Chiharu Ueguchi

Histidine Kinase Homologs That Act as Cytokinin Receptors Possess Overlapping Functions in the Regulation of Shoot and Root Growth in Arabidopsis

Cytokinins are plant hormones that may play essential and crucial roles in various aspects of plant growth and development. Although the functional significance of exogenous cytokinins as to the proliferation and differentiation of cells has been well documented, the biological roles of endogenous cytokinins have remained largely unknown. The recent discovery of the Arabidopsis Histidine Kinase 4 (AHK4)/CRE1/WOL cytokinin receptor in Arabidopsis thaliana strongly suggested that the cellular response to cytokinins involves a two-component signal transduction system. However, the lack of an apparent phenotype in the mutant, presumably because of genetic redundancy, prevented us from determining the in planta roles of the cytokinin receptor. To gain insight into the molecular functions of the three AHK genes AHK2, AHK3, and AHK4 in this study, we identified mutational alleles of the AHK2 and AHK3 genes, both of which encode sensor histidine kinases closely related to AHK4, and constructed a set of multiple ahk mutants. Application of exogenous cytokinins to the resultant strains revealed that both AHK2 and AHK3 function as positive regulators for cytokinin signaling similar to AHK4. The ahk2 ahk4 and ahk3 ahk4 double mutants and the ahk single mutants grew normally, whereas the ahk2 ahk3 double mutants exhibited a semidwarf phenotype as to shoots, such as a reduced leaf size and a reduced influorescence stem length. The growth and development of the ahk2 ahk3 ahk4 triple mutant were markedly inhibited in various tissues and organs, including the roots and leaves in the vegetative growth phase and the influorescence meristem in the reproductive phase. We showed that the inhibition of growth is associated with reduced meristematic activity of cells. Expression analysis involving AHK:β-glucuronidase fusion genes suggested that the AHK genes are expressed ubiquitously in various tissues during postembryonic growth and development. Our results thus strongly suggest that the primary functions of AHK genes, and those of endogenous cytokinins, are triggering of the cell division and maintenance of the meristematic competence of cells to prevent subsequent differentiation until a sufficient number of cells has accumulated during organogenesis.

Expression of Arabidopsis response regulator homologs is induced by cytokinins and nitrate

We examined cytokinin and nitrate responsiveness in gene expression of five distinct response regulator homologs (ARR3–ARR7) in the leaves of nitrogen‐starved Arabidopsis plants. The transcripts accumulated after spraying the shoots with t‐zeatin. The induction of accumulation was highly specific for cytokinins. The transcripts also accumulated by supply of nitrate to the culture medium. These findings suggest that ARRs are involved in inorganic nitrogen signal transduction mediated by cytokinin as in the case of ZmCip1, a response regulator homolog recently identified in maize.

Effects of mutations in heat-shock genes groES and groEL on protein export in Escherichia coli.

Escherichia coli heat‐shock proteins GroES and GroEL are essential cytoplasmic proteins, which have been termed ‘chaperonins’ because of their ability to assist protein assembly of bacteriophage capsids and multimeric enzymes of foreign origin. In this report we show that temperature‐sensitive mutations in groES and groEL genes cause defective export of the plasmid‐encoded beta‐lactamase (Bla) in vivo. Since efficient translocation of proteins across biological membranes is thought to be supported by cytoplasmic factors that protect presecretory molecules from being misfolded, these results suggest that both GroES and GroEL proteins possess a chaperone function by which they facilitate export of Bla. The translocation of other secretory proteins, however, appears to depend minimally on GroE, suggesting that GroE interacts only with a specific class of secreted proteins.

The Escherichia coli nucleoid protein H-NS functions directly as a transcriptional repressor.

The H‐NS protein is a major constituent of the Escherichia coli nucleoid structure and is implicated in the compact organization of the chromosome. Based on recent genetic evidence, this protein appears to influence the transcription of a variety of apparently unlinked genes on the chromosome, although the underlying molecular mechanism is not fully understood. In this study, we carried out a series of in vitro transcription assays including purified H‐NS with special reference to the osmotically inducible proV promoter of the proVWX operon (or proU), whose expression is known to be derepressed by lesions of the hns (osmZ) gene. Here, H‐NS was revealed to selectively inhibit an early step(s) of proV transcription initiation through its direct binding to the promoter region. It was thus demonstrated that H‐NS functions directly as a transcriptional repressor. Under the in vitro conditions used, this in vitro inhibitory effect of H‐NS was affected by changes in the superhelical density of template DNAs and more significantly by the concentration of potassium (K+) ions. These results are also discussed with regard to the mechanism underlying regulation of the proV promoter in response to the medium osmolarity.

Quantitative control of the stationary phase-specific sigma factor, sigma S, in Escherichia coli: involvement of the nucleoid protein H-NS.

In Escherichia coli, recent intensive studies revealed that expression of a certain subset of genes is under the control of the stationary phase‐specific sigma factor, sigma S, which is encoded by the rpoS gene. Since sigma S functions predominantly under certain growth conditions, its activity and/or cellular content has accordingly to be tightly controlled, however, the underlying molecular mechanism is at present unclear. We previously demonstrated that expression of the cbpA gene, encoding an analogue of the DnaJ molecular chaperone, is largely dependent on sigma S function. Here we have found that a mutational lesion of the hns gene, which encodes one of the well‐characterized nucleoid proteins, H‐NS, affects the cellular content of sigma S remarkably and consequently affects the expression of cbpA. Enhanced accumulation of sigma S in hns deletion cells was particularly observed in the logarithmic growth phase and was demonstrated to result from an elevated translational efficiency of rpoS mRNA and also from an increased stability of newly synthesized sigma S. Although H‐NS is known to influence the transcription of a number of apparently unlinked genes on the chromosome, in this study we provide a novel instance in which H‐NS is deeply implicated in post‐transcriptional regulation(s) of the expression of rpoS. As to physiological relevance, it was also demonstrated that hns deletion cells exhibit an extreme thermotolerance even in the logarithmic growth phase, presumably because of the enhanced accumulation of sigma S.

Autoregulatory expression of the Escherichia coli hns gene encoding a nucleoid protein: H-NS functions as a repressor of its own transcription

SummaryThe Escherichia coli nucleoid protein, H-NS (or H1a), appears to influence the regulation of a variety of unrelated E. coli genes and operons. To gain an insight into the regulation of the has gene itself, we constructed in this study a has-lacZ transcriptional fusion gene and inserted a single copy at the attλ locus on the E. coli chromosome. Expression of hns transcription appeared to be moderately regulated in a growth phase-dependent manner. It also emerged that hns transcription is under negative autoregulation, at least in the logarithmic growth phase. The results of in vitro transcription experiments confirmed that H-NS functions as a repressor for its own transcription. Thus, H-NS was shown to exhibit relatively high affinity for the DNA sequence encompassing the hns promoter region, as compared with a non-specific sequence. These results support the view that the nucleoid protein, H-NS, can function as a transcriptional regulator.

Solution structure of the DNA binding domain of a nucleoid-associated protein, H-NS, from Escherichia coli.

The three‐dimensional structure of the C‐terminal domain (47 residues) obtained from the hydrolysis of H‐NS protein with bovine trypsin was determined by NMR measurements and distance geometry calculations. It is composed of an antiparallel β‐sheet, an α‐helix and a 310‐helix which form a hydrophobic core, stabilizing the whole structure. This domain has been found to bind to DNA. Possible DNA binding sites are discussed on the basis of the solution structure of the C‐terminal domain of H‐NS.

RNA interference of the Arabidopsis putative transcription factor TCP16 gene results in abortion of early pollen development

Pollen development is a fundamental and essential biological process in seed plants. Pollen mother cells generated in anthers undergo meiosis, which gives rise to haploid microspores. The haploid cells then develop into mature pollen grains through two mitotic cell divisions. Although several sporophytic and gametophytic mutations affecting male gametogenesis have been identified and analyzed, little is known about the underlying molecular mechanism. In this study, we investigated the function of the TCP16 gene, which encodes a putative transcription factor. Expression analysis of the promoter::GUS fusion gene revealed that TCP16 transcription occurred predominantly in developing microspores. GUS expression began at the tetrad stage and markedly increased in an early unicellular stage. Transgenic plants harboring a TCP16 RNA interference (RNAi) construct generated equal amounts of normal and abnormal pollen grains. The abnormal pollen grains exhibited morphological abnormality and degeneration of genomic DNA. The defective phenotype of the RNAi plants was first detectable at the middle of the unicellular stage. Our results therefore suggest that TCP16, a putative transcription factor, plays a crucial role in early processes in pollen development.

Function of the Escherichia coli nucleoid protein, H-NS molecular analysis of a subset of proteins whose expression is enhanced in a hns deletion mutant

SummaryThe expression of numerous Escherichia coli cellular proteins was previously demonstrated to be greatly enhanced in a hns deletion background, relative to the levels in wild-type cells. In this study, a subset of such proteins, expression of which is affected by HNS, was partially purified, and the genes coding for some of the proteins were identified and characterized. Two of the proteins thus characterized, 19K and 17K, were found to be encoded by previously predicted genes that are located adjacent to, and downstream of, the trpABCDE operon (27.6 min on the E. coli genetic map). The genes coding for the other two proteins, 10 K-L and 10K-S, are located at 77.5 min on the genetic map. Their nucleotide sequences were determined and revealed that they may constitute an operon. To characterize the putative promoters for these genes, a set of promoter-lacZ transcriptional fusion genes was constructed on the E. coli chromosome. The results of such promoter-probe analyses indicated that H-NS represses the expression of these genes at the transcriptional level. Furthermore, H-NS appeared to exhibit relatively strong affinity for the putative promoter sequences in vitro. These results are compatible with the hypothesis that H-NS functions as a transcriptional repressor.

Identification of the DNA binding surface of H-NS protein from Escherichia coli by heteronuclear NMR spectroscopy.

The DNA binding domain of H‐NS protein was studied with various N‐terminal deletion mutant proteins and identified by gel retardation assay and heteronuclear 2D‐ and 3D‐NMR spectroscopies. It was shown from gel retardation assay that DNA binding affinity of the mutant proteins relative to that of native H‐NS falls in the range from 1/6 to 1/25 for H‐NS60–137, H‐NS70–137 and H‐NS80–137, whereas it was much weaker for H‐NS91–137. Thus, the DNA binding domain was defined to be the region from residue A80 to the C‐terminus. Sequential nuclear Overhauser effect (NOE) connectivities and those of medium ranges revealed that the region of residues Q60–R93 in mutant protein H‐NS60–137 forms a long stretch of disordered, flexible chain, and also showed that the structure of the C‐terminal region (residues A95–Q137) in mutant H‐NS60–137 was nearly identical to that of H‐NS91–137. 1H and 15N chemical shift perturbations induced by complex formation of H‐NS60–137 with an oligonucleotide duplex 14‐mer demonstrated that two loop regions, i.e. residues A80–K96 and T110–A117, play an essential role in DNA binding.