Search PubMed⌕ Search

Biomedical subjects

H J Bohnert

Publications and source records attributed to H J Bohnert.

At least 55 records · Page 3Linked to original sources
PubMed1993

SecY, an integral subunit of the bacterial preprotein translocase, is encoded by a plastid genome.

Although the paradigm for the acquisition of photosynthetic organelles is the endocytosis of cyanobacteria-like progenitors by heterotrophic protists, details of this evolutionary process are unclear. The small organellar chromosomes are remnants of the larger bacterial genomes with most genes from the endosymbiont's DNA having been either relocated to the protist's nucleus or entirely lost. As a result of those gene transfers, differences exist between plastids from different algal phyla and higher plants. We report here on the retention of a secY gene in cyanelle (= plastid) DNA of the eukaryotic protist Cyanophora paradoxa. This cyanelle secY encodes a functional protein homologous to SecY of Escherichia coli, identified as a subunit of the preprotein translocase complex. Similarity of the cyanelle and E. coli SecY topology, predicted from sequence information, has been confirmed experimentally through SecY-PhoA fusion protein analysis in E. coli. Cyanelle SecY, expressed in an E. coli secY mutant, substituted for the defective prokaryotic SecY. A plastid-encoded gene for a membrane protein functioning in protein transport across plastid membranes is unprecedented in higher plants. From these results we infer that a functional homolog of the prokaryotic preprotein translocation machinery is retained in some plastids.

Adenosine Triphosphatases↗
PubMed1993

Anion exchange separation and pulsed amperometric detection of inositols from flower petals.

A rapid and specific method for the separation of cyclic sugar alcohols by anion exchange HPLC and subsequent detection by pulsed amperometry is presented. The cyclitols include the inositols and O-methyl inositols. Although examples use plant extracts the method has general application and is suitable for detecting as little as 10 pmol of substance. Carbohydrates are weak acids with pKa values typically above 12. While separation of most monosaccharides on high-performance anion exchange columns in aqueous NaOH is adequate, the ionization of inositols and O-methyl inositols is weak and their separation is poor. Use of a high-capacity anion exchange column separated the inositols well but the more ionized sugars were tightly retained. Automatic switching of the inositol fraction eluting from the lower-capacity column onto the high-capacity column with subsequent detection by pulsed amperometry gave specific and rapid separations of the inositols with a turnaround time of 16 min. As an example of application, we chose the separation and identification of cyclic and acyclic sugar alcohols in flower petals from a variety of plants. There was an apparent correlation between the postharvest longevity of flowers and the content of inositols, particularly D-pinitol.

Chromatography, High Pressure Liquid↗
PubMed1993

Sequence analysis of pre-ferredoxin-NADP(+)-reductase cDNA from Cyanophora paradoxa specifying a precursor for a nucleus-encoded cyanelle polypeptide.

A cDNA clone for pre-ferredoxin-NADP+ reductase (FNR) was obtained by screening a Cyanophora paradoxa expression library with antibodies specific for cyanelle FNR. The 1.4 kb transcript was derived from a single-copy gene. The precursor (41 kDa) and mature forms (34 kDa) of FNR were identified by western blotting of in vitro translation products and cyanelle extracts, respectively. The derived amino acid sequence of the mature form was corroborated by data from N-terminal protein sequencing and yielded identity scores from 58% to 62% upon comparison with cyanobacterial FNRs. Sequence conservation seemed to be even more pronounced in comparison with enzymes from higher plants, but using the neighbor joining method the C. paradoxa sequence was clearly positioned between the prokaryotic and eukaryotic sequences. The transit peptide of 65 or 66 amino acids appeared to be totally unrelated to those from spinach, pea and ice plant but showed overall characteristics of stroma-targeting peptides.

Amino Acid Sequence↗
PubMed1993

Expression of a phosphoenolpyruvate carboxylase promoter from Mesembryanthemum crystallinum is not salt-inducible in mature transgenic tobacco.

The 5' flanking region of a salt-stress-inducible, CAM-specific phosphoenolpyruvate carboxylase (PEPC) gene from the facultative halophyte Mesembryanthemum crystallinum, was fused to the beta-glucuronidase (GUS) reporter gene and introduced into Nicotiana tabacum SR1. The Ppc1 promoter displayed high levels of expression in transgenic tobacco quantitatively and qualitatively similar to a full-length 35S CaMV-GUS construct. Histochemical assays revealed that the full-length Ppc1-GUS fusions expressed GUS activity in all tissues except in root tips. While tobacco is capable of utilizing the Ppc1 cis-acting regulatory regions from M. crystallinum to yield high levels of constitutive expression, this glycophyte fails to direct a stress-inducible pattern of gene expression typical of this promoter in its native, facultative halophytic host.

Base Sequence↗
PubMed1993

The six genes of the Rubisco small subunit multigene family from Mesembryanthemum crystallinum, a facultative CAM plant.

The nucleotide sequences of the entire gene family, comprising six genes, that encodes the Rubisco small subunit (rbcS) multigene family in Mesembryanthemum crystallinum (common ice plant), were determined. Five of the genes are arranged in a tandem array spanning 20 kb, while the sixth gene is not closely linked to this array. The mature small subunit coding regions are highly conserved and encode four distinct polypeptides of equal lengths with up to five amino acid differences distinguishing individual genes. The transit peptide coding regions are more divergent in both amino acid sequence and length, encoding five distinct peptide sequences that range from 55 to 61 amino acids in length. Each of the genes has two introns located at conserved sites within the mature peptide-coding regions. The first introns are diverse in sequence and length ranging from 122 bp to 1092 bp. Five of the six second introns are highly conserved in sequence and length. Two genes, rbcS-4 and rbcS-5, are identical at the nucleotide level starting from 121 bp upstream of the ATG initiation codon to 9 bp downstream of the stop codon including the sequences of both introns, indicating recent gene duplication and/or gene conversion. Functionally important regulatory elements identified in rbcS promoters of other species are absent from the upstream regions of all but one of the ice plant rbcS genes. Relative expression levels were determined for the rbcS genes and indicate that they are differentially expressed in leaves.

Adaptation, Biological↗
PubMed1992

Replacement of a conserved arginine in the assembly domain of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit interferes with holoenzyme formation.

In higher plants the small subunit (S) of ribulose-1,5-bisphosphate carboxylase/oxygenase (ribulose-P2 carboxylase, EC 4.1.1.39) contains a segment of 16 amino acids which is absent from cyanobacterial S. This segment connecting two beta sheets has been shown, by crystallographic analysis, to form a hairpin loop. The quaternary structure of ribulose-P2 carboxylase indicates several S to large subunit (L) interactions. Eleven of 22 residues within the loop form the interface with 20 residues from two different L dimers. Eight of the loop residues are involved in hydrogen bonds, salt links, and hydrophobic interactions. To test the hypothesis, whether this loop had a function in the assembly of L and S into the hexadecameric enzyme, 6 amino acids within the loop were modified by site-directed mutagenesis of the pea rbcS-3A gene. All substituted S were imported by isolated chloroplasts from pea with wild type efficiency. Mutants E54-R, H55-A, P59-A, D63-G, D63-L, and Y66-A were assembly-competent, indicating that changes of side chains at these positions are tolerated. Replacement of arginine 53, whose side chain forms H-bonds with L residues Y226 and G261, with glutamate completely abolished assembly into holoenzyme. We suggest that arginine 53 in S is essential for ribulose-P2 carboxylase quaternary structure in higher plants.

Amino Acid Sequence↗
PubMed1992

Expression of a bacterial mtlD gene in transgenic tobacco leads to production and accumulation of mannitol.

A bacterial gene encoding mannitol-1-phosphate dehydrogenase, mtlD, was engineered for expression in higher plants. Gene constructions were stably incorporated into tobacco plants. The mtlD gene was expressed and translated into a functional enzyme in tobacco, resulting in the synthesis and accumulation of mannitol, which was identified by NMR and mass spectroscopy. Mannitol concentrations exceeded 6 mumol/g (fresh weight) in the leaves and in the roots of some transformants, whereas this sugar alcohol was not detected in these organs of wild-type tobacco plants or of untransformed tobacco plants that underwent the same regeneration scheme. These experiments demonstrate that branch-points in plant carbohydrate metabolism can be generated by which novel gene products can utilize endogenous substrates to divert metabolic energy into novel compounds. Additionally, the system described here allows for physiological studies in which the responses of wild-type and transgenic tobacco to various environmental stimuli can be compared directly. Such studies will facilitate our understanding of the roles of sugar alcohols (e.g., in stress tolerance) in higher plants.

Escherichia coli↗
PubMed1992

A novel methyl transferase induced by osmotic stress in the facultative halophyte Mesembryanthemum crystallinum.

Molecular mechanisms of osmotic stress tolerance were studied in Mesembryanthemum crystallinum (ice plant), a facultative halophyte capable of adjusting to and surviving in highly saline conditions. We screened a subtracted cDNA library enriched for salt stress-induced mRNAs to identify transcripts involved in this plant's adaptation to salinity. One mRNA, Imt1, was found to be up-regulated in leaves and, transiently, in roots. Nuclear run-on assays indicated that this mRNA is transcriptionally regulated. Imt1 encoded a predicted polypeptide of M(r) 40,250 which exhibited sequence similarity to several hydroxymethyl transferases. Expression of the protein in Escherichia coli and subsequent activity assays identified the protein as a novel myoinositol O-methyl transferase which catalyzes the first step in the biosynthesis of the cyclic sugar alcohol pinitol. Pinitol accumulates in salt-stressed M.crystallinum and is abundant in a number of salt- and drought-tolerant plants. The presence of high levels of sugar alcohols correlates with osmotolerance in a diverse range of organisms, including bacteria, fungi and algae, as well as higher plants. The stress-initiated transcriptional induction of IMT1 expression in a facultative halophyte provides strong support for the importance of sugar alcohols in establishing tolerance to osmotic stress in higher plants.

Amino Acid Sequence↗
PubMed1992

Salt stress alters A/T-rich DNA-binding factor interactions within the phosphoenolpyruvate carboxylase promoter from Mesembryanthemum crystallinum.

The common ice plant, Mesembryanthemum crystallinum, shifts from C3 to crassulacean acid metabolism (CAM) photosynthesis in response to osmotic stress. The expression of a number of genes encoding enzymes involved in the CAM pathway increases as a result of increased transcription rates. To begin to investigate the mechanisms responsible for the transcriptional activation, we have characterized the 5' control region of a specific isoform of phosphoenolpyruvate carboxylase gene (Ppc1) that plays a key role in CAM. We have determined the nucleotide sequence of the 5' flanking region of this gene. Ppc1 contains a long 5'-leader sequence with the transcriptional start site located 332/333 nucleotides 5' of the translational initiation codon. Multiple DNA interactions with nuclear factors are detectable within the 5'-flanking region of Ppc1. We have used copper orthophenanthroline footprinting to demonstrate that one particularly abundant factor (designated PCAT-1) binds the Ppc1 promoter at two distinct A/T-rich sites located -128 to -158 and -187 to -205 bp upstream of the transcriptional start site. These binding sites share a loose consensus motif having the sequence AARTAAC(T/A)A(G/T)TTTY. Gel retardation competition experiments with oligonucleotides containing these A/T-rich binding sites suggest that both sites bind the same factor, but with different affinities. Fractionation of crude nuclear extracts by heparin-agarose chromatography indicates that PCAT-1 is more prevalent in extracts prepared from salt-stressed leaf tissue. Additional binding activities that interact with the PCAT-1 binding sites have been detected that either increase or decrease in abundance or binding affinity in response to salt stress.

Base Sequence↗
PubMed1992

Convergent Induction of Osmotic Stress-Responses : Abscisic Acid, Cytokinin, and the Effects of NaCl.

In Mesembryanthemum crystallinum, salt stress induces the accumulation of proline and a specific isoform of the enzyme phosphoenolpyruvate carboxylase (PEPCase) prior to the switch from C(3) to Crassulacean acid metabolism (CAM). To determine whether plant growth regulators initiate or imitate these responses, we have compared the effects elicited by NaCl, abscisic acid (ABA), and cytokinins using PEPCase and proline levels as diagnostic tools. Exogenously applied ABA is a poor substitute for NaCl in inducing proline and CAM-specific PEPCase accumulation. Even though ABA levels increase 8- to 10-fold in leaves during salt stress, inhibition of ABA accumulation does not affect these salt-induced responses. In contrast, the addition of cytokinins (6-benzylaminopurine, zeatin, 2-isopentyladenine) mimic salt by greatly increasing proline and PEPCase amounts. Endogenous zeatin levels remain unchanged during salt stress. We conclude: (a) The salt-induced accumulation of proline and PEPCase is coincident with, but is not attributable to, the rise in ABA or zeatin concentration. (b) For the first time, cytokinins and NaCl are implicated as independent initiators of a sensing pathway that signals leaves to alter PEPCase gene expression. (c) During stress, the sensing of osmotic imbalances leading to ABA, proline, and CAM-specific PEPCase accumulation may be mediated directly by NaCl.

Journal Article↗
PubMed1992

Influence of NaCl on Growth, Proline, and Phosphoenolpyruvate Carboxylase Levels in Mesembryanthemum crystallinum Suspension Cultures.

The facultative halophyte Mesembryanthemum crystallinum responds to salt stress by increasing the levels of phosphoenolpyruvate carboxylase (PEPCase) and other enzymes associated with Crassulacean acid metabolism. A more common response to salt stress in sensitive and tolerant species, including M. crystallinum, is the accumulation of proline. We have established M. crystallinum suspension cultures to investigate whether both these salt-induced responses occur at the cellular level. Leaf-and root-derived cultures maintain 5% of the total soluble amino acids as proline. Cell culture growth slows upon addition of 400 millimolar NaCl, and proline levels increase to 40% of the total soluble amino acids. These results suggest a functional salt-stress and response program in Mesembryanthemum cells. Suspension cultures grown with or without 400 millimolar NaCl have PEPCase levels that compare with those from roots and unstressed leaves. The predominant protein cross-reacting with an anti-PEPCase antibody corresponds to 105 kilodaltons (apparent molecular mass), whereas a second species of approximately 110 kilodaltons is present at low levels. In salt-stressed leaves, the 110 kilodalton protein is more prevalent. Levels of mRNA for both ppc1 (salt stress induced in leaves) and ppc2 (constitutive) genes in salt-treated suspensions cultures are equal to unstressed leaves, and only twice the levels found in untreated suspension cultures. Whereas cells accumulate proline in response to NaCl, PEPCase protein amounts remain similar in salt-treated and untreated cultures. The induction upon salt stress of the 110 kilodalton PEPCase protein and other Crassulacean acid metabolism enzymes in organized tissues is not observed in cell culture and may depend on tissue-dependent or photoautotrophy-dependent programs.

Journal Article↗
PubMed1992

Light Moderates the Induction of Phosphoenolpyruvate Carboxylase by NaCl and Abscisic Acid in Mesembryanthemum crystallinum.

In Mesembryanthemum crystallinum, phosphoenolpyruvate carboxylase is synthesized de novo in response to osmotic stress, as part of the switch from C(3)-photosynthesis to Crassulacean acid metabolism. To better understand the environmental signals involved in this pathway, we have investigated the effects of light on the induced expression of phosphoenolpyruvate carboxylase mRNA and protein in response to stress by 400 millimolar NaCl or 10 micromolar abscisic acid in hydroponically grown plants. When plants were grown in high-intensity fluorescent or incandescent light (850 microeinsteins per square meter per second), NaCl and abscisic acid induced approximately an eightfold accumulation of phosphoenolpyruvate carboxylase mRNA when compared to untreated controls. Levels of phosphoenolpyruvate carboxylase protein were high in these abscisic acid- and NaCl-treated plants, and detectable in the unstressed control. Growth in high-intensity incandescent (red) light resulted in approximately twofold higher levels of phosphoenolpyruvate carboxylase mRNA in the untreated plants when compared to control plants grown in high-intensity fluorescent light. In low light (300 microeinsteins per square meter per second fluorescent), only NaCl induced mRNA levels significantly above the untreated controls. Low light grown abscisic acid- and NaCl-treated plants contained a small amount of phosphoenolpyruvate carboxylase protein, whereas the (untreated) control plants did not contain detectable amounts of phosphoenolpyruvate carboxylase. Environmental stimuli, such as light and osmotic stress, exert a combined effect on gene expression in this facultative halophyte.

Journal Article↗
PubMed1992

Increased Expression of a myo-Inositol Methyl Transferase in Mesembryanthemum crystallinum Is Part of a Stress Response Distinct from Crassulacean Acid Metabolism Induction.

The facultative halophyte Mesembryanthemum crystallinum responds to osmotic stress by switching from C(3) photosynthesis to Crassulacean acid metabolism (CAM). This shift to CAM involves the stress-initiated up-regulation of mRNAs encoding CAM enzymes. The capability of the plants to induce a key CAM enzyme, phosphoenolpyruvate carboxylase, is influenced by plant age, and it has been suggested that adaptation to salinity in M. crystallinum may be modulated by a developmental program that controls molecular responses to stress. We have compared the effects of plant age on the expression of two salinity-induced genes: Gpdl, which encodes the photosynthesis-related enzyme glyceraldehyde 3-phosphate dehydrogenase, and Imtl, which encodes a methyl transferase involved in the biosynthesis of a putative osmoprotectant, pinitol. Imtl mRNA accumulation and the accompanying increase in pinitol in stressed Mesembryanthemum exhibit a pattern of induction distinct from that observed for CAM-related genes. We conclude that the molecular mechanisms that trigger Imtl and pinitol accumulation in response to salt stress in M. crystallinum differ in some respects from those that lead to CAM induction. There may be multiple signals or pathways that regulate inducible components of salinity tolerance in this facultative halophyte.

Journal Article↗
PubMed1992

Epidermis-specific gene expression in Pachyphytum.

Transcripts of exclusively epidermis-specific expression were obtained by differential screening of a cDNA library prepared from isolated epidermis tissue of a succulent plant, Pachyphytum. Six transcripts were selected and characterized by RNA gel blot hybridization. Five cDNAs represented abundant mRNAs found exclusively in the epidermis. In situ hybridizations with three of these transcripts further substantiated their epidermal location. While two transcripts were present in all cells of the epidermis, one transcript was enriched in subsidiary cells of the stomatal complexes. All of the transcripts were highly expressed in the protoderm of the shoot apical meristem. DNA sequence determination indicated that the mRNAs identified represent novel sequences, encoding yet unknown plant functions. These transcripts, their control elements, and their encoded functions should help to advance our understanding of epidermal cell determination and differentiation.

Amino Acid Sequence↗
PubMed1991

An ORF323 with homology to crtE, specifying prephytoene pyrophosphate dehydrogenase, is encoded by cyanelle DNA in the eukaryotic alga Cyanophora paradoxa.

Carotenoids are essential constituents of the light-harvesting and light-protective systems of photosynthetic organisms. The biochemistry of carotenoid biosynthesis in eukaryotes is known, whereas evidence for the genes specifying this biosynthetic pathway is scant. We report here the nucleotide sequence and expression of a gene likely encoding crtE (prephytoene pyrophosphate dehydrogenase). The reaction product of this enzyme is phytoene, a C40 carotenoid precursor common to all organisms. The gene is found in the cyanelle (plastid) DNA of an eukaryotic alga, Cyanophora paradoxa. The expression into protein of cyanelle crtE has been demonstrated in vitro. The identity and similarity scores of CrtE from cyanelles with the corresponding protein from the photosynthetic bacterium Rhodobacter capsulatus are 28.6 and 68.5%, respectively.

Alkyl and Aryl Transferases↗
PubMed1991

rps10, unreported for plastid DNAs, is located on the cyanelle genome of Cyanophora paradoxa and is cotranscribed with the str operon genes.

rps10, encoding the plastid ribosomal protein S10, is a nuclear gene in higher plants and green algae, and is missing from the large ribosomal protein gene cluster of chlorophyll b-type plastids that contains components of the prokaryotic S10, spc and alpha operons. The cyanelle genome of Cyanophora paradoxa is shown to harbor rps10 as another specific feature of its organization. However, this novel plastid gene is not contiguous with the genes of the "S10" operon, but is adjacent to, and cotranscribed with, the str operon, a trait also found in archaebacteria.

Amino Acid Sequence↗