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C J Lamb

Publications and source records attributed to C J Lamb.

At least 55 records · Page 3Linked to original sources
PubMed1990

Hydroxyproline-Rich Glycoprotein Transcripts Exhibit Different Spatial Patterns of Accumulation in Compatible and Incompatible Interactions between Phaseolus vulgaris and Colletotrichum lindemuthianum.

The distribution of transcripts encoding hydroxyproline-rich glycoproteins in hypocotyls of Phaseolus vulgaris L. infected with Colletotrichum lindemuthianum was examined by in situ hybridization to tissue sections. The expression of hypersensitive resistance in an incompatible interaction was accompanied by a massive early accumulation of transcripts in the epidermal, cortical, and perivascular parenchymal tissues immediately adjacent to the inoculation site. In a compatible interaction, there was no accumulation of transcripts in the epidermal and cortical tissues even though fungal hyphae ramified throughout these tissues. However, transcripts accumulated at a later stage in the perivascular tissue directly below the site of infection and in tissue several millimeters from the inoculation site. Thus, there is a spatial and tissue-specific counterpart to the differential timing of transcript accumulation in incompatible versus compatible interactions (AM Showalter, JN Bell, CL Cramer, JA Bailey, CJ Lamb [1985] Proc Natl Acad Sci USA 82: 6551-6555). These differences in the spatial distribution and tissue specificity of transcript accumulation imply the differential induction of signaling systems involved in race:cultivar-specific interactions.

Journal Article↗
PubMed1990

Stress Responses in Alfalfa (Medicago sativa L.): VI. Differential Responsiveness of Chalcone Synthase Induction to Fungal Elicitor or Glutathione in Electroporated Protoplasts.

Protoplasts derived from cell suspensions of alfalfa (Medicago sativa L.) responded to treatment with fungal elicitor (FE) by an increase in endogenous chalcone synthase (CHS) activity but were unresponsive to reduced glutathione (GSH). Preexposure of protoplasts to polyethylene glycol and electroporation resulted in strong responsiveness to GSH but little change in responsiveness to FE. Protoplasts from suspension cultures which had been subcultured more than 12 times lost responsiveness to GSH, but not FE, as assessed by measuring expression of a chimeric gene containing a bean CHS promoter linked to a bacterial chloramphenicol acetyltransferase (CAT) reporter gene. In protoplasts in which putative cis-acting CHS promoter sequences had been coelectroporated in trans with the intact CHS promoter-CAT construct, the extent of CAT expression depended upon the elicitor used (FE or GSH), the age (number of times subcultured) of the cells from which the protoplasts were isolated, and the nature of the coelectroporated CHS promoter sequence. For example, a region of the CHS promoter from -326 to -141 behaved as a trans-activator when coelectroporated with the CAT construct into unelicited protoplasts isolated from newly initiated cell suspensions, but the same region acted as a trans-silencer in the same protoplasts in the presence of FE. This silencer activity was much reduced in GSH-treated protoplasts. The results suggest that there are differences in the signal transduction pathways for elicitation of CHS transcription by FE and GSH, which involve previously indentified cis-elements in the CHS promoter.

Journal Article↗
PubMed1990

Developmental and environmental regulation of a bean chalcone synthase promoter in transgenic tobacco.

Regulatory properties of a 1.4-kilobase promoter fragment of the bean chalcone synthase CHS8 gene were examined by analysis of glucuronidase (GUS) activity in transgenic tobacco containing a CHS8-GUS gene fusion. The promoter was highly active in the root apical meristem and in petals, exclusively in those cells of the inner epidermis that accumulate anthocyanins. The gene fusion was only weakly expressed in other floral organs, mature leaves, and stems. The early stages of seedling development were characterized by an apparent wound induction of the promoter in the endosperm and strong expression in the immature root, which became localized to the apical meristem and perivascular tissue at the root-hypocotyl junction. The promoter became active during lateral root formation in both the new root and damaged tissue of the main root. The gene fusion was also expressed in greening cotyledons and primary leaves but not in the shoot apical meristem. Light modulated expression in the cotyledons and root-shoot junction but had no effect on other aspects of the developmental program. Wounding or fungal elicitor treatment of mature leaves activated the promoter in a well-defined zone adjacent to the stress site. Stress induction occurred in mesophyll and vascular tissues as well as in the epidermis. We conclude that the CHS8 promoter contains cis-elements required to establish temporal and spatial control of flavonoid biosynthesis during development and in response to diverse environmental stimuli.

Acyltransferases↗
PubMed1990

Functional properties of a phenylalanine ammonia-lyase promoter from Arabidopsis.

Phenylalanine ammonia-lyase (PAL) is encoded by a small family of genes in Arabidopsis. We cloned and partially characterized one of these genes, PAL1. The deduced amino acid sequence is highly similar to PAL from bean, parsley, and rice. The promoter contains sequence elements homologous to two putative regulatory elements conserved among several phenylpropanoid genes. The regulation of the PAL1 gene was examined by analysis of beta-glucuronidase (GUS) activity in transgenic Arabidopsis containing PAL1-GUS gene fusions. The PAL1 promoter was activated early in seedling development and in adult plants was strongly expressed in the vascular tissues of roots and leaves, but was not active in the root tip or the shoot apical meristem. In flowers, expression was observed in sepals, anthers, and carpels, but not in petals. Transcripts encoded by the endogenous PAL genes and GUS transcripts from the PAL1-GUS gene fusion were induced by wounding, HgCl2-stress, and light. Analysis of the regulatory properties of 5' deleted promoters showed that the proximal region of the promoter to -290 was sufficient to establish the full tissue-specific pattern of expression and that the proximal region to -540 was responsive to environmental stimuli. Negative and positive elements were located between -1816 and -823 and between -823 and -290, respectively.

Amino Acid Sequence↗
PubMed1989

Differential regulation of phenylalanine ammonia-lyase genes during plant development and by environmental cues.

Phenylalanine ammonia-lyase (PAL) catalyzes the first reaction in the biosynthesis from phenylalanine of a wide variety of phenylpropanoid natural products including lignin, flavonoid pigments, and phytoalexins. In bean (Phaseolus vulgaris L.), PAL is encoded by a family of three genes. We show here by RNase protection with gene-specific probes that these genes are expressed differentially during development and in response to different environmental cues. While all three genes are expressed at high levels in roots, only PAL1 and PAL2 are expressed in shoots and only PAL1 is expressed in leaves. Strikingly, PAL2 is expressed at very high levels in petals, where PAL1 is only very weakly expressed and PAL3 is not expressed. All three genes are induced by mechanical wounding of hypocotyls, but fungal infection only activates PAL1 and PAL3. Illumination of etiolated hypocotyls activates PAL1 and PAL2 but not PAL3. Corresponding differential patterns of synthesis of specific PAL polypeptide isoforms were observed by two-dimensional gel electrophoretic analysis of in vitro translation products encoded by RNA isolated from hypocotyls stimulated by light, wounding, or infection. The specific isoforms encoded by transcripts of the three PAL genes were identified by inhibition of synthesis in vitro with gene-specific anti-sense transcripts followed by comparative two-dimensional gel electrophoretic analysis of the pattern of translation products. These data indicate that selective expression of PAL genes encoding functional variants is governed by a complex set of regulatory networks for developmental and environmental control of phenylpropanoid biosynthesis.

Adaptation, Physiological↗
PubMed1989

Vascular expression of a bean cell wall glycine-rich protein-beta-glucuronidase gene fusion in transgenic tobacco.

In French bean (Phaseolus vulgarisL.), the glycine-rich wall protein GRP 1.8 is specifically synthesized in protoxylem tracheary elements of the vascular system. A 494 bp upstream promoter fragment of the gene encoding GRP 1.8 was isolated and translationally fused to the beta-glucuronidase reporter gene. Transgenic tobacco plants containing this construct expressed the gene in vascular tissue of roots, stems, leaves and flowers. The gene was developmentally expressed during differentiation of both primary and secondary vascular tissue and was also rapidly induced (in < 30 min) after excision-wounding of young stems. This wound response is more rapid than in bean hypocotyls, indicating possible differences between the activation mechanism for glycine-rich protein gene expression in wounded bean and tobacco. Only a subset of cells were found to participate in the wound response. In young stems, the GRP wound induction was localized in pith parenchyma cells adjacent to the wound surface, where vessel regeneration is known to occur. Thus, a promoter fragment of 494 bp, including 427 bp upstream from the transcription start site, contains information for tissue-specific and wound-induced gene regulation. The cell-type specificity of expression suggests that the GRP 1.8 promoter is regulated by very specific developmental and environmental signals.

Journal Article↗
PubMed1989

Developmental and environmental regulation of a phenylalanine ammonia-lyase-beta-glucuronidase gene fusion in transgenic tobacco plants.

A 1.1-kilobase promoter fragment of the bean (Phaseolus vulgaris L.) phenylalanine ammonia-lyase (EC 4.3.1.5) gene PAL2 was translationally fused to the beta-glucuronidase reporter gene and transferred to tobacco by Agrobacterium tumefaciens-mediated leaf disk transformation. The distribution of beta-glucuronidase activity in these transgenic plants is very similar to that of endogenous PAL2 transcripts in bean, with very high levels in petals; marked accumulation in anthers, stigmas, roots, and shoots; and low levels in sepals, ovaries, and leaves. Histochemical analysis of the spatial pattern of beta-glucuronidase activity showed that the PAL2 promoter is highly active in the shoot apical meristem, the zone of cell proliferation immediately adjacent to the root apical meristem, and in the early stages of vascular development at the inception of xylem differentiation. Wounding and light evoke specific changes in the spatial pattern of beta-glucuronidase activity in stems, including induction in the epidermis. These data indicate that the PAL2 promoter transduces a complex set of developmental and environmental cues into an integrated spatial and temporal program of gene expression to regulate the synthesis of a diverse array of phenylpropanoid natural products.

Ammonia-Lyases↗
PubMed1989

Specific localization of a plant cell wall glycine-rich protein in protoxylem cells of the vascular system.

An antibody against glycine-rich protein 1.8 of bean (Phaseolus vulgaris L.) was used for immunogold/silver localization of the protein in different organs of the plant. In hypocotyls, ovaries, and seed coats, the protein was found specifically in xylem cells of the vascular tissue. In hypocotyls, only protoxylem cells were labeled with the antibody, which indicates a remarkable cell-type specificity for accumulation of this cell wall protein. In mature hypocotyls, the protein was restricted to the same subset of xylem cells but was no longer detected on tissue prints, where a positive antibody reaction depends on the transfer of soluble material from plant tissue to the nitrocellulose filter. This indicates that the glycine-rich protein is insolubilized in the cell wall during development. In longitudinal sections of tracheary elements of young hypocotyls and seed coats, the antibody stained a pattern very similar to that of the lignified secondary thickenings of the cell wall, which suggests a close functional relationship between glycine-rich protein and lignin deposition during cell wall biogenesis in protoxylem cells.

Journal Article↗
PubMed1989

Molecular cloning of plant transcripts encoding protein kinase homologs.

Oligonucleotides, corresponding to conserved regions of animal protein-serine/threonine kinases, were used to isolate cDNAs encoding plant homologs in the dicot bean (Phaseolus vulgaris L.) and the monocot rice (Oryzae sativa L.). The C-terminal regions of the deduced polypeptides encoded by the bean (PVPK-1) and rice (G11A) cDNAs, prepared from mRNAs of suspension cultures and leaves, respectively, contain features characteristic of the catalytic domains of eukaryotic protein-serine/threonine kinases, indicating that these cDNAs encode plant protein kinases. The putative catalytic domains are most closely related to cyclic nucleotide-dependent protein kinases and the protein kinase C family, suggesting the plant homologs may likewise transduce extracellular signals. However, outside these domains, PVPK-1 and G11A exhibit no homology either to each other or to regulatory domains of other protein kinases, indicating the plant homologs are modulated by other signals. PVPK-1 corresponds to a 2.4-kb transcript in suspension cultured bean cells. Southern blots of genomic DNA indicate that PVPK-1 and G11A correspond to single copy genes that form part of a family of related plant sequences.

Amino Acid Sequence↗
PubMed1989

Specific expression of a novel cell wall hydroxyproline-rich glycoprotein gene in lateral root initiation.

A gene encoding a novel cell wall hydroxyproline-rich glycoprotein (HRGPnt3) was isolated from a genomic library of tobacco. The deduced protein (620 amino acids, Mr, 65,406) contains an amino-terminal hydrophobic signal peptide, is highly basic, and is rich in proline, although characteristic Ser-Pro4 repeats are found only beyond residue 204. At the carboxyl terminus, there is a 34-amino-acid unit repeated three times. Arginine is the predominant basic amino acid rather than lysine, as in other HRGPs. A second gene homologous to HRGPnt3 was revealed by Southern blot hybridization of tobacco genomic DNA. Northern blot hybridization identified a 1.9-kb transcript present at low levels in roots. To determine the underlying spatial pattern of expression, the HRGPnt3 promoter and the first 27 nucleotides of the open reading frame were fused to the beta-glucuronidase (GUS) reporter gene and transformed back into tobacco. Histochemical localization of GUS activity showed that the HRGPnt3 promoter was transiently induced in the pericycle and endodermis, specifically in the discrete, small subset of cells involved in the initiation of lateral roots. This pattern of expression, in cells destined to form the tip of the emerging lateral root, indicates that the encoded cell wall protein has a specialized structural function, possibly in the mechanical penetration of the cortex and epidermis of the main root, and that the HRGPnt3 promoter responds to an early morphogenetic signal for lateral root induction.

Amino Acid Sequence↗
PubMed1988

Glycine-rich cell wall proteins in bean: gene structure and association of the protein with the vascular system.

A single genomic clone (14 kb) isolated from bean (Phaseolus vulgaris L.) contains two genes that encode glycine-rich proteins. These genes are present as single copies in the genome, are separated by 2.85 kb and encode transcripts of 1.8 kb and 1.0 kb respectively. The encoded proteins contain 60% glycine and have amino-terminal signal peptides. The 1.8 kb transcript is present in young hypocotyls and in ovary tissue. Excision-wounding transiently induced this transcript in old, but not in young hypocotyl tissue. Antibodies raised against regions of the glycine-rich protein 1.8, expressed as a lacZ fusion protein in bacteria, react with a protein of 53 kd in a protein fraction extracted from cell walls of bean ovaries. Tissue imprints of bean ovaries treated with anti-glycine-rich protein antibodies showed that the glycine-rich protein was distributed in a regular pattern of small, highly localized discrete sites. The immunoreactive regions correspond to the pattern of vascular tissue in the pod. In young hypocotyls, glycine-rich protein is present at four pairs of discrete sites symmetrically arranged on the inner side of the vascular ring. These results suggest a close relationship between glycine-rich proteins and development of the vascular system.

Amino Acid Sequence↗
PubMed1988

Cinnamyl-alcohol dehydrogenase, a molecular marker specific for lignin synthesis: cDNA cloning and mRNA induction by fungal elicitor.

Cinnamyl-alcohol dehydrogenase (CAD; EC 1.1.1.195) catalyzes the final step in a branch of phenylpropanoid synthesis specific for production of lignin monomers. We have isolated a full-length cDNA clone encoding CAD, as a molecular marker specific for lignification, by immunoscreening a lambda gt11 library containing cDNAs complementary to mRNA from elicitor-treated cell cultures of bean (Phaseolus vulgaris L.). The clone comprises a single long open reading frame of 1767 base pairs, 31 base pairs of 5' leader, and 152 base pairs of 3' untranslated sequence. The deduced 65-kDa CAD polypeptide has several features that are strongly conserved in alcohol dehydrogenases. Addition of fungal elicitor to cell cultures stimulates CAD transcription, which leads to a remarkably rapid, but transient, accumulation of CAD mRNA, with no detectable lag and maximal levels after 1.5 hr. Southern blot analysis of bean genomic DNA indicates that elicitor-induced CAD is encoded by a single gene. The regulatory significance of the rapid activation of this CAD gene and the possible existence of a second, divergent CAD gene involved in lignification during xylogenesis are discussed.

Alcohol Oxidoreductases↗
PubMed1988

Glutathione and fungal elicitor regulation of a plant defense gene promoter in electroporated protoplasts.

To investigate the mechanisms underlying activation of plant defenses against microbial attack we have studied elicitor regulation of a chimeric gene comprising the 5' flanking region of a defense gene encoding the phytoalexin biosynthetic enzyme chalcone synthase fused to a bacterial chloramphenicol acetyltransferase gene. Glutathione or fungal elicitor caused a rapid, marked but transient expression of the chimeric gene electroporated into soybean protoplasts. The response closely resembled that of endogenous chalcone synthase genes in suspension cultured cells. Functional analysis of 5' deletions suggests that promoter activity is determined by an elicitor-regulated activator located between the "TATA box" and nucleotide position -173 and an upstream silencer between -173 and -326. These cis-acting elements function in the transduction of the elicitation signal to initiate elaboration of an inducible defense response.

Journal Article↗
PubMed1988

Chitinase cDNA cloning and mRNA induction by fungal elicitor, wounding, and infection.

Chitinase, which catalyzes the hydrolysis of beta-1,4 N-acetylglucosamine linkages of the fungal cell wall polymer chitin, is a component of the inducible defenses of plants. We show that chitinase synthesis is stimulated in bean (Phaseolus vulgaris L.) cell suspension cultures treated with fungal cell wall elicitors and in hypocotyls in response to infection with the fungus Colletotrichum lindemuthianum. Chitinase cDNA clones were isolated by antibody screening of a lambdagt11 cDNA library containing sequences complementary to poly A(+) RNA from elicited cells. The identity of these clones was confirmed by nucleotide sequence analysis and comparison of the deduced amino acid sequence with that determined for the amino-terminal sequence of bean chitinase. Elicitor causes a very rapid activation of chitinase transcription with a 10-fold stimulation after 5 minutes and 30-fold increase within 20 minutes. This leads to a marked, transient accumulation of chitinase transcripts with maximum levels 2 hours after elicitor treatment, concomitant with the phase of rapid enzyme synthesis. Chitinase transcripts also markedly accumulate in wounded and infected hypocotyls. Chitinase cDNA sequences hybridize to several genomic fragments suggesting there are several chitinase genes in the bean genome.

Journal Article↗
PubMed1988

Glutathione causes a massive and selective induction of plant defense genes.

The reduced form of glutathione (GSH), when supplied to suspension cultured cells of bean (Phaseolus vulgaris L.) at concentrations in the range 0.01 to 1.0 millimolar, stimulates transcription of defense genes including those that encode cell wall hydroxyproline-rich glycoproteins and the phenylpropanoid biosynthetic enzymes phenylalanine ammonialyase (PAL) and chalcone synthase (CHS) involved in lignin (PAL) and phytoalexin (PAL, CHS) production. Transcriptional activation of these genes leads to marked accumulation of the corresponding transcripts, contributing to a massive change in the overall pattern of protein synthesis which closely resembles that previously observed in response to fungal elicitor. GSH causes a marked increase in extractable PAL activity, whereas the oxidized form of glutathione, constituent amino acids, or other reducing agents are inactive. Possible roles of GSH in signaling biological stress are discussed.

Journal Article↗
PubMed1987

Rapid induction by fungal elicitor of the synthesis of cinnamyl-alcohol dehydrogenase, a specific enzyme of lignin synthesis.

A fivefold increase in the extractable activity of cinnamyl-alcohol dehydrogenase, an enzyme of phenylpropanoid metabolism specific for lignin synthesis, was observed within 10 h of treatment of cell-suspension cultures of bean (Phaseolus vulgaris L.) with a high-molecular-mass elicitor preparation heat-released from mycelial cell walls of the bean pathogen Colletotrichum lindemuthianum. Elicitor caused a rapid, marked but transient increase in the synthesis of cinnamyl-alcohol dehydrogenase with maximum rates 2-3 h after elicitation, concomitant with the phase of rapid increase in enzyme activity. There is a close correspondence between increased polysomal mRNA activity encoding cinnamyl-alcohol dehydrogenase, as measured by incorporation of [35S]methionine into immunoprecipitable enzyme subunits in vitro, and the stimulation of enzyme synthesis in vivo in response to elicitor. This marked increase in polysomal mRNA activity represents an increase as a proportion of total cellular mRNA activity, indicating that elicitor does not stimulate synthesis of this enzyme by selective recruitment from the total pool of cellular mRNA. Elicitor stimulation of cinnamyl-alcohol dehydrogenase activity and enzyme synthesis is more rapid than previously observed for other proteins involved inducible defense mechanisms, such as enzymes of phytoalexin biosynthesis or the apoproteins of cell-wall hydroxyproline-rich glycoproteins.

Alcohol Oxidoreductases↗
PubMed1987

Chalcone isomerase cDNA cloning and mRNA induction by fungal elicitor, wounding and infection.

The environmentally regulated synthesis of phenylpropanoid natural products was studied by examining the expression of the gene encoding chalcone isomerase (CHI). This enzyme catalyzes a step common to the synthesis of flavonoid pigments and isoflavonoid phytoalexins. A lambdagt11 library was constructed using mRNA from cell cultures of bean (Phaseolus vulgaris L.) treated with fungal elicitor. Two positive clones were obtained by screening 10 recombinants with an antiserum to purified bean CHI. The identity of the cloned sequences was confirmed by hybrid-select translation and the production of antigenic polypeptides from transcripts synthesized in vitro. Addition of elicitor to cell cultures resulted in the rapid accumulation of CHI mRNA, with maximum levels achieved 3-4 h after elicitation. CHI mRNA also accumulated during the natural infection of hypocotyls with the fungal pathogen Colletotrichum lindemuthianum, and in mechanically wounded hypocotyls. The kinetics of accumulation of CHI mRNA in response to these environmental signals were strikingly similar to those of mRNAs encoding two other phenylpropanoid pathway enzymes, phenylalanine ammonialyase and chalcone synthase. In contrast to the multi-gene families encoding these two enzymes, chalcone isomerase is encoded by a single gene which is regulated by several environmental stimuli.

Journal Article↗