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P J Gulick

Publications and source records attributed to P J Gulick.

11 recordsLinked to original sources
PubMed2001

The salt stress-inducible protein kinase gene, Esi47, from the salt-tolerant wheatgrass Lophopyrum elongatum is involved in plant hormone signaling.

Protein kinases play a central role in signal transduction in all organisms and to study signal transduction in response to salt stress we have identified and characterized a gene encoding a protein kinase that is induced by salt stress and abscisic acid (ABA) in the salt-tolerant wild wheatgrass Lophopyrum elongatum (Host) A. Love. The product of the early salt stress-induced gene, Esi47, was found to belong to the "novel Arabidopsis protein kinase" group of plant serine/threonine protein kinases. Transient gene expression assays in barley aleurone tissue showed Esi47 to suppress the gibberellin induction of the barley low-pI alpha-amylase gene promoter, thus providing evidence for the role of this protein kinase gene in plant hormone signaling. Esi47 contains a small upstream open reading frame in the 5'-untranslated region of its transcript that is implicated in mediating the repression of the basal level of the gene expression and in regulating the ABA inducibility of the gene, as shown in the transient gene expression assay in maize callus. Three Arabidopsis homologs of Esi47 were identified, and different members of this clade of genes showed differential patterns of regulation by salt stress and ABA in Arabidopsis roots and leaves. At least one of the Arabidopsis homologs contains a small open reading frame in its 5'-untranslated region, indicating that the unusual regulatory mechanism identified in Esi47 may be widely conserved.

5' Untranslated Regions↗
PubMed1998

Characterization of two cDNA clones which encode O-methyltransferases for the methylation of both flavonoid and phenylpropanoid compounds.

Enzymatic O-methylation of phenylpropanoid and flavonoid compounds is believed to be catalyzed by distinct classes of O-methyltransferases [EC 2.1.1.6x]. The O-methylated derivatives of phenylpropanoids and flavonoids play an important role in lignification and as antimicrobial compounds, respectively. Two cDNA clones, OMT1 and OMT2, which differ in three amino acid residues were isolated and characterized from the semiaquatic freshwater weed Chrysosplenium americanum (Saxifragaceae). These two novel cDNA clones encode enzymes which catalyze the 3'-O-methylation of the flavonoid aglycones luteolin and quercetin, although they also catalyze the efficient 3/5-O-methylation of the phenylpropanoids caffeic and 5-hydroxyferulic acids, respectively. Both recombinant proteins were partially purified from an Escherichia coli expression system and their kinetic parameters were compared using two flavonoids and two phenylpropanoids as substrates. Although both gene products methylate caffeic acid and 5-hydroxyferulic acid to a similar extent, they exhibit a threefold higher affinity for and a four- to sixfold increase in turnover of flavonoid compounds. The gene product of OMT1 accepts the flavonoid substrates luteolin and quercetin for methylation at a higher rate than that of OMT2, as indicated by a two- to threefold increase in its Vmax values and turnover ratios. The fact that C. americanum accumulates a variety of highly methylated flavonols and exhibits little lignification suggests that these two flavonoid OMT clones have retained their ability to O-methylate phenylpropanoids as well. These results are discussed in relation to differences in the amino acid sequences of these two clones, as well as with other O-methyltransferases, and the evolutionary divergence of these genes in plants.

Amino Acid Sequence↗
PubMed1996

cDNA cloning and characterization of a 3'/5'-O-methyltransferase for partially methylated flavonols from Chrysosplenium americanum.

Enzymatic O-methylation of plant secondary metabolites is an important mechanism for the inactivation of reactive hydroxyl groups and for the modification of their solubility. A cDNA clone (pFOMT3') encoding the gene for the 3'/5'-O-methylation of partially methylated flavonols was isolated from Chrysosplenium americanum (Saxifragaceae). We used a PCR fragment obtained with degenerate oligonucleotides designed from conserved regions of various O-methyltransferases (OMTs). The pFOMT3' cDNA sequence shows about 67-85% similarity to other plant OMT sequences. The recombinant protein expresses strict specificity for positions 3'/5' (meta) of partially methylated flavonols, but does not accept quercetin or caffeic acid for further methylation. Southern blot analysis of the genomic DNA probed with an OMT sequence suggests the presence of a number of related genes in this species, consistent with the multiple enzymatic methylations involved in the biosynthesis of polymethylated flavonols in this plant.

Amino Acid Sequence↗
PubMed1996

Molecular and biochemical characterization of two nucleoside diphosphate kinase cDNA clones from Flaveria bidentis.

Two nucleoside diphosphate kinase cDNA clones have been isolated from Flaveria bidentis by immunoscreening of an expression library with a polyclonal antibody raised against Flaveria chloraefolia flavonol 3-sulfotransferase (F3-ST). The clones represent members of a small multigene family in this species. The nucleotide sequences of the two cDNA clones show a high degree of sequence similarity to other reported nucleoside diphosphate kinases (NDPKs), including the putative human tumor suppressor gene NM23 and the Drosophila regulatory gene. When these cDNA clones were expressed in Escherichia coli, their gene products exhibited NDPK enzymatic activity. The immunocross reaction of the clones with the antibody raised against the F3-ST suggests a common immuno-epitope and a similarity of a nucleotide binding site for the two proteins.

Amino Acid Sequence↗
PubMed1995

Farnesyl pyrophosphate synthase from white lupin: molecular cloning, expression, and purification of the expressed protein.

Plants produce a variety of sesquiterpenoid compounds with diverse biological functions, whose synthesis is initiated by farnesyl pyrophosphate synthase [EC 2.5.1.1, EC 2.5.1.10]. The lack of availability of molecular tools to analyze this enzyme has, thus far, prevented the study of its expression and regulation in plants. A DNA fragment corresponding to a portion of the farnesyl pyrophosphate synthase gene was amplified by the polymerase chain reaction using was amplified by the polymerase chain reaction using degenerate primers designed from two highly conserved domains (FLV(A/L)DD(I/M)MD and FQIQDDYLD) found in eukaryotic farnesyl pyrophosphate synthase sequences. A clone, pS19, of a 438-bp PCR fragment was used to screen a white lupin root cDNA library. Two full-length cDNA clones (pFPS1 and pFPS2) were isolated and sequenced, and one of them (pFPS2) was expressed in a bacterial system and the enzyme protein encoded by the clone was purified. The 1345-bp insert of pFPS2 contains a 1026-bp open reading frame, encoding a 342-amino-acid peptide with a calculated molecular mass of 39,310 Da. The deduced amino acid sequence of lupin farnesyl pyrophosphate synthase pFPS2 shares 90 and 79% identity with those from Lupinus albus (pFPS1) and Arabidopsis thaliana, respectively, 51% with the yeast enzyme, and 44% identity with those from rat and human. The overexpressed protein, which was purified to near homogeneity, displayed both dimethylallyl transferase and geranyl transferase activities. Polyclonal antibodies raised against the purified protein immunorecognized a ca 39-kDa protein in lupin root extracts.

Alkyl and Aryl Transferases↗
PubMed1992

Coordinate Gene Response to Salt Stress in Lophopyrum elongatum.

Lophopyrum elongatum is a highly salt-tolerant relative of wheat. A previous study showed that the abundance of a number of mRNA species is enhanced or reduced in the roots of the L. elongatum x Triticum aestivum amphiploid by salt stress. Eleven genes with enhanced expression in the roots of salt-stressed L. elongatum plants have been cloned as cDNAs. The clones were used as probes to characterize temporal expression of these genes in roots after initiation of salt (250 mm NaCl) stress. All 11 genes are induced within 2 h after exposure to 250 mm NaCl and reached peak expression after 6 h. The decline of gene expression distinguished two groups, one in which mRNA concentrations returned to basal levels by 24 h and the other in which this occurred between 3 and 7 d. One of the 11 clones was found to be homologous to a multigene family of abscisic acid-induced genes, rab and dhn, identified in other species. We suggest that the coordinate expression of this large number of genes reflects the existence of a highly specific early response to salt stress. We refer to this response as the "early salt stress response."

Journal Article↗
PubMed1990

Selective enrichment of cDNAs from salt-stress-induced genes in the wheatgrass, Lophopyrum elongatum, by the formamide-phenol emulsion reassociation technique.

We present a novel technique for the enrichment of cDNA libraries to enhance the abundance of clones of differentially expressed genes. The technique is relatively simple, requires moderate quantities of poly(A) + RNA and results in preferential enrichment of clones derived from mRNAs that were of low abundance in their original population. This method was used to isolate cDNA clones of salt-stress-induced genes in the roots of Lophopyrum elongatum, a highly salt-tolerant wheatgrass. An excess of sonicated plasmid DNA from a cDNA library from nonstressed roots was hybridized in a formamide-phenol emulsion with inserts from a cDNA library of stressed roots. Clones that were more abundant in, or were unique to, the library of the stressed roots were recovered as double-stranded fragments by virtue of reconstituted restriction-enzyme-digested ends by ligating them to a plasmid vector. The resulting enriched library was screened by differential colony hybridization and clones of eleven different genes that were more strongly expressed in stressed roots than in controls were selected.

Blotting, Northern↗