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 -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.     

Fusions between green fluorescent protein and β-glucuronidase as sensitive and vital bifunctional reporters in plants

By fusing the genes encoding green fluorescent protein (GFP) and -glucuronidase (GUS) we have created a set of bifunctional reporter constructs which are optimized for use in transient and stable expression studies in plants. This approach makes it possible to combine the advantage of GUS, its high sensitivity in histochemical staining, with the advantages of GFP as a vital marker. The fusion proteins were functional in transient expression studies in tobacco using either DNA bombardment or potato virus X as a vector, and in stably transformed Arabidopsis thaliana and Lotus japonicus plants. The results show that high level of expression does not interfere with efficient stable transformation in A. thaliana and L. japonicus. Using confocal laser scanning microscopy we show that the fusion constructs are very suitable for promoter expression studies in all organs of living plants, including root nodules. The use of these reporter constructs in the model legume L. japonicus offers exciting new possibilities for the study of the root nodulation process.     

Pollen-specific gene expression in transgenic plants: coordinate regulation of two different tomato gene promoters during microsporogenesis

To investigate the regulation of gene expression during male gametophyte development, we analyzed the promoter activity of two different genes (LAT52 and LAT59) from tomato, isolated on the basis of their anther-specific expression. In transgenic tomato, tobacco and Arabidopsis plants containing the LAT52 promoter region fused to the beta-glucuronidase (GUS) gene, GUS activity was restricted to pollen. Transgenic tomato, tobacco and Arabidopsis plants containing the LAT59 promoter region fused to GUS also showed very high levels of GUS activity in pollen. However, low levels of expression of the LAT59 promoter construct were also detected in seeds and roots. With both constructs, the appearance of GUS activity in developing anthers was correlated with the onset of microspore mitosis and increased progressively until anthesis (pollen shed). Our results demonstrate co-ordinate regulation of the LAT52 and LAT59 promoters in developing microspores and suggest that the mechanisms that regulate pollen-specific gene expression are evolutionarily conserved.     

Deletion analysis of a 2S seed storage protein promoter of Brassica napus in transgenic tobacco

The promoter and upstream region of the Brassica napus 2S storage protein napA gene were studied to identify cis-acting sequences involved in developmental seed-specific expression. Fragments generated by successive deletions of the 5 control region of the napA gene were fused to the reporter gene -glucuronidase (GUS). These constructs were used to transform tobacco leaf discs. Analyses of GUS activities in mature seeds from the transformed plants indicated that there were both negatively and positively acting sequences in the napin gene promoter. Deletion of sequences between –1101 and –309 resulted in increased GUS activity. In contrast, deletion of sequences between –309 and –211 decreased the expression. The minimum sequence required for seed-specific expression was a 196 bp fragment between –152 and +44. Further 5 deletion of the fragment to –126 abolished this activity. Sequence comparison showed that a G box-like sequence and two sequence motifs conserved between 2S storage protein genes are located between –148 to –120. Histochemical and fluorometric analysis of tobacco seeds showed that the spatial and developmental expression pattern was retained in the deletion fragments down to –152. However, the expression in tobacco seeds differed from the spatial and temporal expression in B. napus. In tobacco, the napA promoter directed GUS activity early in the endosperm before any visible activity could be seen in the heart-shaped embryo. Later, during the transition from heart to torpedo stages, the main expression of GUS was localized to the embryo. No significant GUS activity was found in either root or leaf.     

Mutation of either G box or I box sequences profoundly affects expression from the Arabidopsis rbcS-1A promoter.

A deletion analysis of the Arabidopsis thaliana rbcS-1A promoter defined a 196 bp region (-320 to -125) sufficient to confer light-regulated expression on a heterologous Arabidopsis alcohol dehydrogenase (Adh) reporter gene in transgenic Nicotiana tabacum (tobacco) leaves. This region, which contains DNA sequences I, G and GT boxes, with homology to other ribulose-1,5-bisphosphate carboxylase small subunit (RBCS) gene promoter sequences, directed expression independent of orientation and relative position in the Adh promoter. Site-specific mutagenesis of these conserved sequences and subsequent expression analysis in transgenic tobacco showed that both G box and I box mutations in the context of the full (-1700 to +21) rbcS-1A promoter substantially reduced the expression of Adh and beta-glucuronidase (GUS) reporter genes. The G box has previously been shown to specifically bind in vitro a factor isolated from nuclear extracts of tomato and Arabidopsis. This factor (GBF) is distinct from the factor GT-1 which binds to adjacent GT boxes in the pea rbcS-3A promoter. Multiple mutations in putative Arabidopsis rbcS-1A promoter GT boxes had no pronounced affect on expression, possibly due to a redundancy of these sites. Experiments in which rbcS-1A promoter fragments were fused to truncated 35S CaMV (cauliflower mosaic virus) promoter--GUS reporter constructs showed that cis-acting CaMV promoter elements could partially restore expression to G-box-mutated rbcS-1A sequences.     

Expression of an auxin-inducible promoter of tobacco in Arabidopsis thaliana

The expression of the auxin-inducible Nt103-1 gene of tobacco was studied in Arabidopsis thaliana. For this purpose we introduced a gene fusion between the promoter of the gene and the -glucuronidase reporter gene (GUS) into Arabidopsis thaliana. The expression and location of GUS activity were studied histochemically in time and after incubation of seedlings on medium containing auxins or other compounds. The auxins 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), and 1-naphthylacetic acid (1-NAA) were able to induce GUS activity in the root tips of transgenic seedlings. The auxin transport inhibitor 2,3,5-triiodobenzoic acid was able to induce GUS activity not only in the root tip, but also in other parts of the root. Induction by the inactive auxin analog 3,5-dichlorophenoxyacetic acid was much weaker. Compounds like glutathione and the heavy metal CuSO₄ were weak inducers. GUS activity observed after induction by glutathione was located in the transition zone. Salicylic acid and compounds increasing the concentration of hydrogen peroxide in the cell were also very well able to induce GUS activity in the roots. The possible involvement of hydrogen peroxide as a second messenger in the pathway leading to the induction of the Nt103-1 promoter is discussed.     

Transformation vector based on promoter and intron sequences of a replacement histone H3 gene. A tool for high,constitutive gene expression in plants

This study explored the possibility of using non-viral, plant-based gene sequences to create strong and constitutive expression vectors. Replacement histone H3 genes are highly and constitutively expressed in all plants. Sequences of the cloned alfalfa histone H3.2 gene MsH3g1 were tested. Constructs of the -glucuronidase (GUS) reporter gene were produced with H3.2 gene promoter and intron sequences. Their efficiency was compared with that of the commonly used strong 35S cauliflower mosaic virus promoter in transgenic tobacco plants. Combination of the H3.2 promoter and intron produced significantly higher GUS expression than the strong viral 35S promoter. Histochemical GUS analysis revealed a constitutive pattern of expression. Thus, alfalfa replacement H3 gene sequences can be used instead of viral promoters to drive heterologous gene expression in plants, avoiding perceived risks of viral sequences.     

Gene expression and oxalate oxidase activity of two germin isoforms induced by stress

Wheat germin is a homopentameric 125 kD glycoprotein mainly localized in the cell wall of monocots, and is a specific marker of the onset of growth in germinating seeds. The major objective of this study was to examine the expression and oxalate oxidase activity of two wheat germin isoforms: gf-2.8 and gf-3.8 in transgenic tobacco plants. The transgenic tobacco plants were created with different constructs: 1) one entire excision of gf-2.8 germin promoter and two partially deleted promoter sequences were used to generate 3 independent GUS constructs; 2) the whole gf-2.8 gene construct and the fusion with CaMV 35S promoter; 3) one entire excision of gf-3.8 germin gene and one partially deleted gf-3.8 promoter sequences were used to generate 2 independent GUS constructs; 4) the whole gf-3.8 gene and the fusion with CaMV 35S promoter. Hormonal treatment (auxin and gibberellin), salt treatment, heavy metals (Mn, Fe, Co, Ni, Cu, Zn, Cd, Hg, As) and Al induced high GUS activity in tobacco transformed with entire and one partially deleted of the gf-2.8 gene. The immunoblotting confirmed induction of gf-2.8 gene and its product expressed oxalate oxidase activity in tobacco transformed with the entire gf-2.8 construct. Neither nicotinic acid, salicylic acid, heat shock, cold nor UV-C have enhanced significant GUS activity and germin gf-2.8 synhesis and activity. The germin gf-3.8 constructs with GUS gene and with the entire gf-3.8 sequences gave non-positive response with factors mentioned above. It has been demonstrated that gf-3.8 germin isoform is present as a monomer (M_r 25 kD). The non-active gf-3.8 protein is synthetised in transgenic tobacco plants only under control of the CaMV 35S promoter. Consequently, among two germin isoforms, only the gf-2.8 protein seems to be regulated by hormonal, salt and heavy metal factors. The gf-2.8 oxalate oxidase activity could be then involved in general stress-induced signalling in plant.     

Differential gene expression in nematode-induced feeding structures of transgenic plants harbouring promoter—gusA fusion constructs

Sedentary plant-parasitic nematodes are able to induce specialized feeding structures in the root system of their host plants by triggering a series of dramatic cellular responses. These changes presumably are accompanied by a reprogramming of gene expression. To monitor such changes, a variety of promoter— gus A fusion constructs were introduced into Arabidopsis and tobacco. Transgenic plants were analysed histochemically for GUS activity in the nematode feeding structures after infection with either Heterodera schachtii or Meloidogyne incognita . Promoters of the Cauliflower Mosaic Virus 35S gene, the bacterial nopaline synthase, rooting loci ( rol ) and T- cyt genes and the plant-derived phenylalanine ammonia-lyase I gene, which are highly active in non-infected roots, were all downregulated in the feeding structures as indicated by the strong decrease of GUS activity inside these structures. Less stringent down-regulation was observed with chimeric gus A fusion constructs harbouring truncated rol B and rol C promoter sequences. Similar observations were made with transgenic Arabidopsis lines that carried randomly integrated promoterless gus A constructs to identify regulatory sequences in the plant genome. Most of the lines that were selected for expression in the root vascular cylinder demonstrated local down-regulation in feeding structures after infection with H. schachtii . The reverse pattern of GUS activity, a blue feeding structure amidst unstained root cells, was also found in several lines. However, GUS activity that was entirely specific for the feeding structures was not observed. Our data show that the expression of a large number of genes is influenced during the development of the nematode feeding structures.     

Microprojectile-DNA delivery in conifer species: factors affecting assessment of transient gene expression using the β-glucuronidase reporter gene

The Biolistic^® microprojectile DNA-delivery method was used to test the usefulness in conifers of eight gene constructs based on the 35S promoter, the AMV translational enhancer, and gene fusion between the P-glucuronidase and the neomycin phosphotransferase II genes. The evaluation was done with embryogenic cells of Picea glauca, where the relative strengths of the promoters were 35S-35S-AMVE>35S-AMVE>35S-35S>35S as evaluated by transient gene expression. The fusion gene of GUS and NPT II gave lower levels of transient gene expression than the unfused GUS gene as detected by X-GLU histochemical assays. Experiments comparing the EM promoter of wheat and the 35S-35S-AMVE promoter (with and without fusion between GUS and NPT II) were done in Picea rubens, P. mariana, P. glauca, and Larix x eurolepis. The unfused gene with the 35S-35S-AMVE promoter gave higher levels of transient gene expression than the fused GUS-NPT II gene. The fluorescent MUG assay was more sensitive than the histochemical X-GLU assay to detect the activity of the -glucuronidase gene.Abbreviations AMV alfalfa mosaic virus - AMVE alfalfa mosaic virus translational enhancer - EM protein of mature wheat embryo - GUS P-glucuronidase gene - MUG 4-methylumbelliferyl -D-glucuronide - NPT II neomycin phosphotransferase - X-GLU 5-bromo-4-chloro-3-indolyl -D-glucuronic acid     

Analysis of an ABA-responsive rice gene promoter in transgenic tobacco

We have analyzed in transgenic tobacco the expression of a chimeric gene containing 5 sequences of the rice rab-16B gene fused to the -glucuronidase (GUS) reporter gene. This construct, a translational fusion (–482 to +184) including 14 amino acids of the RAB-16B protein, is expressed only in zygotic and pollen-derived embryos. In zygotic embryos, GUS activity begins to accumulate 10 days after flowering (daf), and increases until seed maturation at 25 daf. Immunological measurements of endogenous abscisic acid (ABA) accumulation in these seeds showed a close parallel between hormone levels and GUS activity. However, GUS activity could not be reproducibly induced by treatment of immature embryos with ABA (10 M). Neither GUS activity nor GUS mRNA could be detected in leaves of transgenic tobacco even after ABA treatment. In contrast, GUS activity could be induced to high levels in pollen-derived embryos by treatment with ABA. Our results show that 482 bp of 5 sequences of the rice rab-16B promoter can confer in transgenic tobacco developmentally regulated expression in embryos but not ABA-responsive expression in vegetative tissues.     

The promoter of rbcS in a C3 plant (rice) directs organ-specific,light-dependent expression in a C4 plant (maize), but does not confer bundle sheath cell-specific expression

The small subunit of ribulose-bisphosphate carboxylase (Rubisco), encoded by rbcS, is essential for photosynthesis in both C3 and C4 plants, even though the cell specificity of rbcS expression is different between C3 and C4 plants. The C3 rbcS is specifically expressed in mesophyll cells, while the C4 rbcS is expressed in bundle sheath cells, and not mesophyll cells. Two chimeric genes were constructed consisting of the structural gene encoding -glucuronidase (GUS) controlled by the two promoters from maize (C4) and rice (C3) rbcS genes. These constructs were introduced into a C4 plant, maize. Both chimeric genes were specifically expressed in photosynthetic organs, such as leaf blade, but not in non-photosynthetic organs. The expressions of the genes were also regulated by light. However, the rice promoter drove the GUS activity mainly in mesophyll cells and relatively low in bundle sheath cells, while the maize rbcS promoter induced the activity specifically in bundle sheath cells. These results suggest that the rice promoter contains some cis-acting elements responding in an organ-pecific and light-inducible regulation manner in maize but does not contain element(s) for bundle sheath cell-specific expression, while the maize promoter does contain such element(s). Based on this result, we discuss the similarities and differences between the rice (C3) and maize (C4) rbcS promoter in terms of the evolution of the C4 photosynthetic gene.     

Fusions between green fluorescent protein and β-glucuronidase as sensitive and vital bifunctional reporters in plants

By fusing the genes encoding green fluorescent protein (GFP) and -glucuronidase (GUS) we have created a set of bifunctional reporter constructs which are optimized for use in transient and stable expression studies in plants. This approach makes it possible to combine the advantage of GUS, its high sensitivity in histochemical staining, with the advantages of GFP as a vital marker. The fusion proteins were functional in transient expression studies in tobacco using either DNA bombardment or potato virus X as a vector, and in stably transformed Arabidopsis thaliana and Lotus japonicus plants. The results show that high level of expression does not interfere with efficient stable transformation in A. thaliana and L. japonicus. Using confocal laser scanning microscopy we show that the fusion constructs are very suitable for promoter expression studies in all organs of living plants, including root nodules. The use of these reporter constructs in the model legume L. japonicus offers exciting new possibilities for the study of the root nodulation process.     

Arabidopsis thaliana small subunit leader and transit peptide enhance the expression ofBacillus thuringiensis proteins in transgenic plants

The expression of the modified gene for a truncated form of thecryIA(c) gene, encoding the insecticidal portion of the lepidopteran-active CryIA(c) protein fromBacillus thuringiensis var.kurstaki (B.t.k.) HD73, under control of theArabidopsis thaliana ribulose-1,5-bisphosphate carboxylase (Rubisco) small subunitats1A promoter with and without its associated transit peptide was analyzed in transgenic tobacco plants. Examination of leaf tissue revealed that theats1A promoter with its transit peptide sequence fused to the truncated CryIA(c) protein provided a 10-fold to 20-fold increase incryIA(c) mRNA and protein levels compared to gene constructs in which the cauliflower mosaic virus 35S promoter with a duplication of the enhancer region (CaMV-En35S) was used to express the samecryIA(c) gene. Transient expression assays in tobacco protoplasts and the whole plant results support the conclusion that the transit peptide plus untranslated sequences upstream of that region are both required for the increase in expression of the CryIA(c) protein. Furthermore, the CaMV-En35S promoter can be used with theArabidopsis ats1A untranslated leader and transit peptide to increase expression of this protein. While subcellular fractionation revealed that the truncated CryIA(c) protein fused to theats1A transit peptide is located in the chloroplast, the increase in gene expression is independent of targeting of the CryIA(c) protein to the chloroplast. The results reported here provide new insight into the role of 5 untranslated leader sequences and translational fusions to increase heterologous gene expression, and they demonstrate the utility of this approach in the development of insect-resistant crops.     

A novel promoter from soybean that is active in a complex developmental pattern with and without its proximal 650 base pairs

We report the isolation of a novel soybean gene, Msg, which is highly expressed in developing soybean pods. The gene shows significant homology to a family of fruit- and flower-specific genes, designated the major latex protein (MLP) homologues, so far reported in only a few species and whose functions are unknown. The MLPs are more distantly related to a group of pathogenesis-related proteins (IPR or PR-10) whose functions are likewise unknown. This is the first report of a MLP homologue in a plant for which there is already an IPR-protein reported. We performed an analysis of the Msg promoter with 14 different promoter fragments ranging from 0.65 kb to 2.26 kb, fused to the uidA (GUS) gene. High transient expression was obtained with all the constructs upon particle bombardment in soybean and green bean pods. Stable Arabidopsis transformants were obtained with the Agrobacterium vacuum infiltration method. The promoter is fully active in Arabidopsis only in plants transformed with the 2.26 kb fragment promoter, expressing GUS in nectaries, nodes, short style and in guard cells of the silique, pedicel and stem but not in mature leaves. Surprisingly, the proximal 650 bp TATA-containing region cannot function on its own in Arabidopsis and can be deleted without a change in expression pattern in both Arabidopsis and soybean. Thus, tissue-specific regions of the complex Msg promoter reside in the distal 5 regions upstream of a dispensable TATA box in contrast to many examples of tissue-specific elements that reside much closer to the TATA box.     

Definition of constitutive gene expression in plants: the translation initiation factor 4A gene as a model

The NeIF-4A10 gene belongs to a family of at least ten genes, all of which encode closely related isoforms of translation initiation factor 4A. The promoter region of NeIF-4A10 was sequenced, and four mRNA 5 ends were determined. Deletions containing 2750, 689 and 188 bp of untranscribed upstream DNA were fused to the GUS reporter gene and introduced into transgenic tobacco. The three constructs mediated GUS expression in all cells of the leaf, stem and shoot apical meristem. Control experiments using in situ hybridization and tissue printing indicated that the observed GUS expression matches the expression patterns of NeIF-4A mRNA and protein. This detailed analysis at the level of mRNA, protein and reporter gene expression shows that NeIF-4A10 is an ideal constitutively expressed control gene. We argue that inclusion of such a control gene in experiments dealing with specifically expressed genes is in many cases essential for the correct interpretation of observed expression patterns.