A truncated version of an ADP-glucose pyrophosphorylase promoter from potato specifies guard cell-selective expression in transgenic plants.

ADP-glucose pyrophosphorylase (AGPase) is a key regulatory enzyme in starch biosynthesis in higher plants. A 3.2-kb promoter of the large subunit gene of the AGPase from potato has been isolated and its activity analyzed in transgenic potato and tobacco plants using a promoter-beta-glucuronidase fusion system. The promoter was active in various starch-containing cells, including guard cells, tuber parenchyma cells, and the starch sheath layer of stems and petioles. No expression was observed in mesophyll cells. Analysis of various promoter derivatives showed that with respect to expression in petioles and stems, essential elements must be located in the 5' distal region of the promoter, whereas elements important for expression in tuber parenchyma cells are located in an internal fragment comprising nucleotides from positions -500 to -1200. Finally, a 0.3-kb 5' proximal promoter fragment was identified that was sufficient to obtain exclusive expression in guard cells of transgenic potato and tobacco plants. The implications of our observations are discussed with respect to starch synthesis in various tissues and the use of the newly identified promoter as a tool for stomatal biology.     

A/T-rich sequences act as quantitative enhancers of gene expression in transgenic tobacco and potato plants

The role of an A/T-rich positive regulatory region (P268, -444 to -177 from the translation start site) of the pea plastocyanin gene (PetE) promoter has been investigated in transgenic plants containing chimeric promoters fused to the -glucuronidase (GUS) reporter gene. This region enhanced GUS expression in leaves of transgenic tobacco plants when fused in either orientation to a minimal pea PetE promoter (-176 to +4) and in roots when fused in either orientation upstream or downstream of a minimal cauliflower mosaic virus 35S promoter (-90 to +5). The region was also able to enhance GUS expression in microtubers of transgenic potato plants when placed in either orientation upstream of a minimal class I patatin promoter (-332 to +14). Dissection of P268 revealed that cis elements responsible for enhancing GUS expression from the minimal PetE promoter were distributed throughout P268. Multiple copies of a 31 bp A/T-rich sequence from within P268 and of a 26 bp random A/T sequence were able to enhance GUS expression from the minimal PetE promoter, indicating that A/T-rich sequences are able to act as quantitative, non-tissue-specific enhancer elements in higher plants. Abbreviations: CaMV, cauliflower mosaic virus; GUS, -glucuronidase; HMG, high-mobility group; MAR, matrix-associated region; MU, methylumbelliferone; SAR, scaffold-associated region.     

Analysis of Arabidopsis PsbQ A Gene Expression in Transgenic Tobacco Reveals Differential Role of Its Promoter and Transcribed Region in Organ-specific and Light-mediated Regulation

Arabidopsis PsbQ, encoding a 16 kDa protein of the oxygen-evolving complex, is regulated by light and is expressed preferentially in leaf tissues. To analyze the components required for light-regulated and organ-specific expression of PsbQA, several promoter constructs were generated and expressed in tobacco. The 2.2 kb promoter could confer organ-specific expression of the reporter gene, whereas regulatory elements for light-dependent induction could not be located within this promoter and the transcribed region extending up to a second exon, represented by a genomic fragment encompassing the gene. The genomic fragment representing the transcribed region, however, could confer light regulation even on a constitutive promoter, as observed by steady-state mRNA analysis in T0 and T1 tobacco plants. The results obtained have led to the conclusion that regulatory elements for organ-specificity mainly reside in the promoter region whereas the transcribed region of the gene has an important role in light regulation.     

Footprinting of the spinach nitrite reductase gene promoter reveals the preservation of nitrate regulatory elements between fungi and higher plants

Nitrite reductase (NiR) is the second enzyme in the nitrate assimilatory pathway reducing nitrite to ammonium. The expression of the NiR gene is induced upon the addition of nitrate. In an earlier study, a 130 bp upstream region of the spinach NiR gene promoter, located between –330 to –200, was shown to be necessary for nitrate induction of -glucuronidase (GUS) expression in tissue-specific manner in transgenic tobacco plant [28]. To further delineate the cis-acting elements involved in nitrate regulation of NiR gene expression, transgenic tobacco plants were generated with 5 deletions in the–330 to –200 region of the spinach NiR gene promoter fused to the GUS gene. Plants with the NiR promoter deleted to –230 showed a considerable increase in GUS activity in the presence of nitrate, indicating that the 30 bp region between –230 to –200 is crucial for nitrate-regulated expression of NiR. In vivo DMS footprinting of the –300 to –130 region of the NiR promoter in leaf tissues from two independent transgenic lines revealed several nitrate-inducible footprints. Footprinting within the –230 to –181 region revealed factor binding to two adjacent GATA elements separated by 24 bp. This arrangement of GATA elements is analogous to cis-regulatory sequences found in the promoters of nitrate-inducible genes of Neurospora crassa, regulated by the NIT2 Zn-finger protein. The –240 to –110 fragment of the NiR promoter, which contains two NIT2 consensus core elements, bound in vitro to a fusion protein comprising the zinc finger domain of the N. crassa NIT2 protein. The data presented here show that nitrate-inducible expression of the NiR gene is mediated by nitrate-specific binding of trans-acting factors to sequences preserved between fungi and higher plants.     

A promoter derived from taro bacilliform badnavirus drives strong expression in transgenic banana and tobacco plants

Taro bacilliform virus (TaBV) is a pararetrovirus of the genus Badnavirus which infects the monocotyledonous plant, taro ( Colocasia esculenta). A region of the TaBV genome spanning nucleotides 6,281 to 12 (T1200), including the 3' end of open reading frame 3 (ORF 3) and the intergenic region to the end of the tRNA(met)-binding site, was tested for promoter activity along with four different 5' deletion fragments (T600, T500, T250 and T100). In transient assays, only the T1200, T600, T500 fragments were shown to have promoter activity in taro leaf, banana suspension cells and tobacco callus. When these three promoters were evaluated in stably transformed, in vitro-grown transgenic banana and tobacco plants, all were found to drive near-constitutive expression of either the green fluorescent protein or beta-glucuronidase (GUS) reporter gene in the stem (or pseudostem), leaves and roots, with strongest expression observed in the vascular tissue. In transgenic banana leaves, the T600 promoter directed four-fold greater GUS activity than that of the T1200, T500 and the maize polyubiquitin-1 promoters. In transgenic tobacco leaves, the levels of GUS expression directed by the three promoters was between four- and ten-fold lower than that of the double Cauliflower mosaic virus 35S promoter. These results indicate that the TaBV-derived promoters may be useful for the high-level constitutive expression of transgenes in either monocotyledonous or dicotyledonous species.     

Fruit developmental regulation of the kiwifruit actinidin promoter is conserved in transgenic petunia plants

We have examined the expression of actinidin, a cysteine protease found in kiwifruit, over the course of fruit development. Protease activity was first seen in fruit that had reached about half their final weight, and rose to high levels at harvest. The 5-flanking region (nucleotides –1301 to +58) of a kiwifruit actinidin gene was fused to the -glucuronidase (GUS)-coding region, and the chimaeric gene was introduced into transgenic petunia plants. Induction of the GUS gene was observed during the later stages of seed pod development, closely resembling the pattern of actinidin induction in fruit tissues of kiwifruit. Some GUS expression was also detected in the vascular system of the receptacle, leaves, stems and roots. A shorter promoter fragment consisting of nucleotides –115 to +58 conferred similar spatial and temporal regulation in some of the transgenic plants.     

Fluorescence microscopy: a powerful technique to detect low GUS activity in vascular tissues

We have previously shown that the Eucalyptus gunnii EgCAD2 promoter was preferentially expressed in vascular tissues in different transgenic plants (poplar, tobacco, Arabidopsis and grapevine). In order to delineate the cis elements governing this vascular expression pattern, promoter deletion analysis was performed allowing us to identify the proximal region [-340/-124] as essential for vascular cambium/xylem-specific expression. In plants transformed with the smallest promoter region [-124/+117], the GUS activity was difficult to detect using conventional bright field microscopy. To overcome this problem, we used fluorescence microscopy, enabling us to show that the [-124/+117] region contained cis-elements driving activity in phloem fibres but not in secondary xylem. The technical improvement of the histochemical detection of GUS activity using fluorescence microscopy enables accurate investigation of low GUS activity in phenol-rich tissues.     

A Comparison of Constitutive Promoters for Expression of Transgenes in Alfalfa (Medicago Sativa)

The activity of constitutive promoters was compared in transgenic alfalfa plants using two marker genes. Three promoters, the 35S promoter from cauliflower mosaic virus (CaMV), the cassava vein mosaic virus (CsVMV) promoter, and the sugarcane bacilliform badnavirus (ScBV) promoter were each fused to the beta-glucuronidase (gusA) gene. The highest GUS enzyme activity was obtained using the CsVMV promoter and all alfalfa cells assayed by in situ staining had high levels of enzyme activity. The 35S promoter was expressed in leaves, roots, and stems at moderate levels, but the promoter was not active in stem pith cells, root cortical cells, or in the symbiotic zones of nodules. The ScBV promoter was active primarily in vascular tissues throughout the plant. In leaves, GUS activity driven by the CsVMV promoter was approximately 24-fold greater than the activity from the 35S promoter and 38-fold greater than the activity from the ScBV promoter. Five promoters, the double 35S promoter, figwort mosaic virus (FMV) promoter, CsVMV promoter, ScBV promoter, and alfalfa small subunit Rubisco (RbcS) promoter were used to control expression of a cDNA from Trichoderma atroviride encoding an endochitinase (ech42). Highest chitinase activity in leaves, roots, and root nodules was obtained in plants containing the CsVMV:ech42 transgene. Plants expressing the endochitinase were challenged with Phoma medicaginis var. medicaginis, the causal agent of spring black stem and leaf spot of alfalfa. Although endochitinase activity in leaves of transgenic plants was 50- to 2650-fold greater than activity in control plants, none of the transgenic plants showed a consistent increase in disease resistance compared to controls. The high constitutive levels of both GUS and endochitinase activity obtained demonstrate that the CsVMV promoter is useful for high-level transgene expression in alfalfa.     

Analysis of regulatory elements involved in stress-induced and organ-specific expression of tobacco acidic and basic β-1,3-glucanase genes

Infection of tobacco by tobacco mosaic virus (TMV) induces coordinate expression of genes encoding acidic and basic -1,3-glucanase isoforms. These genes are differentially expressed in response to other treatments. Salicylate treatment induces acidic glucanase mRNA to a higher level than basic glucanase mRNA. Ethylene treatment and wounding strongly induce the basic glucanase genes but have little effect on genes encoding the acidic isoforms. Furthermore, the basic glucanase genes are constitutively expressed in roots and lower leaves of healthy plants, whereas the acidic glucanase genes are not. In order to investigate how these expression patterns are established, we fused promoter regions of an acidic and a basic glucanase gene to the -glucuronidase (GUS) reporter gene and examined expression of these constructs in transgenic tobacco plants.A fragment of 1750 bp and two 5-truncated fragments of 650 bp and 300 bp of the acidic glucanase promoter were tested for induction of GUS gene expression after salicylate treatment and TMV infection. Upstream sequences of 1750 bp and 650 bp were sufficient for induction of the reporter gene by salicylate treatment and TMV infection, but the activity of the 300 bp fragment was strongly reduced. The results suggest that the 1750 bp upstream sequence of the acidic glucanase gene contains multiple regulatory elements.For the basic glucanase promoter it is shown that 1476 bp of upstream sequences were able to drive expression in response to TMV infection and ethylene treatment, but no response was found to incision wounding. Furthermore, high GUS activity was found in lower leaves and roots of healthy transgenic plants, carrying the 1476 bp basic glucanase promoter/GUS construct. When the promoter was truncated up to position –446 all activity was lost, indicating that the region between –1476 and –446 of the basic glucanase promoter is necessary for organ-specific and developmentally regulated expression as well as for induced expression in response to infection and other stress treatments.     

Tissue-specific and wound-inducible pattern of expression of the mannopine synthase promoter is determined by the interaction between positive and negative cis-regulatory elements

In transgenic tobacco plants the regulatory region of the Agrobacterium genes involved in mannopine synthesis ( pmas ) directs expression in vascular tissues and is induced upon wounding. To identify cis -acting sequences required for the cell-type-specific and wound-inducible expression of this regulatory region, the expression pattern of chimeric genes in which 5' upstream deletions of the pmas1' promoter direct the expression of the β-glucuronidase coding sequence, was analyzed in transgenic tobacco plants. It was found that the pmas1' promoter is specifically expressed in phloem cells and that this tissue-specific pattern of expression is the result of the interaction of at least two negative regulatory elements with a positive element that directs expression in many cell types. It was also found that wound induction is mediated by two different mechanisms, one of them involving a dramatic change in tissue-specific expression.