control. Western blotting revealed that ASM application resulted in up-regulation of ATP synthase, HSP-20, PR-3, and Rubisco in plants exposed to heat stress, and greater accumulation of dehydrin in plants exposed to drought stress. Metabolite profiling identified a number of amino acids, organic acids, and sugars which were differentially accumulated between ASM treated and untreated plants under heat or drought stress, including aspartic acid, glycine, citric acid, malic acid, and the sugars glucose, and fructose. Our results suggested that ASM was effective in improving heat or drought tolerance in creeping bentgrass, mainly through enhancing protein synthesis and metabolite accumulation involved in osmotic adjustment, energy metabolism, and stress signaling.

Amino acid metabolites that regulate G protein signaling during osmotic stress.

All cells respond to osmotic stress by implementing molecular signaling events to protect the organism. Failure to properly adapt can lead to pathologies such as hypertension and ischemia-reperfusion injury. Mitogen-activated protein kinases (MAPKs) are activated in response to osmotic stress, as well as by signals acting through G protein-coupled receptors (GPCRs). For proper adaptation, the action of these kinases must be coordinated. To identify second messengers of stress adaptation, we conducted a mass spectrometry-based global metabolomics profiling analysis, quantifying nearly 300 metabolites in the yeast S. cerevisiae. We show that three branched-chain amino acid (BCAA) metabolites increase in response to osmotic stress and require the MAPK Hog1. Ectopic addition of these BCAA derivatives promotes phosphorylation of the G protein α subunit and dampens G protein-dependent transcription, similar to that seen in response to osmotic stress. Conversely, genetic ablation of Hog1 activity or the BCAA-regulatory enzymes leads to diminished phosphorylation of Gα and increased transcription. Taken together, our results define a new class of candidate second messengers that mediate cross talk between osmotic stress and GPCR signaling pathways.

The SnRK2 kinases modulate miRNA accumulation in Arabidopsis.

MicroRNAs (miRNAs) regulate gene expression and play critical roles in growth and development as well as stress responses in eukaryotes. miRNA biogenesis in plants requires a processing complex that consists of the core components DICER-LIKE 1 (DCL1), SERRATE (SE) and HYPONASTIC LEAVES (HYL1). Here we show that inactivation of functionally redundant members of the SnRK2 kinases, which are the core components of abscisic acid (ABA) and osmotic stress signaling pathways, leads to reduction in miRNA accumulation under stress conditions. Further analysis revealed that the steady state level of HYL1 protein in plants under osmotic stress is dependent on the SnRK2 kinases. Additionally, our results suggest that the SnRK2 kinases physically associate with the miRNA processing components SE and HYL1 and can phosphorylate these proteins in vitro. These findings reveal an important role for the SnRK2 kinases in the regulation of miRNA accumulation and establish a mechanism by which ABA and osmotic stress signaling is linked to miRNA biogenesis.

An insulin-like message from mother

Nematodes experiencing osmotic stress signal to the germ-line to prepare their offspring.

The Physiological Role of Proline and Sodium as Osmotic Stress Signal Components of Some Crop Plants

The increase of Na + and proline content in maize, wheat, broad bean, cotton and parsley plants indicated that the proline content may act as an indicator of Na + uptake and allow the identification of tolerant plants, which effectively exclude Na + from plant either by exclusion or compartmentalization. In maize and cotton plants while Na + increased in shoots and roots, proline content decreased in these organs of these plants. This concomitant with reduction of dry matter of both organs of the maize and cotton plants. Sodium can be considered as osmotic stress marker component in these plants, especially in shoots of cotton. The percent accumulation of proline in both organs of wheat and parsley plants, shoots of broad bean run parallel with the reduction in dry matter of these plants. This strategy of proline and dry matter was concomitant with the increase of Na + content of these plants. Pytohormonal treatment with either GA 3 or Kinetin 200 ppm was mostly decreased the accumulation of both osmotic stress markers (sodium and proline ) at all levels of salinity in shoots and roots of maize, wheat, cotton, broad bean and parsley plants. Phytohormonal application was significant reduced mostly the accumulation both stress markers sodium ion and proline in shoots and roots of maize, wheat, cotton, broad bean and parsley plants. This can be reflected on the accumulation of metabolities which finally affected on the production of dry matter in shoots and roots of the five tested plants.

NLK phosphorylates Raptor to mediate stress-induced mTORC1 inhibition.

The mechanistic target of rapamycin (mTOR) is a central cell growth controller and forms two distinct complexes: mTORC1 and mTORC2. mTORC1 integrates a wide range of upstream signals, both positive and negative, to regulate cell growth. Although mTORC1 activation by positive signals, such as growth factors and nutrients, has been extensively investigated, the mechanism of mTORC1 regulation by stress signals is less understood. In this study, we identified the Nemo-like kinase (NLK) as an mTORC1 regulator in mediating the osmotic and oxidative stress signals. NLK inhibits mTORC1 lysosomal localization and thereby suppresses mTORC1 activation. Mechanistically, NLK phosphorylates Raptor on S863 to disrupt its interaction with the Rag GTPase, which is important for mTORC1 lysosomal recruitment. Cells with Nlk deletion or knock-in of the Raptor S863 phosphorylation mutants are defective in the rapid mTORC1 inhibition upon osmotic stress. Our study reveals a function of NLK in stress-induced mTORC1 modulation and the underlying biochemical mechanism of NLK in mTORC1 inhibition in stress response.

Two SnRK2 protein kinases genes play a negative regulatory role in the osmotic stress response in tomato

Sucrose non-fermenting 1-related protein kinase 2 (SnRK2) proteins positively regulate abscisic acid signaling, and play important roles in plant stress responses. Seven genes encoding SlSnRK2s were isolated from tomato (Solanum lycopersicum cv. Micro Tom), which showed homology to Arabidopsis SnRK2.2/SnRK2.3/SnRK2.6. Transcriptional levels were analyzed in tomato under salinity stress, all seven genes were repressed in the first 24 h under stress treatment. But the transcriptional levels of four SlSnRK2 genes dramatically increased at 72 h, which was not detected in transcriptional level of SlSnRK2.1, SlSnRK2.2 and SlSnRK2.3, indicating SlSnRK2.1, SlSnRK2.2 and SlSnRK2.3 might act differently as other four SlSnRK2 in tomato. SlSnRK2.1 and SlSnRK2.2 were selected for further analysis. The expression patterns of SlSnRK2.1 and SlSnRK2.2 differed among various tissues of tomato plants. A subcellular localization analysis showed that these SlSnRK2s were localized on the cytoplasm and nucleus. Transgenic plants overexpressing SlSnRK2s exhibited a lower germination rate than wild type (WT) under saline conditions. In contrast, RNA-interference lines which down regulate both SlSnRK2.1 and SlSnRk2.2 showed a higher germination rate than that of WT under saline conditions. Furthermore, the activities of peroxidase, catalase and proline content were lower and the malondialdehyde content was higher in overexpressing lines than in WT under salinity and drought conditions. Moreover, overexpression of SlSnRK2s altered the transcriptional levels of the stress-related genes PR5, LHA1, HVA22 and RBOHC under osmotic stress conditions. These results demonstrated that overexpression of SlSnRK2.1 and SlSnRK2.2 decreased osmotic stress tolerance. Therefore, these SlSnRK2s may be involved in osmotic stress signal transduction and may play negative roles in the stress response.

AtDsPTP1 acts as a negative regulator in osmotic stress signalling during Arabidopsis seed germination and seedling establishment.

Dual-specificity protein phosphatases (DsPTPs) target both tyrosine and serine/threonine residues and play roles in plant growth and development. We have characterized an Arabidopsis mutant, dsptp1, which shows a higher seed germination rate and better root elongation under osmotic stress than the wild type. By contrast, its overexpression line, DsPTP1-OE, shows inhibited seed germination and root elongation; and its complemented line, DsPTP1-Com, resembles the wild type and rescues DsPTP1-OE under osmotic stress. Expression of AtDsPTP1 is enhanced by osmotic stress in seed coats, bases of rosette leaves, and roots. Compared with the wild type, the dsptp1 mutant shows increased proline accumulation, reduced malondialdehyde (MDA) content and ion leakage, and enhanced antioxidant enzyme activity in response to osmotic stress. AtDsPTP1 regulates the transcript levels of various dehydration-responsive genes under osmotic stress. Abscisic acid (ABA) accumulation in dsptp1 under osmotic stress is reduced with reduced expression of the ABA-biosynthesis gene NCED3 and increased expression of the ABA-catabolism gene CYP707A4. AtDsPTP1 also regulates the expression of key components in the ABA-signalling pathway. In conclusion, AtDsPTP1 regulates ABA accumulation, and acts as a negative regulator in osmotic stress signalling during Arabidospsis seed germination and seedling establishment.

Overexpression of ShDHN, a dehydrin gene from Solanum habrochaites enhances tolerance to multiple abiotic stresses in tomato

Dehydrins (DHNs) play important roles in plant adaptation to abiotic stress. In this study, a cold-induced SK3-type DHN gene (ShDHN) isolated from wild tomato species Solanum habrochaites was characterized for its function in abiotic stress tolerance. ShDHN was constitutively expressed in root, leaf, stem, flower and fruit. ShDHN was continuously up-regulated during cold stress and showed higher expression level in the cold-tolerant S. habrochaites than in the susceptible S. lycopersicum. Moreover, ShDHN expression was also regulated by drought, salt, osmotic stress, and exogenous signaling molecules. Overexpression of ShDHN in cultivated tomato increased tolerance to cold and drought stresses and improved seedling growth under salt and osmotic stresses. Compared with the wild-type, the transgenic plants accumulated more proline, maintained higher enzymatic activities of superoxide dismutase and catalase, and suffered less membrane damage under cold and drought stresses. Moreover, the transgenic plants accumulated lower levels of H2O2 and O2− under cold stress, and had higher relative water contents and lower water loss rates under dehydration conditions. Furthermore, overexpression of ShDHN in tomato led to the up- or down-regulated expression of several genes involved in ROS scavenging and JA signaling pathway, including SOD1, GST, POD, LOX, PR1 and PR2. Taken together, these results indicate that ShDHN has pleiotropic effects on improving plant adaptation to abiotic stresses and that it possesses potential usefulness in genetic improvement of stress tolerance in tomato.

Arabidopsis reduces growth under osmotic stress by decreasing SPEECHLESS protein.

Plants, which are sessile unlike most animals, have evolved a system to reduce growth under stress; however, the molecular mechanisms of this stress response are not well known. During programmed development, a fraction of the leaf epidermal precursor cells become meristemoid mother cells (MMCs), which are stem cells that produce both stomatal guard cells and epidermal pavement cells. Here we report that Arabidopsis plants, in response to osmotic stress, post-transcriptionally decrease the protein level of SPEECHLESS, the transcription factor promoting MMC identity, through the action of a mitogen-activated protein kinase (MAPK) cascade. The growth reduction under osmotic stress was lessened by inhibition of the MAPK cascade or by a mutation that disrupted the MAPK target amino acids in SPEECHLESS, indicating that Arabidopsis reduces growth under stress by integrating the osmotic stress signal into the MAPK-SPEECHLESS core developmental pathway.

OSCA1 mediates osmotic-stress-evoked Ca2+ increases vital for osmosensing in Arabidopsis.

Water is crucial to plant growth and development. Environmental water deficiency triggers an osmotic stress signalling cascade, which induces short-term cellular responses to reduce water loss and long-term responses to remodel the transcriptional network and physiological and developmental processes. Several signalling components that have been identified by extensive genetic screens for altered sensitivities to osmotic stress seem to function downstream of the perception of osmotic stress. It is known that hyperosmolality and various other stimuli trigger increases in cytosolic free calcium concentration ([Ca(2+)]i). Considering that in bacteria and animals osmosensing Ca(2+) channels serve as osmosensors, hyperosmolality-induced [Ca(2+)]i increases have been widely speculated to be involved in osmosensing in plants. However, the molecular nature of corresponding Ca(2+) channels remain unclear. Here we describe a hyperosmolality-gated calcium-permeable channel and its function in osmosensing in plants. Using calcium-imaging-based unbiased forward genetic screens we isolated Arabidopsis mutants that exhibit low hyperosmolality-induced [Ca(2+)]i increases. These mutants were rescreened for their cellular, physiological and developmental responses to osmotic stress, and those with clear combined phenotypes were selected for further physical mapping. One of the mutants, reduced hyperosmolality-induced [Ca(2+)]i increase 1 (osca1), displays impaired osmotic Ca(2+) signalling in guard cells and root cells, and attenuated water transpiration regulation and root growth in response to osmotic stress. OSCA1 is identified as a previously unknown plasma membrane protein and forms hyperosmolality-gated calcium-permeable channels, revealing that OSCA1 may be an osmosensor. OSCA1 represents a channel responsible for [Ca(2+)]i increases induced by a stimulus in plants, opening up new avenues for studying Ca(2+) machineries for other stimuli and providing potential molecular genetic targets for engineering drought-resistant crops.

Genomic adaptations of the halophilic Dead Sea filamentous fungus Eurotium rubrum.

The Dead Sea is one of the most hypersaline habitats on Earth. The fungus Eurotium rubrum (Eurotiomycetes) is among the few species able to survive there. Here we highlight its adaptive strategies, based on genome analysis and transcriptome profiling. The 26.2 Mb genome of E. rubrum shows, for example, gains in gene families related to stress response and losses with regard to transport processes. Transcriptome analyses under different salt growth conditions revealed, among other things differentially expressed genes encoding ion and metabolite transporters. Our findings suggest that long-term adaptation to salinity requires cellular and metabolic responses that differ from short-term osmotic stress signalling. The transcriptional response indicates that halophilic E. rubrum actively counteracts the salinity stress. Many of its genes encode for proteins with a significantly higher proportion of acidic amino acid residues. This trait is characteristic of the halophilic prokaryotes as well, supporting the theory of convergent evolution under extreme hypersaline stress.

Subcellular localization of the histidine kinase receptors Sln1p, Nik1p and Chk1p in the yeast CTG clade species Candida guilliermondii

Fungal histidine kinase receptors (HKR) sense and transduce many intra- and extracellular signals that regulate a wide range of physiological processes. Candida CTG clade species commonly possess three types of HKR namely Sln1p (type VI), Nik1p (type III) and Chk1p (type X). Although some recent work has demonstrated the potential involvement of HKR in osmoregulation, morphogenesis, sexual development, adaptation to osmotic stresses and drug resistance in distinct Candida species, little data is available in relation to their subcellular distribution within yeast cells. We describe in this work the comparative subcellular localization of class III, VI, and X HKRs in Candida guilliermondii, a yeast CTG clade species of clinical and biotechnological interest. Using a fluorescent protein fusion approach, we showed that C. guilliermondii Sln1p fused to the yellow fluorescent protein (Sln1p-YFP) appeared to be anchored in the plasma membrane. By contrast, both Chk1p-YFP and YFP-Chk1p were localized in the nucleocytosol of C. guilliermondii transformed cells. Furthermore, while Nik1p-YFP fusion protein always displayed a nucleocytosolic localization, we noted that most of the cells expressing YFP-Nik1p fusion protein displayed an aggregated pattern of fluorescence in the cytosol but not in the nucleus. Interestingly, Sln1p-YFP and Nik1p-YFP fusion protein localization changed in response to hyperosmotic stress by rapidly clustering into punctuated structures that could be associated to osmotic stress signaling. To date, this work provides the first insight into the subcellular localization of the three classes of HKR encoded by CTG clade yeast genomes and constitutes original new data concerning this family of receptors. This represents also an essential prerequisite to open a window into the understanding of the global architecture of HKR-mediated signaling pathways in CTG clade species.

Genome-wide transcriptional profiling and enrichment mapping reveal divergent and conserved roles of Sko1 in the Candida albicans osmotic stress response

Candida albicans maintains both commensal and pathogenic states in humans. Here, we have defined the genomic response to osmotic stress mediated by transcription factor Sko1. We performed microarray analysis of a sko1Δ/Δ mutant strain subjected to osmotic stress, and we utilized gene sequence enrichment analysis and enrichment mapping to identify Sko1-dependent osmotic stress-response genes. We found that Sko1 regulates distinct gene classes with functions in ribosomal synthesis, mitochondrial function, and vacuolar transport. Our in silico analysis suggests that Sko1 may recognize two unique DNA binding motifs. Our C. albicans genomic analyses and complementation studies in Saccharomyces cerevisiae showed that Sko1 is conserved as a regulator of carbohydrate metabolism, redox metabolism, and glycerol synthesis. Further, our real time-qPCR results showed that osmotic stress-response genes that are dependent on the kinase Hog1 also require Sko1 for full expression. Our findings reveal divergent and conserved aspects of Sko1-dependent osmotic stress signaling.

Deciphering Dynamic Dose Responses of Natural Promoters and Single cis Elements upon Osmotic and Oxidative Stress in Yeast

Fine-tuned activation of gene expression in response to stress is the result of dynamic interactions of transcription factors with specific promoter binding sites. In the study described here we used a time-resolved luciferase reporter assay in living Saccharomyces cerevisiae yeast cells to gain insights into how osmotic and oxidative stress signals modulate gene expression in a dose-sensitive manner. Specifically, the dose-response behavior of four different natural promoters (GRE2, CTT1, SOD2, and CCP1) reveals differences in their sensitivity and dynamics in response to different salt and oxidative stimuli. Characteristic dose-response profiles were also obtained for artificial promoters driven by only one type of stress-regulated consensus element, such as the cyclic AMP-responsive element, stress response element, or AP-1 site. Oxidative and osmotic stress signals activate these elements separately and with different sensitivities through different signaling molecules. Combination of stress-activated cis elements does not, in general, enhance the absolute expression levels; however, specific combinations can increase the inducibility of the promoter in response to different stress doses. Finally, we show that the stress tolerance of the cell critically modulates the dynamics of its transcriptional response in the case of oxidative stress.

Overexpression of TaSRK2C1, a Wheat SNF1-Related Protein Kinase 2 Gene, Increases Tolerance to Dehydration, Salt, and Low Temperature in Transgenic Tobacco

Protein phosphorylation–dephosphorylations are major signaling events induced by osmotic stress in plants. In this study, a wheat SNF1-related protein kinase 2 (SnRK2) gene, TaSRK2C1, was functionally characterized. The results from the sequence analysis showed that TaSRK2C1 contains conserved domains typified in SnRK2 protein kinases, including the ATP binding site, N-myristoylation site, protein kinase-activating signature, and transmembrane-spanning region. The transcripts of TaSRK2C1 in roots were induced by treatments of dehydration, high salinity, low temperature, and exogenous abscisic acid, which suggest its potential roles relative to osmotic stress signal transductions. The ectopic expression of TaSRK2C1 in tobacco significantly up-regulated the expression levels of three putative central regulators, namely, RD29a, DREB1A, and DREB2, which are involved in responding to osmotic stresses. Thus, higher levels of free proline and soluble carbohydrates in transgenic plants were detected, and conferred tolerance to high salinity, dehydration stress, and low temperature in plants. The overall results in this study indicate that TaSRK2C1 have important functions in plant response and adaptation to osmotic stresses via mediation of signal transductions initiated by distinct abiotic stresses. Manipulating TaSRK2C1 toward improving the osmotic-stress tolerance in crop plants is feasible.

Transporter BetP - Significance of Oligomeric Structure for Catalysis and Regulation

The Na+ coupled betaine uptake system BetP of Corynebacterium glutamicum belongs to the BCCT family of transporters and comprises both a catalytic function (betaine/Na+ cotransport) and a sensory/regulatory function (responding to osmotic stress). Its 2D (electron crystallography) and 3D structure (X-ray crystallography) has been solved. Within a homooligomeric trimer, each BetP protomer harbours both an N- and a C-terminal domain involved in stimulus sensing and intramolecular signal transduction. Factors known so far contributing to the sensory and regulatory function of BetP are (i) the two terminal domains, (ii) K+ ions as an osmostress related stimulus, and (iii) interaction with the surrounding membrane. Due to the trimeric structure of BetP, functional crosstalk between the individual protomers was suggested for both functions of the transporter.We have now investigated in detail the significance of the oligomeric (trimeric) structure of BetP for both transport catalysis (betaine uptake) and sensing/regulation (sensing of osmotic stress and intramolecular signal transduction). For this purpose, we have developed a novel experimental tool, in which each of the three BetP protomers can be addressed individually and its contribution to catalysis and regulation elucidated. Each BetP protomer carries an individual molecular tag as well as mutations in specific functional or regulatory domains of the transporter. Using this tool, we were able to quantify the contribution of oligomerization to the catalytic and the regulatory properties of a secondary carrier for the first time.

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

BackgroundIn 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.ResultsIn 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.ConclusionBased 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.