Gene regulation in intertypic heterokaryons of Solanum tuberosum and Nicotiana tabacum tissue protoplasts

Activities of the ß-glucuronidase (GUS) reporter enzyme were evaluated in transgenic plants, protoplasts, and intertypic heterokaryons of Solanum tuberosum and Nicotiana tabacum. With GUS under control of the promoter of the cauliflower-mosaicvirus 35S RNA gene (CaMV), activities of the enzyme were nearly evenly distributed over the tissues of plants grown in vitro. Activities in microtubers of potato plants with the reporterenzyme under control of the promoter of granule-bound starch synthase (GBSS) from S. tuberosum were higher than in leaves. The CaMV-GUS construct present in leaf protoplasts showed an increased expression in biochemical and cytochemical assays after fusion with wildtype-tuber protoplasts. Such an increase was not observed in the case of the GBSS-GUS constructs. It was concluded that nuclei in plant heterokaryons are being influenced by the fusion partner, but that repressive, trans-acting regulation factors in leaf protoplasts possibly prevent an increase of the expression of the chimeric GBSS-gene in heterokaryons.     

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     

Study on Regulation of 5′ Flanking Regions of Granule-bound Starch Synthase Gene of Potato

Binary vectors were constructed by fusing 0.4, 0.8, 1.6, and 2.9 kb 5' flanking regions of GBSS gene with GUS (β-glucuronidase). Transient GUS expression was observed in in vitro tuber slices bombarded with 0.8 kb GBSS-GUS construct. These constructs were then transferred into potato ( Solanum tuberosum L. cv. Desiree) via Agrobacterium tumefaciens transformation. Transgenic potato plants were confmned by X-Gluc staining and PCR. Using in vitro tuberization system, GUS expressions were assayed with fluorescence, it was shown that 0.8, 1.6 and 2.9 kb GBSS-GUS expressions were higher than 0.4 kb GBSS-GUS. 1.6 and 2.9 kb GBSS-GUS expressions were about 2 to 10-folds higher in tubers than in stems. In cultured shoots, GBSS-GUS expression could be induced by increased sucrose concentration but inhibited by light.     

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     

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.     

Intron position affects expression from thetpi promoter in rice

A series of promoter-GUS fusion constructs containing a portion of the rice triosephosphate isomerase (tpi) promoter, the firsttpi intron, and the gene encoding bacterial -glucuronidase (GUS) were made. These constructs were electroporated into rice protoplasts and transient expression was monitored. Inclusion of the first intron from the ricetpi gene enhanced expression of the GUS gene from thetpi promoter when it was placed 5 of the GUS gene. When thetpi intron was placed in the 3-untranslated region no enhancement of GUS gene expression was observed, indicating the importance of position in intron-mediated enhancement of gene expression.     

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.     

Reporter gene introduction and transient expression in protoplasts of Porphyra yezoensis

The transient expression of foreign genes in the protoplasts of Porphyrayezoensis was examined using three recombinant vectors, pYez-Rub-GUS, pYez-Rub-GFP and pYez-Rub-LUC, which were constructed with the promoter sequence of the ribulose-bisphosphate-carboxylase / oxygenase (Rubisco) gene as a promoter and the bacterial β-glucuronidase (GUS), mutant of green fluorescent protein (S65T-GFP) and firefly luciferase (LUC) genes, respectively, as reporter genes. When the pYez-Rub-GUS was introduced into protoplasts by electroporation, cells stained dark blue by indigotin were observed after the histochemical GUS assay. GUS activity was also detected by quantitative enzyme assays with a chemiluminescent substrate. When the pYez-Rub-GFP was electroporated into protoplasts, the expression of GFP could be detected in vivo observations with fluorescence microscopy. However, the rates of gene expression cells to the total number of cells were different between the GUS and GFP genes. LUC activity was also detected by assay with a chemiluminescent substrate after the introduction of pYez-Rub-LUC into protoplasts, although the activity levels were considerably lower. Relatively high expression rates of introduced GUS genes were observed 3 to 5 days after electroporation. These results show that the promoter sequence of the chloroplast Rubisco gene functions as a promoter of foreign gene expression and that transient expression occurred in protoplasts of P. yezoensis after the introduction of foreign genes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Sucrose-inducible expression of hepatitis B surface antigen using potato granule-bound starch synthase promoter

NT-1 cells of tobacco (Nicotiana tabacum L.) were transformed with pGBSSHBS and pGBSSHER expression cassettes wherein expression of hepatitis B surface antigen (HBsAg) was driven by potato granule-bound starch synthase (GBSS) promoter. The transformed nature of the cells was confirmed by PCR analysis. Expression of HBsAg was confirmed by RT-PCR and Western blotting and levels of expression were assayed by ELISA. Transformed cell lines exhibited a sucrose-inducible pattern of HBsAg expression. NT-1 medium supplemented with 175 mmol L⁻¹ sucrose gave the highest HBsAg expression of 198 ng g⁻¹ FW after 8 days of induction. Different sugars, for example glucose, fructose, and palatinose, were also tested to study the inducible nature of GBSS promoter. The results demonstrate that potato GBSS promoter can be used in heterologous host systems like tobacco NT-1 cell suspension cultures for sucrose-inducible expression of recombinant proteins.     

The promoter of barley trypsin-inhibitor BTI-CMe,discriminates between wheat and barley endosperm protoplasts in transient expression assays

Several promoter fragments from the barley gene coding for trypsin inhibitor, BTI-CMe, have been fused to the -glucuronidase (GUS) reporter gene and these chimeric constructs used for transient expression in protoplasts. Transfection of developing endosperm protoplasts from barley (cv Bomi) show a maximum GUS expression of about 50% of that driven by the cauliflower mosaic virus 35S promoter, while in wheat endosperm protoplasts expression is less than 10%. No significant expression is found in transfected leaf protoplasts from barley, wheat or tobacco (<2% of the 35S control). All the information required for endosperm and barley specificity is present in the 343 bp proximal to the translation initiation site.Abbreviations MS Murashige and Skoog medium - PEG polyethyleneglycol - GUS -glucuronidase - MU methylumbelliferone - MUG 4-methylumbelliferyl--D glucuronide - pp protoplasts     

Introduction and expression of foreign DNA in isolated spinach chloroplasts by electroporation

An electroporation-mediated method for the study of foreign gene expression within chloroplasts has been developed. The chloroplast expression vector pHD203-GUS, which consists of coding regions for β-glucuronidase (GUS) and chloramphenicol acetyltransferase (CAT) separated by a double psbA promoter fragment from pea (in opposite orientation) was electroporated into spinach chloroplasts and the transient gene expression was examined. Conditions for the expression of the reporter genes have been optimized. Both CAT and GUS activities were detected in chloroplasts electroporated with pHD203-GUS, but not with nuclear expression vector pBI221 or negative control pUC18. No GUS activity was detected when pHD203-GUS was electroporated into spinach protoplasts. Dot immunoblot analysis using anti-GUS antibody confirmed the existence of GUS protein in chloroplasts electroporated with chloroplast-specific vector but not the negative controls, excluding the possibilities of endogenous GUS or bacterial contamination. The expression of GUS protein in treated chloroplasts was further confirmed by Western blot analysis.     

Characterization of the regulatory elements of the maize P-rr gene by transient expression assays

The maize P-rr gene conditions floral-specific flavonoid pigmentation, especially in the kernel pericarp and cob. We analyzed the P-rr promoter by transient expression assays, in which segments of the P-rr promoter were fused to the GUS reporter gene and introduced into maize cells by particle bombardment. A basal P-rr promoter fragment (–235 to +326) gave low, but significant, levels of GUS reporter gene expression. Interestingly, two widely spaced segments containing enhancer-like activity were found. When tested individually, both the proximal (–1252 to –236) and distal (–6110 to –4842) segments boosted expression of the basal P-rr promoter::GUS construct about five-fold. A 1.6 kb segment of the P-rr promoter (–1252 to +326) containing the proximal enhancer and the 5-untranslated leader driving the GUS reporter gene showed preferential expression in BMS and embryogenic suspension cell cultures vs. endosperm-derived suspension cell cultures. These results demonstrate the application of transient assay techniques for the identification of regulatory elements responsible for floral-specific regulation of the complex P-rr gene promoter in maize.     

Green fluorescent protein: an in vivo reporter of plant gene expression

Summary Protoplasts were isolated from H89, an embryogenic sweet orange (Citrus sinensis (L.) Osbeck cv. Hamlin) suspension culture, and electroporated with p35S-GFP, a plasmid carrying the gene for the green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoria. p35S-GFP was constructed by replacing the GUS coding sequence of pBI221 with a functional GFP gene, thereby placing the GFP gene under the control of the CaMV 35S promoter. Protoplasts were viewed by incident-light fluorescence microscopy twentyfour h after electroporation. 20–60% of the protoplasts emitted an intense green light when illuminated with blue (450–490 nm) light.Abbreviations GUS -glucuronidase - LUC luciferase - NPTII neomycinphosphotransferase - CaMV cauliflower mosaic virus - MUG 4-methylumbelliferyl -D-glucuronide     

β-glucuronidase gene expression and mRNA stability in oat protoplasts

Summary Protoplasts derived from oat (Avena sativa L.) suspension culture cells (7 days after subculturing) were electroporated with plasmid DNA containing the Escherichia coli uidA gene encoding the ß-glucuronidase reporter enzyme. Consistently high enzyme activity was observed with electroporation conditions of 500 F and 1125 volts/cm. Enzyme activity and mRNA accumulation time courses were determined. The maximum enzyme activity was detected at 24 hours after electroporation, while the maximum mRNA level was detected at 12 hours after electroporation. ß-glucuronidase mRNA was in vitro synthesized with and without a 5 methylated cap and then electroporated into protoplasts. Only capped mRNA produced significant enzyme activity. By electroporating radiolabeled, in vitro synthesized mRNA, the ß-glucuronidase mRNA half-life was estimated to be 35 minutes in oat protoplasts.Abbreviations GUS ß-glucuronidase - mRNA messenger RNA - ICP insecticidal crystal proteins - OCS octopine synthase - CAT chloramphenicol acetyltransferase - nt nucleotide - kb kilobase - MSOD3 Murashige and Skoog media with zero 2,4-dichlorophenoxy acetic acid and 3% sucrose - MU 4-methyl umbelliferone; ATA: aurintricarboxylic acid     

Direct gene transfer to protoplasts of two genotypes of Asparagus officinalis L. by electroporation

Callus-derived protoplasts of two genotypes of asparagus (Asparagus officinalis L.) were electroporated to introduce the -glucuronidase gene (GUS). The level of GUS transient gene expression and the viability of the protoplasts were influenced by the voltage and duration of the electric pulse. The transient expression level was enhanced by increasing the plasmid DNA concentration and by the presence of polyethylene glycol (PEG) in the electroporation medium. A considerable increase in GUS activity was observed in presence of both PEG and upon heat-shock treatments compared to PEG treatment alone. An optimal level of GUS activity was obtained after electroporation with a capacitive discharge of 500 V/cm and 94 ms duration in both genotypes. The two genotypes differed in their responses to in vitro culture and also showed variable levels of transient expression. The present technique was found to be suitable to obtain transgenic plants as the histochemical GUS assay revealed GUS activity in the protoplast-derived micro-colonies as well as in callus tissues.Abbreviations CaMV cauliflower mosaic virus - CAT chloramphenicol acetyltransferase - CPW salts Cocking-Power-White salts - MES 2-[N-Morpholino]ethanesulfonic acid - MU 4-methyl umbelliferone - MUG 4-methyl umbelliferyl glucuronide - F microfarad - NAA naphthaleneacetic acid - NOS nopaline synthase - NPT II neomycin phosphotransferase - X-Gluc 5-bromo-4-chloro-3-indolyl--D-glucuronide