Are Histidine Kinases of Arbuscular Mycorrhizal Fungi Involved in the Response to Ethylene and Cytokinins?

Signals are exchanged at all stages of the arbuscular mycorrhizal (AM) symbiosis between fungi and their host plants. Root-exuded strigolactones are well-known early symbiotic cues, but the role of other phytohormones as interkingdom signals has seldom been investigated. Here we focus on ethylene and cytokinins, for which candidate receptors have been identified in the genome of the AM fungus Rhizophagus irregularis. Ethylene is known from the literature to affect asymbiotic development of AM fungi, and in the present study, we found that three cytokinin forms could stimulate spore germination in R. irregularis. Heterologous complementation of a Saccharomyces cerevisiae mutant strain with the candidate ethylene receptor RiHHK6 suggested that this protein can sense and transduce an ethylene signal. Accordingly, its N-terminal domain expressed in Pichia pastoris displayed saturable binding to radiolabeled ethylene. Thus, RiHHK6 displays the expected characteristics of an ethylene receptor. In contrast, the candidate cytokinin receptor RiHHK7 did not complement the S. cerevisiae mutant strain or Medicago truncatula cytokinin receptor mutants and seemed unable to bind cytokinins, suggesting that another receptor is involved in the perception of these phytohormones. Taken together, our results support the hypothesis that AM fungi respond to a range of phytohormones and that these compounds bear multiple functions in the rhizosphere beyond their known roles as internal plant developmental regulators. Our analysis of two phytohormone receptor candidates also sheds new light on the possible perception mechanisms in AM fungi. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Searching for Osmosensing Determinants in Poplar Histidine-Aspartate Kinases.

Previous works have shown the existence of protein partnership, belonging to a MultiStep Phosphorelay (MSP), potentially involved in osmosensing in Populus. The first actor of this signalling pathway belongs to the histidine-aspartate kinase (HK) family, which also includes the yeast osmosensor Sln1, as well as the Arabidopsis putative osmosensor AHK1. In poplar, the homologous AHK1 protein corresponds to a pair of paralogous proteins, HK1a and HK1b, exhibiting an extracellular domain (ECD), as in Sln1 and AHK1. An ECD alignment of AHK1-like proteins, from different plant species, showed a particularly well conserved ECD and revealed the presence of a cache domain. This level of conservation suggested a functional role of this domain in osmosensing. Thus, we tested this possibility by modelling assisted mutational analysis of the cache domain of the Populus HK1 proteins. The mutants were assessed for their ability to respond to different osmotic stress and the results point to an involvement of this domain in HK1 functionality. Furthermore, since HK1b was shown to respond better to stress than HK1a, these two receptors constituted a good system to search for osmosensing determinants responsible for this difference in efficiency. With domain swapping experiments, we finally demonstrated that the cache domain, as well as the second transmembrane domain, are involved in the osmosensing efficiency of these receptors.

Black Poplar (Populus nigra L.) Root Extracellular Trap, Structural and Molecular Remodeling in Response to Osmotic Stress.

The root extracellular trap (RET) consists of root-associated, cap-derived cells (root AC-DCs) and their mucilaginous secretions, and forms a structure around the root tip that protects against biotic and abiotic stresses. However, there is little information concerning the changes undergone by the RET during droughts, especially for tree species. Morphological and immunocytochemical approaches were used to study the RET of black poplar (Populus nigra L.) seedlings grown in vitro under optimal conditions (on agar-gelled medium) or when polyethylene glycol-mediated (PEG6000-infused agar-gelled medium) was used to mimic drought conditions through osmotic stress. Under optimal conditions, the root cap released three populations of individual AC-DC morphotypes, with a very low proportion of spherical morphotypes, and equivalent proportions of intermediate and elongated morphotypes. Immunolabeling experiments using anti-glycan antibodies specific to cell wall polysaccharide and arabinogalactan protein (AGP) epitopes revealed the presence of homogalacturonan (HG), galactan chains of rhamnogalacturonan-I (RG-I), and AGPs in root AC-DC cell walls. The data also showed the presence of xylogalacturonan (XGA), xylan, AGPs, and low levels of arabinans in the mucilage. The findings also showed that under osmotic stress conditions, both the number of AC-DCs (spherical and intermediate morphotypes) and the total quantity of mucilage per root tip increased, whereas the mucilage was devoid of the epitopes associated with the polysaccharides RG-I, XGA, xylan, and AGPs. Osmotic stress also led to reduced root growth and increased root expression of the P5CS2 gene, which is involved in proline biosynthesis and cellular osmolarity maintenance (or preservation) in aerial parts. Together, our findings show that the RET is a dynamic structure that undergoes pronounced structural and molecular remodeling, which might contribute to the survival of the root tip under osmotic conditions.

Cytokinin and Ethylene Cell Signaling Pathways from Prokaryotes to Eukaryotes.

Cytokinins (CKs) and ethylene (ET) are among the most ancient organic chemicals on Earth. A wide range of organisms including plants, algae, fungi, amoebae, and bacteria use these substances as signaling molecules to regulate cellular processes. Because of their ancestral origin and ubiquitous occurrence, CKs and ET are also considered to be ideal molecules for inter-kingdom communication. Their signal transduction pathways were first historically deciphered in plants and are related to the two-component systems, using histidine kinases as primary sensors. Paradoxically, although CKs and ET serve as signaling molecules in different kingdoms, it has been supposed for a long time that the canonical CK and ET signaling pathways are restricted to terrestrial plants. These considerations have now been called into question following the identification over recent years of genes encoding CK and ET receptor homologs in many other lineages within the tree of life. These advances shed new light on the dissemination and evolution of these hormones as both intra- and inter-specific communication molecules in prokaryotic and eukaryotic organisms.

New Insight into HPts as Hubs in Poplar Cytokinin and Osmosensing Multistep Phosphorelays: Cytokinin Pathway Uses Specific HPts.

We have previously identified proteins in poplar which belong to an osmosensing (OS) signaling pathway, called a multistep phosphorelay (MSP). The MSP comprises histidine-aspartate kinases (HK), which act as membrane receptors; histidine phosphotransfer (HPt) proteins, which act as phosphorelay proteins; and response regulators (RR), some of which act as transcription factors. In this study, we identified the HK proteins homologous to the Arabidopsis cytokinin (CK) receptors, which are first partners in the poplar cytokinin MSP, and focused on specificity of these two MSPs (CK and OS), which seem to share the same pool of HPt proteins. Firstly, we isolated five CK HKs from poplar which are homologous to Arabidopsis AHK2, AHK3, and AHK4, namely, HK2, HK3a, HK3b, HK4a, HK4b. These HKs were shown to be functional kinases, as observed in a functional complementation of a yeast HK deleted strain. Moreover, one of these HKs, HK4a, was shown to have kinase activity dependent on the presence of CK. Exhaustive interaction tests between these five CK HKs and the 10 HPts characterized in poplar were performed using two-hybrid and BiFC experiments. The resulting partnership was compared to that previously identified between putative osmosensors HK1a/1b and HPt proteins. Finally, in planta coexpression analysis of genes encoding these potential partners revealed that almost all HPts are coexpressed with CK HKs in four different poplar organs. Overall, these results allowed us to unravel the common and specific partnerships existing between OS and CK MSP in Populus.

Setting-up a fast and reliable cytokinin biosensor based on a plant histidine kinase receptor expressed in Saccharomyces cerevisiae.

Cytokinins (CK) have been extensively studied for their roles in plant development. Recently, they also appeared to ensure crucial functions in the pathogenicity of some bacterial and fungal plant pathogens. Thus, identifying cytokinin-producing pathogens is a prerequisite to gain a better understanding of their role in pathogenicity. Taking advantage of the cytokinin perception properties of Malus domestica CHASE Histidine Kinase receptor 2 (MdCHK2), we thereby developed a selective and highly sensitive yeast biosensor for the application of cytokinin detection in bacterial samples. The biosensor is based on the mutated sln1Δ Saccharomyces cerevisiae strain expressing MdCHK2. The biosensor does not require any extraction or purification steps of biological samples, enabling cytokinin analysis directly from crude bacterial supernatants. For the first time, the production of cytokinin was shown in the well-known plant pathogenic bacteria Erwinia amylovora and was also revealed in human pathogens Staphylococcus aureus and Streptococcus agalactiae. Importantly, this biosensor was shown to be an efficient tool for unraveling certain steps in cytokinin biosynthesis by micro-organisms since this it was successfully used to unveil the role of ygdH22, a LOG-like gene, that is probably involved in cytokinin biosynthesis pathway in Escherichia coli. Overall, we demonstrated that our biosensor displays several advantages including time- and cost-effectiveness by allowing a rapid and specific detection of cytokinins in bacterial supernatants These results also support its scalability to high-throughput formats.

CHASE-Containing Histidine Kinase Receptors in Apple Tree: From a Common Receptor Structure to Divergent Cytokinin Binding Properties and Specific Functions.

Cytokinin signaling is a key regulatory pathway of many aspects in plant development and environmental stresses. Herein, we initiated the identification and functional characterization of the five CHASE-containing histidine kinases (CHK) in the economically important Malus domestica species. These cytokinin receptors named MdCHK2, MdCHK3a/MdCHK3b, and MdCHK4a/MdCHK4b by homology with Arabidopsis AHK clearly displayed three distinct profiles. The three groups exhibited architectural variations, especially in the N-terminal part including the cytokinin sensing domain. Using a yeast complementation assay, we showed that MdCHK2 perceives a broad spectrum of cytokinins with a substantial sensitivity whereas both MdCHK4 homologs exhibit a narrow spectrum. Both MdCHK3 homologs perceived some cytokinins but surprisingly they exhibited a basal constitutive activity. Interaction studies revealed that MdCHK2, MdCHK4a, and MdCHK4b homodimerized whereas MdCHK3a and MdCHK3b did not. Finally, qPCR analysis and bioinformatics approach pointed out contrasted expression patterns among the three MdCHK groups as well as distinct sets of co-expressed genes. Our study characterized for the first time the five cytokinin receptors in apple tree and provided a framework for their further functional studies.

Functional Divergence of Poplar Histidine-Aspartate Kinase HK1 Paralogs in Response to Osmotic Stress.

Previous works have shown the existence of protein partnerships belonging to a MultiStep Phosphorelay (MSP) in Populus putatively involved in osmosensing. This study is focused on the identification of a histidine-aspartate kinase, HK1b, paralog of HK1a. The characterization of HK1b showed its ability to homo- and hetero-dimerize and to interact with a few Histidine-containing Phosphotransfer (HPt) proteins, suggesting a preferential partnership in poplar MSP linked to drought perception. Furthermore, determinants for interaction specificity between HK1a/1b and HPts were studied by mutagenesis analysis, identifying amino acids involved in this specificity. The HK1b expression analysis in different poplar organs revealed its co-expression with three HPts, reinforcing the hypothesis of partnership participation in the MSP in planta. Moreover, HK1b was shown to act as an osmosensor with kinase activity in a functional complementation assay of an osmosensor deficient yeast strain. These results revealed that HK1b showed a different behaviour for canonical phosphorylation of histidine and aspartate residues. These phosphorylation modularities of canonical amino acids could explain the improved osmosensor performances observed in yeast. As conserved duplicates reflect the selective pressures imposed by the environmental requirements on the species, our results emphasize the importance of HK1 gene duplication in poplar adaptation to drought stress.

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.

Screening for changes in leaf and cambial proteome of Populus tremula × P. alba under different heat constraints.

Young poplar plants were exposed to different heat regimes, a rapid heat constraint at 42°C (heat shock HS) alone or preceded by a stepwise increase in temperature (heat gradient HG). Proteomics analyses were carried out on both leaf and cambial tissues. The responses of both tissues were compared and linked to morphological and physiological observations. Both heat treatments negatively affected the photosynthetic rate while increasing the stomatal conductance. In the leaf, the HS impacted some photosynthetic proteins, and particularly induced an increase in abundance of proteins of the oxygen evolving complexes. On the other hand, the HG reduced carbohydrate metabolism and induced mainly an increase in germin-like proteins. In the cambial zone, the HS caused a decrease in sucrose synthase content and in enzymes related to protein synthesis. The main effect of HG was the accumulation of thaumatin-like proteins as well as an increase in the abundance of proteins involved in carbohydrate metabolism. Further, both tissues underwent changes in the content of heat shock proteins, but more importantly, of peroxiredoxins. The results show more sustainable changes in leaf and cambial proteomes in response to HS compared to HG.

Poplar under drought: comparison of leaf and cambial proteomic responses.

The forest ecosystem is of particular importance from an economic and ecological perspective. However, the stress physiology of trees, perennial and woody plants, is far from being fully understood. For that purpose, poplar plants were exposed to drought; the plants exhibited commonly reported drought stress traits in the different plant tissues. Leafy rooted cuttings of poplar were investigated through a proteomic approach in order to compare the water constraint response of two plant tissues, namely leaf and cambium. Sampling was realized during the drought period at 2 time points with increased drought intensity and 7 days after rewatering. Our data show that there is a difference in the moment of response to the water constraint between the two tissues, cambium being affected later than leaves. In leaves, drought induced a decrease in rubisco content, and an increase in the abundance of light harvesting complex proteins as well as changes in membrane-related proteins. In the cambial tissue, the salient proteome pattern change was the decrease of multiple proteins identified as bark storage proteins. After rewatering, almost all changes in cambial proteome disappeared whereas a significant number of leaf proteins appeared to be differentially regulated only during the recovery from drought.

Acute metal stress in Populus tremula x P. alba (717-1B4 genotype): leaf and cambial proteome changes induced by cadmium 2+.

The comprehension of metal homeostasis in plants requires the identification of molecular markers linked to stress tolerance. Proteomic changes in leaves and cambial zone of Populus tremula x P. alba (717-1B4 genotype) were analyzed after 61 days of exposure to cadmium (Cd) 360 mg/kg soil dry weight in pot-soil cultures. The treatment led to an acute Cd stress with a reduction of growth and photosynthesis. Cd stress induced changes in the display of 120 spots for leaf tissue and 153 spots for the cambial zone. It involved a reduced photosynthesis, resulting in a profound reorganisation of carbon and carbohydrate metabolisms in both tissues. Cambial cells underwent stress from the Cd actually present inside the tissue but also a deprivation of photosynthates caused by leaf stress. An important tissue specificity of the response was observed, according to the differences in cell structures and functions.

Leaf proteome analysis of eight Populus xeuramericana genotypes: genetic variation in drought response and in water-use efficiency involves photosynthesis-related proteins.

Genetic variation of leaf proteome in drought response was investigated among eight Populus xeuramericana genotypes contrasting for their leaf carbon isotope discrimination (Delta), an estimate of intrinsic water-use efficiency. Plants were grown in open field on two similar plots. Drought was induced by an 86-day irrigation cessation on one plot, whereas a second plot remained regularly irrigated. Using 2-DE, 863 reproducible spots were detected; about 60% presented at least one significant effect i.e. treatment, genotype and/or genotype by treatment interaction effect. A significant genotype by treatment interaction was detected for 62 reliably identified proteins among which, about 65% consisted in chloroplast-associated proteins either involved in the Calvin cycle or in the electron-transport chains. The other proteins were involved in oxidative stress, amino acid or protein metabolisms. Correlations between protein abundance and Delta variations were found for 45 reliably identified proteins. The abundance of ribulose-1,5-bisphosphate carboxylase/oxygenase activase isoforms scaled negatively with Delta regardless of the treatment, suggesting that a large intrinsic water-use efficiency could be due to higher abundance of ribulose-1,5-bisphosphate carboxylase/oxygenase activase. Under control condition, abundance of enzymes involved in carbon fixation was also negatively correlated with Delta, whereas abundance of enzymes involved in photorespiration or respiration was positively correlated with Delta.

Genetic variation and drought response in two Populus x euramericana genotypes through 2-DE proteomic analysis of leaves from field and glasshouse cultivated plants.

Genotype and water deficit effects on leaf 2-DE protein profiles of two Populus deltoides x Populus nigra, cv. 'Agathe_F' and 'Cima', were analysed over a short-term period of 18 days in glasshouse using 4-month-old rooted cuttings and over a long-lasting period of 86 days in open field using 4-year-old rooted cuttings. Leaf proteomes were analyzed using two-dimensional gel electrophoresis, and proteins were identified after database searching from MS peptide spectra. A reliable genotype effect was observed in the leaf proteome over experiment locations, water regimes and sampling dates. Quantitative differences between genotypes were found. Most of them corresponded to proteins matching isoforms or post-translational modification variants. However, 'Cima' displayed the highest abundance of antioxidant enzymes. In response to water deficit, about 10% of the reproducible spots significantly varied regardless of the experiment location, among which about 25% also displayed genotype-dependent variations. As a whole, while 'Cima' differed from 'Agathe_F' by increased abundance of enzymes involved in photorespiration and in oxidative stress, 'Agathe_F' was mainly differentiated by increased abundance of enzymes involved in photosynthesis.

Physiological characterization and identification of genes differentially expressed in response to drought induced by PEG 6000 in Populus canadensis leaves.

We report here about the physiological and molecular responses of Populus canadensis (clone Dorskamp) to drought. The stress was applied to young rooted cuttings by PEG 6000 application over 30 days. This stress induces a decrease in predawn leaf water potential. After 10 days of stress, there was a decrease in stomatal conductance and a slight retardation of leaf growth, but the osmotic potential remained constant. Using the differential display technique, we searched for genes differentially expressed in response to drought at this date. Thirty-six differentially expressed leaf cDNAs were detected between stressed and control conditions. Thirty-four cDNAs clones were successfully cloned and 23 were found to share high identity with Arabidopsis thaliana and Populus trichocarpa genes. The transcriptional regulation of 21 genes was examined by reverse RNA dot blot, confirming an increase in expression for 16 of them after 10 days of treatment. Among these 16 genes, most of them are involved in a different cellular metabolic pathway. These differentially expressed genes are also involved and/or regulated by other treatments such as salt, withholding water or auxin application. The maintenance of growth observed during the first 10 days of the stress period could be due to the regulation of these genes and can be a common response between herbaceous plants and trees.

Osmotic stress sensing in Populus: components identification of a phosphorelay system.

To study the Populus response to an osmotic stress, we have isolated one cDNA encoding a histidine-aspartate kinase (HK1) and four cDNAs encoding histidine-containing phosphotransfer proteins (HPts), HPt1-4. The predicted HK1 protein shares a typical structure with ATHK1 and SLN1 osmosensors. The 4 HPTs are characterized by the histidine phosphotransfer domain. We have shown that HK1 is upregulated during an osmotic stress in hydroponic culture. We have detected an interaction between HK1 and HPt2, using the yeast two-hybrid system. These results suggest the existence of a multi-step phosphorelay pathway probably involved in osmotic stress sensing in Populus.