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PubMed1993

Developmental and tissue-specific expression of a tomato anionic peroxidase (tap1) gene by a minimal promoter, with wound and pathogen induction by an additional 5'-flanking region.

The tomato anionic peroxidase genes (tap1 and tap2) are induced by wounding and pathogen attack. The 5'-flanking region of tap1 confers wound- and pathogen-inducible beta-glucuronidase (GUS) expression in tobacco plants transformed with a tap1/GUS chimeric fusion gene construct. A series of nested 5' promoter deletions in the tap1/GUS fusion gene construct was created, and introduced into tobacco protoplasts via polyethylene glycol-mediated DNA transfer. A -202 construct (where the transcriptional start site is denoted +1) and larger tap1 promoter constructs showed constitutive GUS expression. A 2-fold increase in GUS expression over the high constitutive levels was observed with -358 bp and larger tap1 constructs when protoplasts were incubated with elicitor preparations from Verticillium albo-atrum. In tobacco plants transformed with the tap1 promoter deletion/GUS fusion gene constructs, wounding caused induction of GUS expression by 20 h that increased 6- to 18-fold by 72 h. The region between -202 and -358 of the tap1 promoter conferred wound responsiveness. GUS was also found to be expressed in the epidermis and trichomes in the aerial parts of transgenic plants. High-level GUS expression was observed in the nodal region of stems that was associated with the leaf traces. GUS that was absent in very young flower buds was found in the subsequent developmental stages in the pistils, ovaries and anthers. The developmentally regulated tissue-specific expression of GUS was found with all constructs containing the -202 and larger promoters whereas wound and pathogen induction required -358 or larger promoter. These results suggest that the tap1 gene, which was heretofore thought to be expressed only upon wounding or pathogen attack, plays a role in normal developmental processes of the plant and this gene acquired additional 5'-flanking promoter for the purpose of responding to wounding and fungal attack.

Base Sequence↗

PubMed1996

Tissue-specific expression of the rolA gene mediates morphological changes in transgenic tobacco.

The spatial and temporal activity of the entire and individual promoter domains of the rolA gene of Agrobacterium rhizogenes was investigated and correlated with the distinctive features of the phenotypes of transgenic tobacco plants. The GUS assay was performed in the presence of an oxidative catalyst during the development of transgenic plants expressing chimeric genes containing the beta-glucuronidase coding sequence under the control of the different promoter domains. In situ hybridization was also used on transgenic plants harbouring rolA under the control of the entire or deleted promoter. This paper demonstrates for the first time that the entire rolA promoter, composed of domains, A, B and C, is silent in seeds, then activated at the onset of germination in the cotyledons and in the elongation zone of the radicle and is finally expressed throughout the vegetative and floral phases. Domains B + C, which were sufficient to induce wrinkled leaves and short internodes, were active in all the stem tissues, but only in the companion cells of the phloem strands of the leaves. Domain C, which specified a dwarf phenotype with normal leaves, was weakly expressed in the stem vascular bundles and in the leaf internal phloem. These results indicate that the vascular bundles are the primary targets for the generation of the short internode phenotype. Furthermore, the local expression of rolA in the stem vascular bundles induced a size reduction of the surrounding parenchyma cells, suggesting the existence of some diffusible factor(s) associated with the expression of the rolA gene.

Bacterial Proteins↗

PubMed1992

Activation of a truncated PR-1 promoter by endogenous enhancers in transgenic plants.

PR-1 genes are induced by various environmental stimuli such as pathogen attack or exposure of the plants to certain chemicals. To examine the regulation of these genes, the 5' flanking regions of the PR-la gene and of two PR-1 pseudogenes were joined by a transcriptional fusion to the Escherichia coli beta-glucuronidase (GUS) gene. These constructs were stably integrated into the tobacco genome and independent primary transformants were monitored for the expression of the reporter gene. Unexpectedly, out of 55 transformants analysed, four plants exhibited considerable GUS activities without any inductive treatment of the plants. Expression of the endogenous PR-1 genes, however, could not be detected in these plants. Primer extension analyses revealed correct initiation of the PR1/GUS hybrid transcripts from the PR-1a TATA box. When the plants were analysed at the cellular level, clear differences regarding the tissue specificity of expression of the reporter gene were observed. These results strongly suggest that the PR1/GUS hybrid promoter expression cassettes may be activated when integrated in the vicinity of heterologous enhancer elements dispersed in the tobacco genome. In order to support this hypothesis, domain B of the enhancer of the 35S RNA promoter from cauliflower mosaic virus (CaMV) was fused to various PR1/GUS hybrid genes upstream as well as downstream from the RNA start site. These constructs were stably introduced into the tobacco genome. In any primary transformant analysed, strong GUS activities were observed with the PR1/GUS hybrid RNAs originating from the normal transcription start site of the PR-1a gene. The tissue specificity of gene expression was identical to that described previously for the CaMV 35S domain B enhancer element. Thus, modulations of the transcriptional activity of the PR-1 promoter can be achieved by heterologous enhancers in transgenic plants and may be encountered upon random integration of PR-1 promoter constructs into the tobacco genome.

Base Sequence↗

PubMed1996

Coordinated activation of as-1-type elements and a tobacco glutathione S-transferase gene by auxins, salicylic acid, methyl-jasmonate and hydrogen peroxide.

The molecular mechanism of signal transduction pathways which mediate the action of phytohormones are poorly understood. Recently, we and others have shown that the as -1 type cis-acting elements can respond to auxin and salicylic acid, two well-characterized signaling molecules in plants. In the present work, we have examined a comprehensive set of physiological and abiotic agents and found that auxin, salicylic acid and methyl-jasmonate are three effective inducers of the as-1-type elements in transgenic tobacco. Using a cell suspension culture containing a synthetic promoter-GUS fusion, we demonstrated rapid and sensitive induction of the as-1-type element by these phytohormones. Furthermore, a tobacco glutathione S-transferase gene, GNT35, that contains an as-1-type binding site in its promoter is also inducible by auxin, salicylic acid and methyl-jasmonate with similar kinetics. As Ulmasov et al. have recently reported, we found that the as-1-type elements can also respond to weak/inactive analogues of auxin and salicylic acid. In addition, we show that hydrogen peroxide can also effectively activate the expression of GNT35 as well as the as-1-type element in a cell suspension culture, but not with whole seedlings. These results are discussed with respect to the possible mechanism(s) through which a single cis element may respond to a diverse array of molecules.

Acetates↗

PubMed1996

Structure of the dehydrin tas14 gene of tomato and its developmental and environmental regulation in transgenic tobacco.

We have isolated a genomic clone encoding tomato TAS14, a dehydrin that accumulates in response to mannitol, NaCl or abscisic acid (ABA) treatment. A fragment of tas14 gene containing the region from -2591 to +162 fused to beta-glucuronidase gene drives ABA- and osmotic stress-induced GUS expression in transgenic tobacco. Histochemical analysis of salt-, mannitol- and ABA-treated plants showed GUS activity mainly localized to vascular tissues, outer cortex and adventitious root meristems, coinciding with the previously observed distribution of TAS14 protein in salt-stressed tomato plants. In addition, GUS activity was also observed in guard cells, trichomes and leaf axils. Developmentally regulated gus expression was studied in unstressed plants and found to occur not only in embryos, but also in flowers and pollen. Tas14 expression in floral organs was confirmed by northern blots of tomato flowers.

Abscisic Acid↗

PubMed1996

The 22 bp W1 element in the pea lectin promoter is necessary and, as a multimer, sufficient for high gene expression in tobacco seeds.

The pea lectin (Psl) gene encodes an abundant seed protein. Its seed-specific expression pattern is conserved in transgenic tobacco plants. Progressive 5' promoter deletions resulted in a gradual decrease of transcriptional activity in tobacco seed. A fragment of 115 bp still conferred seed-specific expression albeit at a low level. This fragment contains a 22 bp element (W1), which has been demonstrated to be important for seed-specific expression when coupled as a trimer to a heterologous TATA box (de Pater et al., Plant Cell 5:877-886, 1993). Here we show that deletion of W1 in the natural promoter context resulted in a strongly decreased level of gene expression. A 4 bp mutation of W1 reduced the expression of truncated derivatives of the Psl promoter. A single copy of W1 coupled to the TATA box of the CaMV 35S promoter directed low gene expression in seeds and leaves. Multimerization enhanced the expression in seeds up to 100-fold, to levels found with the Psl promoter, whereas the expression level in leaves remained low. These results demonstrate that the W1 element is an essential control element in the Psl promoter. When taken out of its natural context and multimerized, it is sufficient for high expression in seeds.

Base Sequence↗

PubMed1995

Identification of several soybean cytosolic glutamine synthetase transcripts highly or specifically expressed in nodules: expression studies using one of the corresponding genes in transgenic Lotus corniculatus.

A DNA fragment containing sequences hybridizing to the 5' region of GS15, a gene encoding soybean cytosolic glutamine synthetase, was isolated from a soybean genomic library. Mapping and partial sequence analysis of the genomic clone revealed that it encodes a cytosolic GS gene, GS21, which is different from GS15. In parallel, a number of cDNA clones encoding cytosolic GS were isolated using the coding region of pGS20 as a probe (pGS20 is a cDNA clone which corresponds to a transcript of the GS15 gene). Two new full-length cDNAs designated pGS34 and pGS38 were isolated and sequenced. In the 5' non-coding region a strong homology was found between the two clones and the GS21 gene. However, none of these sequences were identical, which suggests that there are at least three members in this group of genes. In order to determine their relative levels of transcription, specific sequences from pGS34, pGS38 and GS21 were used in an RNAse protection assay. This experiment clearly showed that GS21 and the gene encoding pGS38 are specifically expressed in young or mature nodules, whereas the gene encoding pGS34 is highly transcribed in nodules and constitutively expressed at a lower level in other soybean organs. In order to further analyse the molecular mechanisms controlling GS21 transcription, different fragments of the promoter region were fused to the Escherichia coli reporter gene encoding beta-glucuronidase (GUS) and the constructs were introduced into Lotus corniculatus via Agrobacterium rhizogenes-mediated transformation. Analysis of GUS activity showed that the GS21 promoter-GUS constructs were expressed in the vasculature of all vegetative organs. This result is discussed in relation to species-specific metabolic and developmental characteristics of soybean and Lotus.

Amino Acid Sequence↗

PubMed1995

Light-regulated expression of the Arabidopsis thaliana ferredoxin gene requires sequences upstream and downstream of the transcription initiation site.

The effect of light on the expression of the Arabidopsis thaliana ferredoxin gene (fedA) was studied in mature tobacco plants. In light-treated leaves of tobacco plants transformed with a full-length ferredoxin gene, fedA-specific mRNA levels were more than twenty fold higher than in dark-treated controls. This indicates that all components for regulation of the Arabidopsis ferredoxin gene are present in tobacco. To identify light-regulatory elements in the fedA gene, we have tested a set of chimeric genes containing various parts of the fedA gene for light-dependent expression in mature tobacco plants. A fedA promoter-GUS fusion gene was not light-responsive, indicating that the 5'-upstream promoter region is not sufficient for light regulation. Fusion genes in which different transcribed regions of the fedA gene were expressed from the CaMV 35S promoter showed only limited light regulation, if any at all. This indicates that, like the fedA upstream region, the region downstream of the transcription start site is also not sufficient for full light regulation. The combined results suggest that for full light-regulated expression of the fedA gene, both the promoter region and sequences downstream of the transcription start site are required.

Arabidopsis↗

PubMed1993

Activity of the promoter of the Lhca3.St.1 gene, encoding the potato apoprotein 2 of the light-harvesting complex of Photosystem I, in transgenic potato and tobacco plants.

We have isolated cDNA and genomic clones for the potato (Solanum tuberosum) apoprotein 2 of the light harvesting complex of Photosystem I, designated Lhca3.St.1. The protein shows all characteristics of the family of chlorophyll a/b-binding proteins. Potato Lhca3.1 gene expression occurs predominantly in leaves, and is transcriptionally regulated by light. One gene copy is present per haploid genome. The sequence of the 5' upstream region was determined. Most boxes identified in the promoter sequences of genes whose expression is light-regulated recur in the Lhca3.St.1 sequence. Functional analyses of the Lhca3.St.1 promoter and two deletion derivatives in transgenic potato transformed with a promoter-GUS fusion show high promoter activity in leaves and other green parts of the plant, which depends on light. Activity is absent in roots and potato tubers. The 500 bp promoter fragment is as active as the full 2.0 kb sequence, showing that all regulatory elements are present on the smallest deletion derivative. In transgenic tobacco (Nicotiana tabacum) plants carrying the largest promoter derivative a similar distribution of activity is found. Promoter activity is not restricted to the phloem, but also prominent in the xylem of the young stem, which contrasts with promoters of other photosynthesis-associated genes.

Amino Acid Sequence↗

PubMed1995

Analysis of the lupin Nodulin-45 promoter: conserved regulatory sequences are important for promoter activity.

The promoter from the Lupinus angustifolius late nodulin gene, Nodulin-45, has been analysed to identify cis-elements and trans-acting factors. Various regions of the Nodulin-45 promoter, fused to the luciferase reporter gene, were introduced into Lotus roots using an Agrobacterium rhizogenes, transformation procedure. The transgenic roots were then nodulated. The promoter region A (-172 to +13, relative to the transcription start site) was capable of directing low-level expression of the reporter gene and in a nodule-enhanced manner when compared to roots. The addition of region C (-676 to -345) resulted in a significant increase in the expression within the nodule, whilst a low level of root expression was maintained. The C region, which confers this high-level nodule expression, contains the nodule consensus motifs AAAGAT and CTCTT. When region C was ligated to a minimal promoter element from the unrelated asparaginase gene rather than the Nodulin-45 A region, nodule-enhanced expression was still apparent, but at a much lower level. Mutation of the AAAGAT element in this construct resulted in a further significant decrease of expression. Gel retardation assays revealed that a factor from lupin nodule nuclear extracts interacted with two sequences of the C region. The binding of the factor to both of these regions could be removed by the addition of an oligonucleotide containing the AT-rich binding site for the soybean factor NAT2. This suggests that the lupin factor identified here is a NAT2 homologue. No factor binding was observed to the AAAGAT or CTCTT elements present in the C region.

Base Sequence↗

PubMed1995

The promoter of coconut foliar decay-associated circular single-stranded DNA directs phloem-specific reporter gene expression in transgenic tobacco.

A full-length double-stranded DNA copy of the single-stranded circular DNA associated with coconut foliar decay virus (CFDV) was constructed. Full-length CFDV DNA and smaller fragments were transcriptionally fused to the beta-glucuronidase reporter gene and examined for promoter activity in vivo. In stably transformed tobacco plants, the CFDV DNA promoter confered a tissue-specific expression pattern in that the reporter gene was specifically expressed in the phloem tissue of the vascular system in stem, leaves and flower. These results are in agreement with the previously reported association of CFDV DNA with the phloem of its coconut host plant.

Base Sequence↗

PubMed1996

Differential regulation of small heat-shock genes in plants: analysis of a water-stress-inducible and developmentally activated sunflower promoter.

We have isolated two sunflower genes, Ha hsp18.6 G2 and Ha hsp17.7 G4, that encode small heat shock proteins (sHSPs). RNAse A protection experiments, carried out with RNA probes transcribed from each gene and hybridized to sunflower total RNA, allowed us to distinguish their mRNA accumulation patterns. In sunflower, Ha hsp17.7 G4 mRNAs accumulated during zygotic embryogenesis at 25 degrees C. In vegetative tissues, these mRNAs accumulated in response to either heat shock (42 degrees C), abscisic acid (ABA), or mild water stress treatments. In all cases, the mRNAs were transcribed from the same initiation site. In contrast, Ha hsp 18.6 G2 mRNAs accumulated only in response to heat-shock. This result demonstrates differential regulation of these two sHSP genes. The complex regulation depicted by the Ha hsp 17.7 G4 promoter has been further analyzed in transgenic tobacco, using G4::GUS translational fusions. Developmental induction of Ha hsp 17.7 G4 during zygotic embryogenesis was faithfully reproduced in the transgenic plants. 5'-distal sequences (between -1132 and -395) were required to confer a preferential spatial expression of GUS activity in the cotyledons. More proximal sequences (from -83 to +163) conferred to the chimeric genes most of the developmental regulation, and the responses to ABA and heat shock characteristic of the Ha hsp17.7 G4 promoter. The water stress response of this gene was not reproduced in transgenic tobacco and, thus, could be uncoupled from its regulation during embryogenesis.

Abscisic Acid↗

PubMed1994

Coordinate expression of antibody subunit genes yields high levels of functional antibodies in roots of transgenic tobacco.

To explore the feasibility of employing antibodies to obtain disease resistance against plant root pathogens, we have studied the expression of genes encoding antibodies in roots of transgenic plants. A model monoclonal antibody was used that binds to a fungal cutinase. Heavy and light chain cDNAs were amplified by PCR, fused to a signal sequence for secretion and cloned behind CaMV 35S and TR2' promoters in a single T-DNA. The chimeric genes were cloned both in tandem and in a divergent orientation. The roots of tobacco plants transformed with these constructs produced antibodies that were able to bind antigen in an ELISA. Immunoblotting showed assembly to a full-size antibody. In addition, a F(ab')2-like fragment was observed, which is probably formed by proteolytic processing. Both antibody species were properly targeted to the apoplast, but the full-size antibody was partially retained by the wall of suspension cells. The construct with divergent promoters showed a better performance than the construct with promoters in tandem. It directed the accumulation of functional antibodies to a maximum of 1.1% of total soluble protein, with half of the plants having levels higher than 0.35%. The high efficiency of this construct probably results from coordinated and balanced expression of light and heavy chain genes, as evidenced by RNA blot hybridization.

Amino Acid Sequence↗

PubMed1994

Molecular characterization of rice genes specifically expressed in the anther tapetum.

In situ localization of mRNA was carried out on two cDNAs (Osc4 and Osc6) that had been isolated from rice anthers at the microspore stage. The mRNA corresponding to each cDNA was shown to be localized only in the tapetal cells of the rice immature anthers, but not in the microspores or the mature pollen. The corresponding genomic clone, Osg6B, was isolated, and its 5'-upstream region was found to regulate beta-glucuronidase expression in the tapetum of transgenic tobacco. A set of 5' deletions was also generated and a 1095 bp 5' region was revealed to be necessary for activation of the Osg6B promoter in transgenic tobacco.

Base Sequence↗

PubMed1994

Identification of domains in an Arabidopsis acyl carrier protein gene promoter required for maximal organ-specific expression.

Deletions were made in the promoter of the acyl carrier protein (ACP) Acll.2 gene from Arabidopsis to investigate the nature of the cis-acting elements that direct its expression. These constructs, which included the untranslated leader region, were fused to a reporter gene coding for beta-glucuronidase (GUS) and transformed into tobacco. Quantitative fluorometric analysis of GUS activity in transgenic plants showed that expression in young leaves drops to a basal level when a 85 bp domain, from -320 to -236 relative to transcription initiation, is deleted. Maximum promoter activity in roots also depends on this domain, but two other regions are also important. In total, deletion of the sequences from -466 to -55 caused an ca. 80-fold reduction in Acl1.2 promoter activity in roots. The -320 to -236 domain forms a complex with a protein factor found in leaves and roots, which was not detectable in seeds. The formation of this protein-DNA complex was abolished by mutation of a bZIP core motif, ACGT, found within the context AAGACGTAG, which is dissimilar to the other bZIP-binding sites thus far characterized in plants. Previously we showed that Acl1.2 promoter activity is highest in seeds [2]. Here we find, in contrast to leaves and roots, that deletion to position -236 has no effect on GUS levels in seeds. However, nearly a 100-fold drop was observed when the -235 to -55 region was removed. Hence, this 180 bp domain contains all the cis-acting information necessary for Acl1.2 promoter activity in seeds. The same region is necessary for Acl1.2 activity in the receptacle, stigma, tapetum and pollen of the flower, as demonstrated by histochemical staining.

Acyl Carrier Protein↗

PubMed1994

The expression of a chimeric Phaseolus vulgaris nodulin 30-GUS gene is restricted to the rhizobially infected cells in transgenic Lotus corniculatus nodules.

In Phaseolus vulgaris there is a nodulin family, Npv30, of ca. 30 kDa, as detected in an in vitro translation assay [2]. We isolated a gene (npv30-1) for one of the members of this family. The nucleotide sequence of the promoter of npv30-1 contains nodule-specific motifs common to other late nodulin genes. The promoter was fused to the GUS reporter gene; this chimeric fusion was introduced into Lotus corniculatus via Agrobacterium rhizogenes transformation. GUS activity was only detected in the infected cells of the nodules of transgenic plants. By contrast, the expression of a 35S-GUS construct was restricted to the uninfected cells and the vascular tissue.

Base Sequence↗

PubMed1996

Identification of tissue-specific, dehydration-responsive elements in the Trg-31 promoter.

Trg-31 is a pea gene which is rapidly induced in response to dehydration stress. Trg-31 belongs to a family of membrane intrinsic proteins that play a role in facilitating inter-membrane transport. In order to understand the mechanism of regulation of Trg-31 expression we defined the cis-acting elements of the Trg-31 promoter and the trans-acting factors that interact with them. Deletion analysis of the Trg-31 promoter in transgenic tobacco plants indicated the presence of two positive regulatory region: (1) between the -1412 to -1066 nucleotides and (2) between the -559 to -391 nucleotides, with respect to the transcription initiation site. A negative regulatory element was also identified between -1066 and -559 nucleotides. All three regulatory regions specifically regulated Trg-31 induction in leaf tissue and had little or no effect on Trg-31 induction in root tissue. Gel-mobility shift assays indicated the presence of leaf nuclear proteins that interacted with the -1412 to -1066 and -1066 to -559 regions and an 'ABRE-like' sequence at -74 in the 5' promoter region of Trg-31.

Aquaporins↗

PubMed1993

Induction of a tomato anionic peroxidase gene (tap1) by wounding in transgenic tobacco and activation of tap1/GUS and tap2/GUS chimeric gene fusions in transgenic tobacco by wounding and pathogen attack.

The anionic peroxidase genes of tomato, tap1 and tap2, are induced by wounding in tomato fruits and by elicitor treatment in cell suspension cultures. These homologous genes code for anionic peroxidases that are postulated to cause polymerization of the phenolic residues into wall polymers in wound-healing and pathogen-infected tissues. An expression construct containing the entire TAP1 gene with its 5' and 3' flanking sequences was introduced into tobacco by Agrobacterium tumefaciens-mediated gene transfer. Also, constructs containing the 5' upstream regions of tap1 and tap2 including sequences coding for their respective putative leader peptides fused translationally to the beta-glucuronidase (GUS) reporter gene were made and introduced into tobacco. Northern blot analysis of transcripts from wound-healing leaf tissues of transformants containing tap1 showed that the introduced gene was being transcribed in the heterologous host. The induction of tap1 transcripts in the wound-healing transgenic tobacco tissues was observed by 48 h and increased over time period of 84 h. Wounding also led to expression of GUS in tap1/GUS and tap2/GUS transformants and GUS activity was localized to the wound site. Activation of the tap1 and tap2 promoters in wound-healing transgenic tobacco tissues showed a GUS expression profile that correlated with the postulated role for anionic peroxidases in phenolic polymerization in suberizing tissues. Inoculation of tap1/GUS and tap2/GUS transformant leaves with fungal conidia from Fusarium solani f. sp. pisi caused expression of GUS in locally inoculated regions, and GUS expression increased over a period of four days.

Blotting, Northern↗