Cytokinin receptor-dependent and receptor-independent pathways in the dehydration response of Arabidopsis thaliana

Cytokinin signaling in Arabidopsis thaliana utilizes a multi-step two-component signaling (TCS) system comprised of sensor histidine kinases (AHKs), histidine phosphotransfer proteins (AHPs), and response regulators (ARRs). Recent studies have suggested that the cytokinin TCS system is involved in a variety of other signaling and metabolic pathways. To further explore a potential function of the cytokinin TCS in the Arabidopsis dehydration stress response, we investigated the expression of all type-A ARR genes and a type-C ARR, ARR22, in both wild type and ahk single, double, and triple mutants in response to dehydration compared to cytokinin as well as dehydration tolerance of ahk mutants. We found that drought significantly induced the expression of a subset of ARR genes, ARR5, ARR7, ARR15, and ARR22. The results of expression analyses in ahk single, double, and triple mutants demonstrated that the cytokinin receptors AHK2 and AHK3 are redundantly involved in dehydration-inducible expression of ARR7, but not that of ARR5, ARR15, or ARR22. Dehydration tolerance assays showed that ahk2 and ahk3 single mutants exhibited enhanced dehydration tolerance compared with that of wild-type plants and ahk4 mutants, and that ahk2 ahk3 double mutants exhibited stronger drought tolerance than that of ahk3 ahk4, which exhibited more enhanced drought tolerance than that of wild-type plants and ahk single mutants. Taken together, these results demonstrate that while the cytokinin receptors AHK2 and AHK3 are critically involved in the dehydration tolerance response, both cytokinin receptor-dependent pathway and receptor-independent pathway occur in the dehydration response regulating ARR gene expression. In addition, preincubating ahk2, ahk3, ahk4, and the wild-type plants with cytokinin induced enhanced dehydration stress tolerance in these plants, demonstrating that cytokinins are involved in regulating plant response to dehydration stress.     

Genome-wide identification and expression analysis of two component system genes in Cicer arietinum

The two-component system (TCS) plays an important role in signal transduction pathways, cytokinin signaling and stress resistance of prokaryotes and eukaryotes. It is comprised of three types of proteins in plants; histidine kinases (HKs), histidine phosphotransfer proteins (HPs) and response regulators (RRs). Chickpea (Cicer arietinum L.) is one of the most important legume crops worldwide with special economic value in semi-arid tropics. Availability of complete genome sequence of chickpea presents a valuable resource for comparative analysis among angiosperms. In current study, Arabidopsis thaliana and Oryza sativa were used as reference plant species for comparative genomics analysis with C. arietinum. A genome-wide computational survey enabled us to identify putative members of TCS protein family including 18HKs, 26 RRs (7 type-A, 7 type-B, 2 type C and 10 pseudo) and 7 HPs (5 true and 2pseudo) genes in chickpea. The predicted TCS genes displayed family specific intron/exon organization and were randomly distributed across all the eight chromosomes. Comparative phylogenetic and evolutionary analysis suggested a variable conservation of TCS genes in relation to mono/dicot model plants and segmental duplication was the principal route of expansion for this family in chickpea. The promoter regions of TCS genes exhibited several abiotic stress-related cis-elements indicating their involvement in abiotic stress response. The expression analysis of TCS genes demonstrated stress (drought, heat, osmotic and salt) specific differential expression. Current study provides insight into TCS genes in C. arietinum, which will be helpful for further functional analysis of these genes in response to different abiotic stresses.     

Function of the pseudo phosphotransfer proteins has diverged between rice and Arabidopsis

The phytohormone cytokinin plays a significant role in nearly all aspects of plant growth and development. Cytokinin signaling has primarily been studied in the dicot model Arabidopsis, with relatively little work done in monocots, which include rice (Oryza sativa) and other cereals of agronomic importance. The cytokinin signaling pathway is a phosphorelay comprised of the histidine kinase receptors, the authentic histidine phosphotransfer proteins (AHPs) and type-B response regulators (RRs). Two negative regulators of cytokinin signaling have been identified: the type-A RRs, which are cytokinin primary response genes, and the pseudo histidine phosphotransfer proteins (PHPs), which lack the His residue required for phosphorelay. Here, we describe the role of the rice PHP genes. Phylogenic analysis indicates that the PHPs are generally first found in the genomes of gymnosperms and that they arose independently in monocots and dicots. Consistent with this, the three rice PHPs fail to complement an Arabidopsis php mutant (aphp1/ahp6). Disruption of the three rice PHPs results in a molecular phenotype consistent with these elements acting as negative regulators of cytokinin signaling, including the induction of a number of type-A RR and cytokinin oxidase genes. The triple php mutant affects multiple aspects of rice growth and development, including shoot morphology, panicle architecture, and seed fill. In contrast to Arabidopsis, disruption of the rice PHPs does not affect root vascular patterning, suggesting that while many aspects of key signaling networks are conserved between monocots and dicots, the roles of at least some cytokinin signaling elements are distinct.     

Proteomic analysis of wheat embryos with 2-DE and liquid-phase chromatography (ProteomeLab PF-2D) — A wider perspective of the proteome

Cereal embryos are a model system to study desiccation tolerance due to their ability to survive extreme water loss during late embryogenesis. To identify proteins accumulating in mature embryos which can be used as potential markers for dehydration tolerance, we compared the embryo proteome from two durum wheat genotypes (Triticum durum Desf.), Mahmoudi (salt and drought sensitive) and Om Rabia3 (salt and drought tolerant). Total protein extracts from wheat embryos were analyzed by using conventional 2-DE and ProteomeLab PF-2D. Analysis using different pH ranges showed that a larger number of fractions were solved by LC, than by conventional 2-DE at extreme technical pHs (pH 4.0–5.0 and pH 6.5–8.0). In contrast, at intermediate pHs (pH 5.0–6.5), resolution was better in 2-DE gels. The two techniques were used in parallel to analyze total protein extracts from embryos of the two wheat varieties. Several proteins belonging to the seed storage family, LEA-type/heat shock proteins, enzyme metabolism and radical scavengers were identified by analysis of trypsin digested peptides via mass spectrometry. These proteins accumulate in different amounts in embryos of tolerant and sensitive wheat varieties. The differences in expression pattern were further validated by enzyme activity, western blotting analysis and correlated with their corresponding mRNA expression by RT-PCR analyses for the corresponding protein. We suggest that the differential expression pattern could be used as a basis for a biochemical screen of tolerance/sensitivity to drought and salt stress in wheat embryos and germplasm.     

Vibrio cholerae Response Regulator VxrB Controls Colonization and Regulates the Type VI Secretion System

Two-component signal transduction systems (TCS) are used by bacteria to sense and respond to their environment. TCS are typically composed of a sensor histidine kinase (HK) and a response regulator (RR). The Vibrio cholerae genome encodes 52 RR, but the role of these RRs in V. cholerae pathogenesis is largely unknown. To identify RRs that control V. cholerae colonization, in-frame deletions of each RR were generated and the resulting mutants analyzed using an infant mouse intestine colonization assay. We found that 12 of the 52 RR were involved in intestinal colonization. Mutants lacking one previously uncharacterized RR, VCA0566 (renamed VxrB), displayed a significant colonization defect. Further experiments showed that VxrB phosphorylation state on the predicted conserved aspartate contributes to intestine colonization. The VxrB regulon was determined using whole genome expression analysis. It consists of several genes, including those genes that create the type VI secretion system (T6SS). We determined that VxrB is required for T6SS expression using several in vitro assays and bacterial killing assays, and furthermore that the T6SS is required for intestinal colonization. vxrB is encoded in a four gene operon and the other vxr operon members also modulate intestinal colonization. Lastly, though ΔvxrB exhibited a defect in single-strain intestinal colonization, the ΔvxrB strain did not show any in vitro growth defect. Overall, our work revealed that a small set of RRs is required for intestinal colonization and one of these regulators, VxrB affects colonization at least in part through its regulation of T6SS genes.     

Evolution of prokaryotic two-component system signaling pathways: gene fusions and fissions

Two-component systems (TCSs) are common signal transduction systems, typically comprising paired histidine protein kinase (HK) and response regulator (RR) proteins. In many examples, it appears RR and HK genes have fused, producing a "hybrid kinase " We have characterized a set of prokaryotic genes encoding RRs, HKs, and hybrid kinases, enabling characterization of gene fusion and fission. Primary factors correlating with fusion rates are the presence of transmembrane helices in HKs and the presence of DNA-binding domains in RRs, features that require correct (and separate) spatial location. In the absence of such features, there is a relative abundance of fused genes. The order of paired HK and RR genes and the nucleotide distance between encoded domains also correlate with apparent gene fusion rates. We propose that localization requirements and relative positioning of encoded domains within TCS genes affect the function (and therefore retention) of hybrid kinases resulting from gene fusion.     

Comparative Genomic Analysis of Two-Component Signal Transduction Systems in Probiotic Lactobacillus casei

Lactobacillus casei has traditionally been recognized as a probiotic, thus needing to survive the industrial production processes and transit through the gastrointestinal tract before providing benefit to human health. The two-component signal transduction system (TCS) plays important roles in sensing and reacting to environmental changes, which consists of a histidine kinase (HK) and a response regulator (RR). In this study we identified HKs and RRs of six sequenced L. casei strains. Ortholog analysis revealed 15 TCS clusters (HK–RR pairs), one orphan HKs and three orphan RRs, of which 12 TCS clusters were common to all six strains, three were absent in one strain. Further classification of the predicted HKs and RRs revealed interesting aspects of their putative functions. Some TCS clusters are involved with the response under the stress of the bile salts, acid, or oxidative, which contribute to survive the difficult journey through the human gastrointestinal tract. Computational predictions of 15 TCSs were verified by PCR experiments. This genomic level study of TCSs should provide valuable insights into the conservation and divergence of TCS proteins in the L. casei strains.     

Building interacting partner predictors using co-varying residue pairs between histidine kinase and response regulator pairs of 48 bacterial two-component systems

The two‐component system (TCS) is a signal transduction system that involves a histidine kinase (HK) and a response regulator (RR). Although up to hundreds of TCSs may operate in parallel in a bacterial cell, the high‐fidelity of a TCS signaling is well maintained, minimizing irrelevant crosstalk between TCSs. When a HK gene and a RR gene in a given TCS system exist in neighboring positions, it is almost certain that their protein products (i.e., HK and RR) are interacting partners. However, large bacterial genomes often have multiple HK genes and/or cognate RR genes that are not neighboring positions. In many partially assembled genomes, some HK genes and RR genes belong to different contigs. In these cases, it is not clear which HK(s) and RR(s) interact. By combining information‐theoretic and graph‐theoretic approaches, we developed a computational method identifying co‐evolving residue pairs between HKs and cognate RRs and predicting the interacting HK:RR pairs for each TCS. In addition, we built a TCSppWWW webserver ( http://compath.org/platcom/tcs ) that takes query sequences of pairing candidates and predicts their HK:RR pairing using precomputed models. The current release of TCSppWWW provides predictors for 48 TCSs using over 20,000 protein sequences from about 900 bacterial genomes. Three different types of predictors using Random Forest, RBF Network, and Naïve Bayes are provided. Once a set of HK and RR candidate sequences are submitted, TCSppWWW aligns query sequences to the precomputed multiple sequence alignment of HK:RR pairs, extracts co‐evolving column positions, then returns prediction results with prediction margin and additional information. Proteins 2011. © 2010 Wiley‐Liss, Inc.     

Importance of pre‐anthesis anther sink strength for maintenance of grain number during reproductive stage water stress in wheat

Reproductive stage water stress leads to spikelet sterility in wheat. Whereas drought stress at anthesis affects mainly grain size, stress at the young microspore stage of pollen development is characterized by abortion of pollen development and reduction in grain number. We identified genetic variability for drought tolerance at the reproductive stage. Drought‐tolerant wheat germplasm is able to maintain carbohydrate accumulation in the reproductive organs throughout the stress treatment. Starch depletion in the ovary of drought‐sensitive wheat is reversible upon re‐watering and cross‐pollination experiments indicate that the ovary is more resilient than the anther. The effect on anthers and pollen fertility is irreversible, suggesting that pollen sterility is the main cause of grain loss during drought conditions in wheat. The difference in storage carbohydrate accumulation in drought‐sensitive and drought‐tolerant wheat is correlated with differences in sugar profiles, cell wall invertase gene expression and expression of fructan biosynthesis genes in anther and ovary (sucrose : sucrose 1‐fructosyl‐transferase, 1‐SST; sucrose : fructan 6‐fructosyl‐transferase, 6‐SFT). Our results indicate that the ability to control and maintain sink strength and carbohydrate supply to anthers may be the key to maintaining pollen fertility and grain number in wheat and this mechanism may also provide protection against other abiotic stresses.     

Expression profiling of rice cultivars differing in their tolerance to long-term drought stress

Understanding the molecular basis of plant performance under water-limiting conditions will help to breed crop plants with a lower water demand. We investigated the physiological and gene expression response of drought-tolerant (IR57311 and LC-93-4) and drought-sensitive (Nipponbare and Taipei 309) rice (Oryza sativa L.) cultivars to 18 days of drought stress in climate chamber experiments. Drought stressed plants grew significantly slower than the controls. Gene expression profiles were measured in leaf samples with the 20 K NSF oligonucleotide microarray. A linear model was fitted to the data to identify genes that were significantly regulated under drought stress. In all drought stressed cultivars, 245 genes were significantly repressed and 413 genes induced. Genes differing in their expression pattern under drought stress between tolerant and sensitive cultivars were identified by the genotype x environment (G x E) interaction term. More genes were significantly drought regulated in the sensitive than in the tolerant cultivars. Localizing all expressed genes on the rice genome map, we checked which genes with a significant G x E interaction co-localized with published quantitative trait loci regions for drought tolerance. These genes are more likely to be important for drought tolerance in an agricultural environment. To identify the metabolic processes with a significant G x E effect, we adapted the analysis software MapMan for rice. We found a drought stress induced shift toward senescence related degradation processes that was more pronounced in the sensitive than in the tolerant cultivars. In spite of higher growth rates and water use, more photosynthesis related genes were down-regulated in the tolerant than in the sensitive cultivars.     

Proteome analysis of outer membrane vesicles from a clinical Acinetobacter baumannii isolate

Two-component systems (TCSs), typically consisting of a histidine kinase (HK) and a cognate response regulator (RR), are the most common signaling systems in bacteria. Besides paired genes encoding TCSs, there also exists unpaired HKs and orphan RRs. In Streptomyces coelicolor , 13 orphan RRs have been annotated. Because of lack of cognate HKs, little is known as yet about the regulation of orphan RRs. Bioinformatic analysis revealed that several orphan RRs had high amino acid sequence identities with RRs from typical TCSs in S. coelicolor . Among them, the orphan RR SCO3818 and RR SCO0204, which paired with HK SCO0203, showed the highest identity (65%), suggesting that the two RRs might both be under the regulation of SCO0203. Following studies showed that SCO0203 could phosphorylate not only SCO0204 but also SCO3818. Deletion of either sco0203 or sco3818 led to enhanced production of blue-pigmented antibiotic actinorhodin, which indicated a functional correlation between SCO0203 and SCO3818. These results suggested that SCO3818 might be regulated by SCO0203. This is the first report describing the regulation of an orphan RR by an HK. Moreover, this is also the first identification of cross-talk between different TCS components in S. coelicolor .