Plant histidine kinases: an emerging picture of two-component signal transduction in hormone and environmental responses.

In the Arabidopsis thaliana genome, 11 genes encode bacterial-type two-component histidine kinases. Genetic and biochemical analyses indicate that five two-component histidine kinase-like proteins (ETR1, ETR2, EIN4, ERS1, and ERS2) function as ethylene receptors. A hybrid histidine kinase, CRE1 (also known as AHK4), acts as a cytokinin receptor, and a set of response regulators may be involved in cytokinin signal transduction. In addition to CRE1, histidine kinases CKI1 and CKI2 are likely to play important roles in cytokinin signaling. A database search of the entire Arabidopsis genome sequence has identified two additional homologs of CRE1. Arabidopsis seems to employ a hybrid histidine kinase, ATHK1, as an osmosensor. Plants widely use two-component systems in the detection of, and signal transduction by, the growth regulators ethylene and cytokinin, as well as in their responses to environmental stimuli.

Possible His to Asp phosphorelay signaling in an Arabidopsis two-component system.

We have so far cloned a cDNA encoding a hybrid-type histidine kinase (ATHK1), three cDNAs encoding phosphorelay intermediates (ATHP1-3), and four cDNAs encoding response regulators (ATRR1-4) from Arabidopsis thaliana. To determine which molecules constitute a His to Asp phosphorelay pathway, we examined protein-protein interactions between them using a pairwise yeast two-hybrid analysis, as an initial step. We detected a specific interaction between ATHK1 and ATHP1. We further examined protein-protein interactions between ATHP1-3 and other histidine kinases. We detected interactions between ETR1 and all ATHPs, and between CKI1 and ATHP1 or ATHP2. Interestingly, ERS1 could not interact with any ATHPs. We also examined protein-protein interactions between ATHP1-3 and ATRR1-4. The results indicated that ATHP2 could interact with ATRR4, and that ATHP3 could interact with ATRR1 or ATRR4. However, ATHP1 could not interact with any ATRRs. On the basis of these results, we discuss the possible phosphorelay networks in an Arabidopsis two-component system.

Semiquantitative immunoblot analysis of nm23-H1 and -H2 isoforms in adenocarcinomas of the lung: prognostic significance.

Total amounts of nm23 protein and relative levels of H1 and H2 isoforms were studied in 27 fresh-frozen samples of pulmonary adenocarcinoma and adjacent non-neoplastic tissues that were obtained at surgery. Semiquantitative immunoblotting with a monoclonal antibody (Pan-242) against nm23 protein demonstrated both isoforms, recognized as 20.5 kDa for H1 and 18.5 kDa for H2, to be present in all cases. Both H1 and H2 levels in neoplastic tissues were higher than in the corresponding non-neoplastic samples. Expression of H2 was usually greater than of H1. The H2/H1 ratio varied from 1.9 to 14.1 (mean value 5.2) in non-neoplastic tissues and 1.0-5.9 (mean value 2.5) in neoplastic tissues, although this ratio did not correlate with any prognostic factor like tumor size, nodal status or distant metastasis (TNM tumor stage). H1 and H2 levels were significantly lower (mean values 4.3 and 2.4) in well-differentiated than in moderately and poorly differentiated adenocarcinomas (8.3 and 3.0) (P < 0.03 and P < 0.05, respectively). These data indicate that H1 and H2 isoform levels correlate with histological differentiation, but not the metastatic potential or stage of pulmonary adenocarcinoma.

Two-component systems in plant signal transduction.

In plants, two-component systems play important roles in signal transduction in response to environmental stimuli and growth regulators. Genetic and biochemical analyses indicate that sensory hybrid-type histidine kinases, ETR1 and its homologs, function as ethylene receptors and negative regulators in ethylene signaling. Two other hybrid-type histidine kinases, CKI1 and ATHK1, are implicated in cytokinin signaling and osmosensing processes, respectively. A data base search of Arabidopsis ESTs and genome sequences has identified many homologous genes encoding two-component regulators. We discuss the possible origins and functions of these two-component systems in plants.

A transmembrane hybrid-type histidine kinase in Arabidopsis functions as an osmosensor.

Water deficit and the resulting osmotic stress affect plant growth. To understand how plant cells monitor and respond to osmotic change from water stress, we isolated a cDNA from dehydrated Arabidopsis plants. This cDNA encodes a novel hybrid-type histidine kinase, ATHK1. Restriction fragment length polymorphism mapping showed that the ATHK1 gene is on chromosome 2. The predicted ATHK1 protein has two putative transmembrane regions in the N-terminal half and has structural similarity to the yeast osmosensor synthetic lethal of N-end rule 1 (SLN1). The ATHK1 transcript was more abundant in roots than other tissues under normal growth conditions and accumulated under conditions of high or low osmolarity. Histochemical analysis of beta-glucuronidase activities driven by the ATHK1 promoter further indicates that the ATHK1 gene is transcriptionally upregulated in response to changes in external osmolarity. Overexpression of the ATHK1 cDNA suppressed the lethality of the temperature-sensitive osmosensing-defective yeast mutant sln1-ts. By contrast, ATHK1 cDNAs in which conserved His or Asp residues had been substituted failed to complement the sln1-ts mutant, indicating that ATHK1 functions as a histidine kinase. Introduction of the ATHK1 cDNA into the yeast double mutant sln1Delta sho1Delta, which lacks two osmosensors, suppressed lethality in high-salinity media and activated the high-osmolarity glycerol response 1 (HOG1) mitogen-activated protein kinase (MAPK). These results imply that ATHK1 functions as an osmosensor and transmits the stress signal to a downstream MAPK cascade.

Characterization of genes for two-component phosphorelay mediators with a single HPt domain in Arabidopsis thaliana.

Three cDNAs that encode two-component phosphorelay-mediator-like proteins were cloned from Arabidopsis thaliana. Putative proteins (ATHP1-3) contain an HPt (Histidine-containing Phospho transfer)-like domain with a conserved histidine and some invariant residues that are involved in phosphorelay. Growth retardation of YPD1-disrupted yeast cells was reversed with ATHPs, which indicates that ATHPs function as phosphorelay mediators in yeast cells. The ATHP genes are expressed more in roots than in other tissues, similar to the expression of genes for a sensor histidine kinase, ATHK1, and response regulators ATRR1-4. These results suggest that ATHPs function as two-component phosphorelay mediators between sensor histidine kinase and response regulators in Arabidopsis.

Stress-responsive expression of genes for two-component response regulator-like proteins in Arabidopsis thaliana.

Four cDNAs that encode two-component response regulator-like proteins were cloned from Arabidopsis thaliana. Putative proteins (ATRR1-4) contain a receiver domain with a conserved aspartate residue - a possible phosphorylation site - at the N-terminal half. ATRR2 lacks the C-terminal half; the others contain a C-terminal domain abundant in acidic amino acids or proline residues. ATRR1 and ATRR2 are expressed more in roots than in other tissues and are induced by low temperature, dehydration and high salinity. Levels of ATRR3 and ATRR4 were not affected by stress treatments. These results suggest that ATRRs play distinct physiological roles in Arabidopsis, and that some are involved in stress responses.

Role of arabidopsis MYC and MYB homologs in drought- and abscisic acid-regulated gene expression.

In Arabidopsis, the induction of a dehydration-responsive gene, rd22, is mediated by abscisic acid (ABA) and requires protein biosynthesis for ABA-dependent gene expression. Previous experiments established that a 67-bp DNA fragment of the rd22 promoter is sufficient for dehydration- and ABA-induced gene expression and that this DNA fragment contains two closely located putative recognition sites for the basic helix-loop-helix protein MYC and one putative recognition site for MYB. We have carefully analyzed the 67-bp region of the rd22 promoter in transgenic tobacco plants and found that both the first MYC site and the MYB recognition site function as cis-acting elements in the dehydration-induced expression of the rd22 gene. A cDNA encoding a MYC-related DNA binding protein was isolated by DNA-ligand binding screening, using the 67-bp region as a probe, and designated rd22BP1. The rd22BP1 cDNA encodes a 68-kD protein that has a typical DNA binding domain of a basic region helix-loop-helix leucine zipper motif in MYC-related transcription factors. The rd22BP1 protein binds specifically to the first MYC recognition site in the 67-bp fragment. RNA gel blot analysis revealed that transcription of the rd22BP1 gene is induced by dehydration stress and ABA treatment, and its induction precedes that of rd22. We have reported a drought- and ABA-inducible gene that encodes the MYB-related protein ATMYB2. In a transient transactivation experiment using Arabidopsis leaf protoplasts, we demonstrated that both the rd22BP1 and ATMYB2 proteins activate transcription of the rd22 promoter fused to the beta-glucuronidase reporter gene. These results indicate that both the rd22BP1 (MYC) and ATMYB2 (MYB) proteins function as transcriptional activators in the dehydration- and ABA-inducible expression of the rd22 gene.

Novel drought-inducible genes in the highly drought-tolerant cowpea: cloning of cDNAs and analysis of the expression of the corresponding genes.

Ten cDNAs of genes that were induced by dehydration stress were cloned by differential screening from the highly drought-tolerant legume, cowpea (Vigna unguiculata), a major crop in West Africa. The clones were collectively named CPRD (cowpea clones responsive to dehydration). Northern blot analysis revealed that nine of the CPRD genes were induced by dehydration stress, but the timing of induction of mRNA synthesis varied among the CPRD genes. We analyzed the effects of other environmental stresses on the expression of the CPRD8, CPRD14 and CPRD22 genes, and we found that these genes were strongly induced by high-salinity stress but not by cold or heat stress. Drought-stressed cowpea plants accumulated abscisic acid (ABA) to a level that was 160 times higher than that in unstressed plants. The CPRD8 and CPRD22 genes were induced to a significant extent by the application of exogenous ABA but the CPRD14 gene was not. These results indicate the existence of at least two signal-transduction pathways between the detection of water stress and the expression of CPRD genes in cowpea. Sequence analysis of CPRD8 and CPRD22 cDNAs revealed that they encoded putative proteins that were related to old yellow enzyme and group 2 LEA proteins, respectively. The protein encoded by CPRD14 exhibited sequence homology to dihydroflavonol-4-reductase (DFR) and vestitone reductase (VR). Old yellow enzyme, DFR and VR have not been identified as drought-inducible proteins in other plants, whereas LEA genes have been well characterized as drought-inducible genes. The various gene products might function to protect cells from environmental stress.

A transcriptional activation domain of ATMYB2, a drought-inducible Arabidopsis Myb-related protein.

Trans-activation activity of ATMYB2, a drought-inducible Myb-related protein in Arabidopsis thaliana, was analyzed using a transient assay of Arabidopsis leaf protoplasts. ATMYB2 activated the transcription of a reporter gene from the MYB-binding site in a sequence-specific manner. Deletion of the C-terminal region of ATMYB2 reduced the trans-activation of the reporter gene, indicating that the acidic region at the C-terminus of ATMYB2 is required for transcriptional activation. The domain exchange analysis with the yeast GAL4 revealed that the C-terminal acidic region of ATMYB2 contains a sufficient domain for trans-activation. These results indicate that ATMYB2 acts as a transcriptional activator and that the C-terminal acidic region of ATMYB2 can function as a transcriptional activation domain.

Molecular cloning and characterization of a gene that encodes a MYC-related protein in Arabidopsis.

In plants, MYC-related proteins function as transcription factors involved in anthocyanin production and trichome development. We cloned a gene, Atmyc1, and its corresponding cDNA, that encodes for a MYC-related protein from Arabidopsis thaliana. The putative protein has a basic/helix-loop-helix motif at the C-terminus and a highly homologous region with that of the maize B/R family at the N-terminus. The promoter region of Atmyc1 contains a Sph box (CATGCATG) that is known as a cis-regulatory element conferring seed-specific expression. In fact, Atmyc1 transcripts were more abundant in developing seeds than in stems and leaves where trichomes are normally expressed. Restriction fragment length polymorphism mapping demonstrated that Atmyc1 is located on the upper region of chromosome 4, which clearly indicates that Atmyc1 is distinct from the ttg (transparent testa glabrous) locus that affects both trichome development and anthocyanin biosynthesis.

Cloning of cDNAs and the molecular evolution of a bovine MHC class II DRA gene.

Two overlapping cDNA clones coding for bovine major histocompatibility complex (MHC) class II antigen DRA chain were isolated and characterized. The full-length cDNA clone, MR1, encoded a primary translated product of 253 amino acids, 24 of which were deduced to be a signal peptide and 229 which formed a mature polypeptide. The amino acid sequences deduced from this clone resembled those of class II A molecules from other species in both size and structure, but no potential consensus site of N-linked glycosylation comparable to those in the human, mouse, rat and swine proteins was found in the alpha 2 domain, as well as ovine and equine DRA molecules. Comparison of amino acid sequences encoded by class II A genes among several species and a dendrogram constructed from these data places the DRA gene and the DQA/DYA genes on two distinct branches of a phylogenetic tree, with bovine DRA and ovine DRA being most similar on the DRA branch.

Two genes that encode Ca(2+)-dependent protein kinases are induced by drought and high-salt stresses in Arabidopsis thaliana.

Two cDNA clones, cATCDPK1 and cATCDPK2, encoding Ca(2+)-dependent, calmodulin-independent protein kinases (CDPK) were cloned from Arabidopsis thaliana and their nucleotide sequences were determined. Northern blot analysis indicated that the mRNAs corresponding to the ATCDPK1 and ATCDPK2 genes are rapidly induced by drought and high-salt stress but not by low-temperature stress or heat stress. Treatment of Arabidopsis plants with exogenous abscisic acid (ABA) had no effect on the induction of ATCDPK1 or ATCDPK2. These findings suggest that a change in the osmotic potential of the environment can serve as a trigger for the induction of ATCDPK1 and ATCDPK2. Putative proteins encoded by ATCDPK1 and ATCDPK2 which contain open reading frames of 1479 and 1488 bp, respectively, are designated ATCDPK1 and ATCDPK2 and show 52% identity at the amino acid sequence level. ATCDPK1 and ATCDPK2 exhibit significant similarity to a soybean CDPK (51% and 73%, respectively). Both proteins contain a catalytic domain that is typical of serine/threonine protein kinases and a regulatory domain that is homologous to the Ca(2+)-binding sites of calmodulin. Genomic Southern blot analysis suggests the existence of a few additional genes that are related to ATCDPK1 and ATCDPK2 in the Arabidopsis genome. The ATCDPK2 protein expressed in Escherichia coli was found to phosphorylate casein and myelin basic protein preferentially, relative to a histone substrate, and required Ca2+ for activation.

An Arabidopsis myb homolog is induced by dehydration stress and its gene product binds to the conserved MYB recognition sequence.

An Arabidopsis cDNA (Atmyb2) that contains a sequence that encodes a transcription factor, which is a homolog of MYB, was cloned from a cDNA library prepared from dehydrated Arabidopsis rosette plants. A gene (Atmyb2) corresponding to the Atmyb2 cDNA was also cloned and its nucleotide sequence was determined. RNA gel blot analysis showed that the Atmyb2 mRNA was induced by dehydration and disappeared upon rehydration. The Atmyb2 mRNA also accumulated upon salt stress and with the onset of treatment with abscisic acid. A beta-glucuronidase reporter gene driven by the Atmyb2 promoter was induced by dehydration and salt stress in transgenic Arabidopsis plants. These observations indicate that Atmyb2 is responsive to dehydration at the transcriptional level. The putative protein (ATMYB2) encoded by Atmyb2 has 274 amino acids, a molecular mass of 32 kD, and a putative DNA binding domain that shows considerable homology to plant MYB-related proteins, such as maize C1. A fusion protein that included ATMYB2 was expressed in Escherichia coli, and it bound specifically to oligonucleotides that contained a consensus MYB recognition sequence (TAACTG), such as is found in the simian virus 40 enhancer and the maize bronze-1 promoter. Binding was sequence specific, as indicated by a gel mobility shift experiment. These results suggest that a MYB-related transcription factor is involved in the regulation of genes that are responsive to water stress in Arabidopsis.

Nucleotide sequence of a gene from Arabidopsis thaliana encoding a myb homologue.

A gene encoding a proto-oncogene, a myb-related gene named Atmyb1, was cloned from Arabidopsis thaliana, and its nucleotide sequence was determined. The Atmyb1 gene contains an intron of 494 bp, and there are no highly homologous sequences present in the A. thaliana genome, but evidence was found that other myb-related genes exist. In the 5' flanking region, we found several typical cis-acting elements found in plant promoters. Sequence comparisons revealed that the ATMYB1 protein has a putative DNA-binding domain with two repeats of tryptophan clusters, which is common in MYB-related proteins in plants, while animal MYB-related proteins contain DNA-binding domains with three repeats of tryptophan clusters. The putative DNA-binding domain of the ATMYB1 protein has higher homology with that of the human c-MYB protein than with those of other plant MYB proteins.