The effects of promoter on transient expression in conifer cell lines

Summary Protoplasts from suspension cultures of somatic embryos of white spruce (Picea glauca Moench Voss) were electroporated with plasmids containing the chimeric genes for chloramphenicol acetyl transferase (CAT) or -glucuronidase (GUS), under control of one of three promoters. Transient CAT gene expression of approximately equal magnitude resulted when the CAT gene was fused to either the cauliflower mosaic virus (CaMV) 35S promoter or the nopaline synthase (NOS) promoter. When the CAT gene was fused to a tandem repeat CaMV 35S promoter (pPBI-363), CAT enzyme activity compared to NOS or 35S promoters increased up to eightfold (cell line WS-34), and were up to 100-fold greater than control (electroporated without plasmid). Comparatively, protoplasts of black spruce (Picea mariana Mill) and jack pine (Pinus banksiana Lamb.), electroporated with pPBI-363, produced increases in CAT activity compared to control of 90-fold and 70-fold, respectively. White spruce (WS-34) protoplasts were subsequently electroporated with the GUS gene fused to the tandem repeat CaMV 35S promoter. Comparatively, GUS enzyme activity increased up to tenfold compared to GUS fused to a CaMV 35S promoter. The results indicated that transient expression of the CAT and GUS genes was influenced by the type of promoter and cell line used, as well as by electroporation conditions.NRCC No. 30498     

Transient gene expression in electroporated Solanum protoplasts

Electroporation was used to evaluate parameters important in transient gene expression in potato protoplasts. The protoplasts were from leaves of wild potato Solanum brevidens, and from leaves, tubers and suspension cells of cultivated Solanum tuberosum cv. Désirée. Reporter enzyme activity, chloramphenicol acetyl transferase (CAT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter, depended on the field strength and the pulse duration used for electroporation. Using field pulses of 85 ms duration, the optimum field strengths for maximum CAT activity were: S. brevidens mesophyll protoplasts –250 V/cm; Désirée mesophyll protoplasts –225 V/cm; Désirée suspension culture protoplasts –225 V/cm; and Désirée tuber protoplasts –150 V/cm. The optimum field strengths correlated inversely with the size of the protoplasts electroporated; this is consistent with biophysical theory. In time courses, maximum CAT activity (in Désirée mesophyll protoplasts) occurred 36–48 h after electroporation. Examination at optimised conditions of a chimaeric gene consisting of a class II patatin promoter linked to the -glucuronidase (gus) gene, showed expression (at DNA concentrations between 0–10 pmol/ml) comparable to the CaMV 35S promoter in both tuber and mesophyll protoplasts. At higher DNA concentrations (20–30 pmol/ml) the patatin promoter directed 4–5 times higher levels of gus expression. Implications and potential contributions towards studying gene expression, in particular of homologous genes in potato, are discussed.     

Factors affecting transient gene expression in electroporated Glycine max protoplasts

Electroporation was used to evaluate parameters affecting transient gene expression in Glycine max protoplasts. Protoplast viability and reporter enzyme activity for chloramphenicol acetyl transferase (CAT) and ß-glucuronidase (GUS) depended on the field strength employed. Maximum CAT and GUS activity was obtained when a field strength of 500 V/cm at 1000 F and a protoplast concentration of 1–3 × 10⁶/ml was used. Transformation efficiencies up to approximately 1.6% GUS positive protoplasts were obtained. Transient gene expression increased with increasing plasmid DNA concentration and with the time after electroporation, reaching a maximum after 48 hr. Addition of polyethylene glycol at 5.6% and heat shock (5 rain at 45 °C) given to the protoplasts before adding DNA further enhanced the transformation efficiency. Under the optimized experimental conditions, CAT and GUS activity increased simultaneously, thereby indicating that the increased expression is caused by DNA uptake by more cells rather than greater DNA uptake by the same cells. Our results demonstrate that both GUS and CAT can be used as efficient screenable markers for transformation studies in soybean.Abbreviations CAT chloramphenicol acetyl transferase - GUS ß-glucuronidase - PEG polyethylene glycol     

Antisense regulation of the rice waxy gene expression using a PCR-amplified fragment of the rice genome reduces the amylose content in grain starch

The waxy gene encodes a granule-bound starch synthase. A 1.0-kb portion of the sequence of the rice waxy gene, which includes the region between exon 4 and exon 9, was inserted in an antisense orientation between the 35 S promoter and the GUS gene of pBI221. The resultant plasmid, pWXA23, was introduced into rice protoplasts by electroporation. GUS activity was clearly detected in derived callus lines, suggesting that the antisense component of the fusion gene was also expressed. Transgenic rice plants were regenerated from these callus lines and their GUS activity was confirmed. Some of the rice seeds from these transformants showed a significant reduction in the amylose content of grain starch, even though they had become polyploid. These results suggest that even when intron sequences are included, antisense constructs can bring about a reduced level of expression of a target gene. The utility of GUS, included as a reporter gene, for the simple detection of expression of an antisense gene, was apparent from these results.     

Genotype- and promoter-induced variability in transient β-glucuronidase expression in pea protoplasts

Summary Leaf mesophyll protoplasts isolated from pea (Pisum sativum L.) genotypes Century and PI244253 showed transient expression of -glucuronidase (GUS) when electroporated with plasmid DNA containing various promoter-leader sequence constructs driving the GUS gene. The optimum conditions for transient expression were: using protoplasts isolated from leaf material that had been kept in the dark for 90 h; electroporating at 250 V and 960 F; and using 125 g of calf thymus carrier DNA and 75 of plasmid DNA. PI244253 had 5 to 20 times the GUS activity levels of Century. Similar levels of transient expression were obtained using either the nopaline synthase or cauliflower mosaic virus 35S (35S) promoters. These levels were lower than that obtained using a duplicated 35S promoter derivative. The presence of an untranslated coat protein mRNA leader sequence from alfalfa mosaic virus between each promoter and the GUS gene resulted in increased GUS activity. Leaf mesophyll protoplasts and root protoplasts of PI244253 did not differ in levels of transient expression.NRCC No. 30910     

Factors affecting transient gene expression in protoplasts isolated from very slowly growing embryogenic callus cultures of wheat (Triticum aestivum L.)

Protoplasts isolated from embryogenic (Mustang and Chinese Spring) and non-embryogenic (Mit) calli of wheat (Triticum aestivum L.) genotypes transiently expressed -glucuronidase (GUS) activity when electroporated with a plasmid containing the GUS gene and driven by an enhanced 35S promoter and a TMV leader sequence. Conditions for the maximum expression of GUS activity were: electroporation of the freshly isolated protoplasts at 250 Vcm^-1 and 250 F for 2 s using 50 g/ml of plasmid DNA; incubation of the protoplasts with the plasmid before the pulse for 2 h; and a 15-min recovery period on ice after the pulse. In general, a higher GUS activity was obtained in protoplasts of non-embryogenic (NE) callus origin than in those of embryogenic (E) callus origin. Only GUS constructs containing a duplicate 35S promoter derivative resulted in a significant level of GUS expression. The presence of the TMV viral leader sequence in the pAGUS1-TN2 plasmid construct resulted in a significant increase of GUS activity in the electroporated protoplasts of both callus types. On the other hand, protoplasts electroporated with the Adh1 promoter and intron showed a threefold less GUS activity than those electroporated with pAGUS1-TN2. Optimized conditions for DNA uptake and expression were very similar for protoplasts of both callus types. The importance of these findings for the successful regeneration of transgenic and fertile wheat plants is discussed.     

Regulation of the maizerab17 gene promoter in transgenic heterologous systems

The maizerab17 gene is expressed in different plant parts in response to ABA and osmotic stress (J. Vilardellet al., Plant Mol Biol 14 (1990) 423–432). Here we demonstrate that 5 upstream sequences of therab17 gene confer the appropriate patterns of expression on the chloramphenicol acetyl transferase (CAT) reporter gene in transgenic tobacco plants, as well as in protoplasts derived from cultured rice cells. Specifically, a CAT construct containing a large 5 upstream fragment ofrab17 (–1330/+29) results in high levels of CAT activity in embryos, leaves and roots of transgenic plants subjected to water stress or ABA treatment. Transient expression assays in rice protoplasts transfected with CAT genes fused torab17 promoter deletions indicate that a 300 bp DNA fragment (–351/–102) is sufficient to confer ABA responsiveness upon the reporter gene. Furthermore, a 100 bp sequence (–219/–102) is capable of conferring ABA responsiveness upon a minimal promoter derived from the 35S CaMV promoter. Gel retardation experiments indicate that maize nuclear proteins bind to this fragment. This region of 100 bp contains a sequence (ACGTGGC) which has been identified as an abscisic acid response element in studies of other ABA-responsive plant genes.     

The sequence surrounding the translation initiation codon of the pea plastocyanin gene increases translational efficiency of a reporter gene

The 5-upstream region of the pea plastocyanin gene (petE) directed 5–10-fold higher levels of -glucuronidase (GUS) activity than the cauliflower mosaic virus 35S promoter in transgenic tobacco plants, although the levels of GUS mRNA were similar. The sequence (AAAAAUGG) around the translation initiation codon of petE enhanced translation of the GUS mRNA 10-fold compared to translation from the GUS translation initiation codon in transgenic tobacco plants and transfected protoplasts.     

Effect of deletions in the cauliflower mosaic virus polyadenylation sequence on the choice of the polyadenylation sites in tobacco protoplasts

Summary Deletions were made in the cauliflower mosaic virus polyadenylation sequence which was cloned downstream of the -glucuronidase gene (gus). The populations of mRNAs generated in tobacco mesophyll protoplasts by transient expression with the various constructs were analysed using a polymerase chain reaction procedure. When no deletion was present in the sequence, the mRNA appeared to be polyadenylated at two major polyadenylation sites. A deletion upstream from the AATAAA sequence made the population of polyadenylated mRNAs very heterogenous at their 3 ends. A deletion downstream of the AATAAA sequence had no effect on the choice of the site. Alternative polyadenylation sites were used when the native polyadenylation site was deleted. These results are discussed in relation to data obtained with other polyadenylation sequences from both plants and animals.     

Mitochondrial and chloroplast targeting sequences in tandem modify protein import specificity in plant organelles

Protein targeting to plant mitochondria and chloroplasts is usually very specific and involves targeting sequences located at the amino terminus of the precursor. We challenged the system by using combinations of mitochondrial and chloroplast targeting sequences attached to reporter genes. The sequences coding for the presequence of the mitochondrial F₁-ATPase -subunit and the transit peptide of the chloroplast chlorophyll a/b-binding protein, both from Nicotiana plumbaginifolia, were fused together in both combinations, then linked to the reporter genes, chloramphenicol acetyl transferase (CAT) and -glucuronidase (GUS), and introduced into tobacco. Analysis of CAT and GUS activities and proteins in the subcellular fractions revealed that the chloroplast transit peptide alone was not sufficient to target the reporter proteins to chloroplasts. However, when the mitochondrial -presequence was inserted downstream of the chloroplast sequence, import of CAT and GUS into chloroplasts was observed. Using the reciprocal system, the mitochondrial presequence alone was able to direct transport of CAT and, to a lesser extent, GUS to mitochondria; the GUS targeting to mitochondria was increased when the chloroplast targeting sequence was linked downstream of the mitochondrial presequence. Immuno-detection experiments using subcellular fractions confirmed the results observed by enzymatic assays. These results indicate the importance of the amino-terminal position of the targeting sequence in determining protein import specificity and are considered within the hypothesis of a co-translational protein import.     

Efficiency of physical (light) or chemical (ABA,tetracycline, CuSO4 or 2‐CBSU)‐stimulus‐dependent gus gene expression in tobacco cell suspensions

In this study, the efficiency of inducible promoters to switch on gene expression in the presence of inducer or to switch it off in its absence was evaluated in tobacco cell suspensions transformed with the gus gene coding sequence. Either plant (pats1A, pSalT, pIn2‐2) or microbial (pMre, pTet) inducible promoters were used to drive gus expression. The inducers were light, abscisic acid, 2‐CBSU, CuSO₄, tetracycline, respectively. For each construct (inducible promoter‐gus coding sequence), the optimal induction conditions were determined (inducer concentration, induction time, and age of cells in culture cycle before induction). The efficiency of the inducible promoter was then evaluated under optimal induction conditions. GUS‐expression levels obtained under non‐inducing and inducing conditons were systematically compared. Thirty or forty percent of the clones transformed with the pSalT‐gus or pTet‐gus construct, respectively, showed high induction rates (>1000) and GUS activities of the same order as those obtained with a constitutive system. However, basal GUS levels were always high for the pTet‐gus cell lines. Seventy or eighty‐five percent of the cell lines transformed with the pMre‐gus or pln2‐2‐gus construct, respectively, had induction rates of 1.5 to 1000. The pats1A‐gus construct gave very low induction rates—55% of cell lines had induction rates less than 1.5. Only the pSalt‐gus construct gave both the highest induction rates and basal GUS‐levels equivalent to the endogenous GUS background. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 1–13, 1999.     

Cotton α-Globulin Promoter: Isolation and Functional Characterization in Transgenic Cotton,Arabidopsis, and Tobacco

Globulins are the most abundant seed storage proteins in cotton and, therefore, their regulatory sequences could potentially provide a good source of seed-specific promoters. We isolated the putative promoter region of cotton -globulin B gene by gene walking using the primers designed from a cotton staged embryo cDNA clone. PCR amplified fragment of 1108 bp upstream sequences was fused to gusA gene in the binary vector pBI101.3 to create the test construct. This was used to study the expression pattern of the putative promoter region in transgenic cotton, Arabidopsis, and tobacco. Histochemical GUS analysis revealed that the promoter began to express during the torpedo stage of seed development in tobacco and Arabidopsis, and during cotyledon expansion stage in cotton. The activity quickly increased until embryo maturation in all three species. Fluorometric GUS analysis showed that the promoter expression started at 12 and 15 dpa in tobacco and cotton, respectively, and increased through seed maturation. The strength of the promoter expression, as reflected by average GUS activity in the seeds from primary transgenic plants, was vastly different amongst the three species tested. In Arabidopsis, the activity was 16.7% and in tobacco it was less than 1% of the levels detected in cotton seeds. In germinating seedlings of tobacco and Arabidopsis, GUS activity diminished until it was completely absent 10 days post imbibition. In addition, absence of detectable level of GUS expression in stem, leaf, root, pollen, and floral bud of transgenic cotton confirmed that the promoter is highly seed-specific. Analysis of GUS activity at individual seed level in cotton showed a gene dose effect reflecting their homozygous or hemizygous status. Our results show that this promoter is highly tissue-specific and it can be used to control transgene expression in dicot seeds.     

Timing of Transposition of Ac Mobile Element in Potato

The timing of excision of maize transposable element Ac was studied using visual histochemical assay based on Ac excision restoring activity of -glucuronidase (GUS). The Solanum tuberosum L. cv. Bintje was used for Agrobacterium-mediated transformation with pTT230 plasmid harbouring Ac-interrupted gus A gene and npt II gene as a selectable marker gene. Twenty-eight out of 72 kanamycin resistant calli did not express any GUS activity, 31 calli showed partial GUS expression and 13 out of assayed calli revealed strong expression of gus A gene. Plants were regenerated from calli without and/or with partial expression of gus A gene. The regenerated transformants which did not express GUS during the callus phase often contained many small GUS expressing spots on leaves. A phenotypic selection assay for excision of Ac has been also used. This non-detectable excision of Ac in callus tissue could be followed by a "late" timing excision during leaf development. After transformation with pTT224 plasmid harbouring Ac-interrupted hpt II gene and npt II gene transgenic calli containing Ac within the hygromycin resistance gene were derived and hygromycin sensitive plants were regenerated from them. Protoplasts isolated from leaves of transgenic regenerated plants were selected on hygromycin. Hygromycin resistant minicalli showed to harbour multiple copies of Ac and mark out low uniqueness of integration sites.     

Tagging genomic sequences that direct transgene expression by activation of a promoter trap in plants

As part of a gene tagging strategy to study the developmental regulation of patterns of plant gene expression, a promoterlessuidA (gus A) gene, encoding the -glucuronidase (GUS) reporter, was introduced into populations of tobacco,Arbidopsis and potato byAgrobacterium-mediated gene transfer. The objective was to generate random functional fusions following integration of thegusA gene downstream of native gene promoters. We describe here a detailed analysis of levels and patterns ofgusA activation in diverse organs and cell types in those populations.gusA activation occurred at high frequency in all three species, and unique patterns of fusion gene expression were found in each transgenic line. The frequency ofgusA activation was differentially blased in different organs in the three species. Fusion gene activity was identified in a wide range of cell types in all organs studied, and expression patterns were stably transmissible to the T₂ and T₃ progeny. Developmentally-regulated and environmentally-inducible expression ofgusA is described for one transgenic line. Phenotypic variants were detected in the transgenic population. These results demonstrate the potential of T-DNA insertion as a means of creating functional tags of genes expressed in a wide spectrum of cell types, and the value of the approach as a complement to standard T-DNA insertional mutagenesis and transposon tagging for developmental studies is discussed.     

The Expression of a Cytosolic Cyclophilin Promoter from Periwinkle in Transgenic Tobacco Plants

The cloning of a 465 bp fragment from the 5-flanking region of the gene encoding a cytosolic cyclophilin from periwinkle was achieved through inverse polymerase chain reaction. The DNA fragment was fused to a gusA-intron marker then introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. Histochemical analysis of the transgenic shoot cultures demonstrated that the construct was able to drive GUS expression in stomata guard cells, but not in mesophyll cells when shoots were still attached to the callus from which they were initiated. In separated transgenic shoots and in seedlings, GUS was expressed in external and internal phloem and root hairs, respectively. GUS activity in transgenic tobacco seedlings was also investigated by fluorimetric assays. Treatments with NaCl or ABA decreased promoter activity whereas treatment with yeast extracts increased it.     

Chimeric gene expression using maize intron in cultured cells of breadwheat

High voltage electrical pulses were used to introduce the CAT reporter gene into cultured protoplasts of breadwheat,Triticum aestivum. Four DNA constructs harboring the CAT gene and the 35S or mannipine synthase promoter were tested for levels of CAT activity 40–45 hr after electroporation of protoplasts. One construct, containing a maize intron sequence between 35S and CAT sequences, conferred 30 to 185 fold greater CAT activity over the other three constructs. Data from these experiments suggest that a maize intron or sequences with similar effects may be required in DNA constructs for efficient heterologous gene expression in cultured cells of breadwheat.Abbreviations CAT Chloramphenicol acetyl transferase - NPT II neomycin phosphotransferase - 35S the 35S promoter of Cauliflower Mosaic Virus - PEG Polyethylene glycol - MES 2-[N-morpholino] ethanesulfonic acid     

Efficient transient expression and transformation of PEG-mediated gene uptake into mesophyll protoplasts of pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.)

PEG-mediated transformation was used for gene delivery and evaluation of various parameters affecting the transient expression of a gene for ß-glucuronidase (gus) in mesophyll protoplasts of Capsicum annuum. Transient expression was found to be dependent on PEG concentration and exposure time of plasmid DNA to protoplasts as well as the amount of plasmid DNA. Maximum GUS activity was obtained when protoplasts were applied to 40% concentration and molecular weight was 6,000 of PEG solution with 30 min of exposure time. Protoplasts of pepper were transformed with a vector, pCAMBIA::Ac, which contained a pCAMBIA1302 T-DNA vector carrying a maize transposable element, Ac (activator), a selection marker HPT (hygromycin phosphotransferase), and a GFP-coding region driven by the 35S promoter in the presence of PEG. Approximately 30% of the protoplasts expressed GFP. Visibly transformed colonies were obtained from protoplasts after 2 months of culture and GFP was expressed. Southern hybridization confirmed the presence of Ac in the pepper genome.