Beta-glucuronidase Fusion Research Articles

The Arabidopsis thaliana ABC transporter AtMRP5 controls root development and stomata movement.

In the present study, we investigated a new member of the ABC transporter superfamily of Arabidopsis thaliana, AtMRP5. AtMRP5 encodes a 167 kDa protein and exhibits low glutathione conjugate and glucuronide conjugate transport activity. Promotor- beta-glucuronidase fusion constructs showed that AtMRP5 is expressed mainly in the vascular bundle and in the epidermis, especially guard cells. Using reverse genetics, we identified a plant with a T-DNA insertion in AtMRP5 (mrp5-1). mrp5-1 exhibited decreased root growth and increased lateral root formation. Auxin levels in the roots of mrp5-1 plants were increased. This observation may indicate that AtMRP5 works as an auxin conjugate transporter or that mutant plants are affected in ion uptake, which may lead to changes in auxin concentrations. Experiments on epidermal strips showed that in contrast to wild type, the sulfonylurea glibenclamide had no effect on stomatal opening in mrp5-1 plants. This result strongly suggests that AtMRP5 may also function as an ion channel regulator.

The 5' untranslated region of the At-P5R gene is involved in both transcriptional and post-transcriptional regulation.

The steady-state level of transcripts coding for the pyrroline-5-carboxylate reductase of Arabidopsis (At-P5R) increased under salt and heat stress, mainly because of an enhanced mRNA stability. However, the At-P5R protein level was not induced, and its translation was inhibited at initiation stage and probably also at later stages. Replacement of the 5' untranslated region (5'UTR) and beta-glucuronidase (gus) fusion analysis revealed that the first 92 bp region of the At-P5R 5'UTR was sufficient to mediate transcript stabilization and translation inhibition during salt and heat stresses. Furthermore, the first 92 bp region of the At-P5R 5'UTR was also involved in transcription efficiency in a promoter-dependent manner. The results demonstrated that the stress regulation of At-P5R is complex and involves the 5'UTR which acts at three levels, partly in opposing directions.

The H1 histone variant of tomato, H1-S, is targeted to the nucleus and accumulates in chromatin in response to water-deficit stress

Water deficit has a significant impact on patterns of gene expression. Based on the deduced amino acid sequence, it has been proposed that the drought and abscisic acid-induced gene (his1-s) of tomato (Lycopersicon esculentum Mill.) encodes an H1 histone variant. To study the role of H1-S it is important to understand the expression characteristics of the protein. To identify the his1-s product in vivo the his1-s cDNA was fused to a (His)6 tag and overexpressed in Escherichia coli. The H1-S fusion protein was used to generate an antibody that recognized a protein with an apparent molecular weight of 31 kDa that accumulates in response to water deficit in the whole plant and detached leaves. A time course of his1-s expression showed that protein accumulation is delayed compared to the mRNA accumulation in both the whole plant and detached leaves. Cellular fractionation, immunofluorescence and H1-S::beta-glucuronidase fusion analyses in transgenic tissues were used to determine the cellular localization of H1-S. The results showed that H1-S accumulates in nuclei and is associated with chromatin of wilted tomato leaves. The drought- and abscisic acid-induced gene his1-s encodes a linker-histone subtype specifically accumulated in the nuclei and chromatin of tomato leaves subjected to water-deficit conditions. Although the molecular mechanism of H1-S function is still unclear, the expression characteristics of H1-S are consistent with a potential role of this protein in the regulation of gene expression in response to water deficit.

IFL1, a gene regulating interfascicular fiber differentiation in Arabidopsis, encodes a homeodomain-leucine zipper protein.

Arabidopsis inflorescence stems develop extraxylary fibers at specific sites in interfascicular regions. The spatial specification of interfascicular fiber differentiation is regulated by the INTERFASCICULAR FIBERLESS1 (IFL1) gene because mutation of that gene abolishes the formation of normal interfascicular fibers in Arabidopsis stems. To understand further the role of IFL1 in the specification of fiber differentiation, we cloned the IFL1 gene by using a positional cloning strategy. Sequence analysis showed that the IFL1 gene encodes a transcription factor that has the same features as a family of homeodomain-leucine zipper (HD-ZIP) proteins found only in plants. The predicted IFL1 protein is composed of three distinct domains, including a 60-amino acid HD at the N terminus followed by a 28-amino acid ZIP motif and a 724-amino acid C-terminal region. A nuclear targeting assay showed that IFL1 is able to direct a beta-glucuronidase fusion protein into the nucleus, which is consistent with IFL1's presumed function as a transcription factor. Gene expression analysis demonstrated that the IFL1 gene is expressed in the interfascicular regions in which fibers differentiate, which is consistent with its role in the control of interfascicular fiber differentiation. Furthermore, the IFL1 gene was shown to be expressed in the vascular regions, indicating its possible role in the regulation of vascular tissue formation. This possibility is supported by the observation that differentiation of both xylary fibers and vessel elements is altered in the vascular bundles of ifl1 mutants. Our results provide direct evidence that an HD-ZIP protein plays a role in the spatial control of fiber differentiation.

Two cytosolic cyclophilin genes of Arabidopsis thaliana differently regulated in temporal- and organ-specific expression.

We have previously isolated two closely related genes (ATCYP1 and ATCYP2) each encoding a cytosolic cyclophilin of Arabidopsis thaliana. Here we tested expression patterns of these two genes by Northern analysis and by histochemical analysis with transgenic plants carrying the promoter: beta-glucuronidase (GUS) fusion. The results showed that ATCYP1 is predominantly transcribed in vascular tissue and flowers, but ATCYP2 is at higher levels in younger leaves. The different expression patterns seemed to be conferred by the quite different promoter structures carrying various cis elements. Our finding suggests that the two cyclophilins have different roles in Arabidopsis thaliana cells.

HSF3, a new heat shock factor from Arabidopsis thaliana, derepresses the heat shock response and confers thermotolerance when overexpressed in transgenic plants.

Organisms synthesize heat shock proteins (HSPs) in response to sublethal heat stress and concomitantly acquire increased tolerance against a subsequent, otherwise lethal, heat shock. Heat shock factor (HSF) is essential for the transcription of many HSP genes. We report the isolation of two HSF genes, HSF3 and HSF4, from an Arabidopsis cDNA library. Transgenic Arabidopsis plants were generated containing constructs that allow expression of HSF3 and HSF4 or the respective translational beta-glucuronidase (GUS) fusions. Overexpression of HSF3 or HSF3-GUS, but not of HSF4 or HSF4-GUS, causes HSP synthesis at the non-heat-shock temperature of 25 degrees C in transgenic Arabidopsis. In transgenic plants bearing HSF3/HSF3-GUS, transcription of several heat shock genes is derepressed. Electrophoretic mobility shift assays suggest that derepression of the heat shock response is mediated by HSF3/HSF3-GUS functioning as transcription factor. HSF3/HSF3-GUS-overexpressing Arabidopsis plants show an increase in basal thermotolerance, indicating the importance of HSFs and HSF-regulated genes as determinants of thermoprotective processes. Plants transgenic for HSF3/HSF3-GUS exhibit no other obvious phenotypic alterations. Derepression of HSF activity upon overexpression suggests the titration of a negative regulator of HSF3 or an intrinsic constitutive activity of HSF3. We assume that stable overexpression of HSFs may be applied to other organisms as a means of derepressing the heat shock response.

Transcriptional activation of the tobacco retrotransposon Tto1 by wounding and methyl jasmonate.

The tobacco retrotransposon Tto1, one of a few active retrotransposons of plants, has been shown to be activated by tissue culture. Its transposition is regulated mainly at the transcriptional level. It is shown here that expression of Tto1 can be induced in leaves of tobacco by wounding stress. Exogenous supply of methyl jasmonate, which is known to be a potent inducer of certain wound-responsive genes in plants, also induces Tto1 RNA expression. Tto1 RNA was detected within 2 to 4 h after wounding/cutting treatment, and increased levels of Tto1 RNA were observed during subsequent incubation periods for 48 h. Expression of Tto1 RNA after cutting treatment was induced more significantly in young expanding leaves rather than in older mature leaves, suggesting that developmental or physiological factors may be required for the strong response to Tto1 transcription to wounding stimuli. Experiments with transgenic tobacco plants carrying the Tto1-LTR: beta-glucuronidase fusion gene (LTR:GUS) revealed that Tto1 actually contains cis-regulatory regions in response to wounding and methyl jasmonate. These findings are discussed in relation to the mechanism of transcriptional activation and the evolutionary role played by retrotransposons.

Promoter trap markers differentiate structural and positional components of polar development in Arabidopsis.

To investigate mechanisms involved in establishing polar organization in Arabidopsis embryos and seedlings, we used promoter trapping to identify molecular markers (beta-glucuronidase fusion genes) expressed in spatially restricted patterns along the apical-basal axis. Three markers were identified that are expressed, respectively, in the embryonic and seedling root tip (POLARIS), cotyledons and shoot and root apices (EXORDIUM), and root cap (COLUMELLA). Each marker was crossed into the mutants hydra and emb30, which are defective in embryonic and seedling morphogenesis. All three markers were expressed in hydra mutants in patterns similar to those observed in phenotypically wild-type embryos and seedlings. In emb30 mutants, the EXORDIUM marker was expressed in cotyledons but not in the expected position of shoot and root meristems, and the marker COLUMELLA was not expressed at all, which is consistent with the view that the emb30 mutant, but not hydra, lacks shoot and root meristems. However, POLARIS was expressed in the basal part of hydra embryos lacking an embryonic root and in the basal parts of both hydra and emb30 seedlings. Expression of POLARIS is inducible by exogenous auxin and suppressed by cytokinin but is unaffected by inhibitors of polar auxin transport or cell division. We conclude that POLARIS differentiates positional aspects of polar development from structural aspects.

Molecular characterization of the AP19 gene family in Arabidopsis thaliana: components of the Golgi AP-1 clathrin assembly protein complex.

AP19 is the smallest polypeptide component of AP-1, the clathrin associated protein complex found in clathrin-coated vesicles of the Golgi apparatus. Two genomic clones that encode homologues of AP19 were isolated from Arabidopsis thaliana (AAP19-1 and AAP19-2). Analysis of their nucleotide sequences predict proteins of 162 and 163 amino acids with mr of 18,913 and 18,758 respectively. Amino acid sequence comparisons with mammalian, yeast and plant clathrin associated sequences indicates that the Arabidopsis genes encode polypeptides that are more closely related to the AP19 proteins associated with clathrin-coated Golgi vesicles than to AP17, which is part of the AP-2 complex of endocytic clathrin-coated pits. Ribonuclease protection assays showed that both genes are expressed in all Arabidopsis tissues throughout development. Constitutive transcription of AAP19-1 was confirmed in transgenic Arabidopsis seedlings and plants containing an AAP19-1 promoter::beta-glucuronidase (GUS) fusion by ribonuclease protection assays and GUS histochemical staining.

Expression analysis of an Arabidopsis C2H2 zinc finger protein gene

C2H2 zinc finger protein genes encode nucleic acid-binding proteins involved in the regulation of gene activity. AtZFP1 (Arabidopsis thaliana zinc finger protein 1) is one member of a small family of C2H2 zinc finger-encoding sequences previously characterized from Arabidopsis. The genomic sequence corresponding to the AtZFP1 cDNA has been determined. Molecular analysis demonstrates that AtZFP1 is a unique, intronless gene which encodes a 1100 nucleotides mRNA highly expressed in roots and stems. A construct in which 2.5 kb of AtZFP1 upstream sequences is linked to the beta-glucuronidase gene was introduced into Arabidopsis by Agrobacterium-mediated transformation of roots. Histochemical analysis of transgenic Arabidopsis carrying the AtZFP1 promoter: beta-glucuronidase fusion shows good correlation with RNA blot hybridization analysis. This transgenic line will be a useful tool for analyzing the regulation of AtZFP1 to further our understanding of its function.

Isolation of a Polyubiquitin Promoter and Its Expression in Transgenic Potato Plants

A polyubiquitin clone (ubi7) was isolated from a potato (Solanum tuberosum) genomic library using a copy-specific probe from a stress-induced ubiquitin cDNA. The genomic clone contained a 569-bp intron immediately 5' to the initiation codon for the first ubiquitin-coding unit. Two chimeric beta-glucuronidase (GUS) fusion transgenes were introduced into potato. The first contained GUS fused to a 1156-bp promoter fragment containing only 5' flanking and 5' untranslated sequences from ubi7. The second transgene contained GUS translationally fused to the carboxy terminus of the first ubiquitin-coding unit and thus included the intron present in the 5' untranslated region of the polyubiquitin gene. Both ubi7-GUS transgenes were activated by wounding in tuber tissue and in leaves by application of exogenous methyl jasmonate. They were also expressed constitutively in the potato tuber peel (outer 1-2 mm). Both transgenes were actively expressed in mature leaves. Exceptionally high levels of expression were observed in senescent leaves. Transgenic clones containing the ubi7 intron and the first ubiquitin-coding unit showed GUS expression levels at least 10 times higher than clones containing GUS fused to the intronless promoter.

Different sets of cis-elements contribute to the expression of a catalase gene from castor bean during seed formation and postembryonic development in transgenic tobacco.

Deletion analysis of the promoter region of a gene for catalase, cat2, from castor bean (Ricinus communis) was performed to identify the cis-regulatory elements responsible for the expression of a beta-glucuronidase (GUS) fusion gene during seed formation and postembryonic development in transgenic tobacco. The analysis showed that multiple cis-elements contribute to the activity of the cat2 promoter during seed formation and postembryonic development. The 5'-upstream regions from -1,241 to -816 bp, from -720 to -682 bp, and from -632 to -535 bp, relative to the site of initiation of translation of cat2, contributed positively to the activity of the cat2 promoter during both stages. By contrast, the region from -816 to -720 bp had a negative effect at both stages. The region from -682 to -632 bp contributed positively to the activity during seed formation but negatively during postembyonic development. Histochemical analysis revealed that the multiple cis-elements determined not only the level of expression of the chimeric gene but also the tissue-specificity of such expression. For example, the region from -1,241 to -816 bp allowed expression of the chimeric gene in the axis of the embryo of the dry seed, as well as in the cortex of the middle part of the hypocotyl and at the base of epicotyl in the young seedling.

Molecular dissection of GT-1 from Arabidopsis.

We isolated and characterized an Arabidopsis cDNA encoding the DNA binding protein GT-1. This protein factor, which contains 406 amino acids, is highly homologous to the previously described tobacco DNA binding protein GT-1a/B2F but is 26 amino acids longer. Recombinant Arabidopsis GT-1, which was obtained from in vitro translation, bound to probes consisting of four copies of pea small subunit of ribulose bisphosphate carboxylase rbcS-3A box II and required the same GGTTAA core binding site as the binding activity of an Arabidopsis nuclear protein preparation. However, unlike the truncated tobacco GT-1a prepared from Escherichia coli extracts, the full-length Arabidopsis GT-1 bound to pea rbcS-3A box III and Arabidopsis chlorophyll a/b binding protein CAB2 light-responsive elements, both of which contain GATA motifs. Deletion and mutational analyses suggested that the predicted trihelix region of GT-1 is essential for DNA binding. Moreover, GT-1 binds to target DNA as a dimer, and its C-terminal region contains a putative dimerization domain that enhances the binding activity. Transient expression of the GT-1::beta-glucuronidase fusion protein in onion cells revealed the presence of a nuclear localization signal(s) within the first 215 amino acids of GT-1.

In vivo random beta-glucuronidase gene fusions in Arabidopsis thaliana.

Vectors were constructed for the isolation of random transcriptional and translational beta-glucuronidase gene fusions in plants. This system is based on the random integration of the transferred DNA (T-DNA) into the plant nuclear genome. The Escherichia coli beta-glucuronidase coding sequence without promoter, and also devoid of its ATG initiation site in the translational gene fusion vector, was inserted in the T-DNA with its 5' end at a distance of 4 base pairs from the right T-DNA border sequence. Transgenic plants can be selected by using a chimeric (P35S-nptII-3' ocs) kanamycin-resistance gene present in the same T-DNA. Subsequent screening of these for beta-glucuronidase expression allows the identification of clones harboring a fusion of the beta-glucuronidase coding sequence with plant 5' regulatory sequences. After transformation of Arabidopsis thaliana C24 root explants, beta-glucuronidase expression was detected in 54% and 1.6% of the plants transformed with the transcriptional and translational fusion vectors, respectively. Several different patterns of tissue-specific beta-glucuronidase expression were identified. The plant upstream sequence of a beta-glucuronidase fusion that is specifically expressed in the phloem of all organs was cloned and sequenced. After introduction in A. thaliana C24 and Nicotiana tabacum SR1, this sequence mediates the same highly phloem-specific beta-glucuronidase expression pattern as in the original transgenic plant from which it was isolated. These data demonstrate that this system facilitates the isolation and analysis of plant DNA sequences mediating regulated gene expression.

LpsZ, a lipopolysaccharide gene involved in symbiosis of Rhizobium meliloti

lpsZ+ is an allele that allows exo (exopolysaccharide-deficient) mutants of Rhizobium meliloti to invade nodules by modifying rhizobial lipopolysaccharide. We have cloned and sequenced the lpsZ gene. The predicted LpsZ protein has a molecular weight of 48,589 and is probably localized in the cytoplasm. A beta-glucuronidase fusion in the lpsZ gene indicates that lpsZ is not regulated by oxygen or nitrogen.