Overexpression of phytochrome A enhances the light-induced formation of adventitious shoots on horseradish hairy roots

A full-length cDNA of the gene for phytochrome A from Arabidopsis thaliana was fused with the 35S promoter of cauliflower mosaic virus (CaMV35S-PHYA) and introduced into horseradish (Armoracia rusticana Gaert., Mey. et Scherb.) hairy roots. The phytochrome level in hairy roots transformed with CaMV35SPHYA was about three times greater than that in normal hairy roots and the rate of light-induced formation of adventitious shoots was also higher in the hairy roots transformed with CaMV35SPHYA. These results indicate that the light-induced formation of adventitious shoots on horseradish hairy roots is closely related to the phytochrome level. Received: 11 August 1998 / Revision received: 21 October 1998 / Accepted: 20 November 1998     

Comparison of four constitutive promoters for the expression of transgenes in the tropical nitrogen-fixing tree Allocasuarina verticillata

Allocasuarina verticillata is an actinorhizal tree that lives in symbiotic association with a nitrogen fixing actinomycete called Frankia. In the search for promoters that drive strong constitutive expression in this tropical tree, we studied the organ specificity of four different constitutive promoters (CaMV 35S, e35S, e35S-4ocs and UBQ1 from Arabidopsis thaliana) in stably transformed A. verticillata plants. The ß-glucuronidase (gus) gene was used as a reporter and expression studies were carried out by histochemical analyses on shoots, roots and actinorhizal nodules. While the 35S promoter was poorly expressed in the shoot apex and lateral roots, both the e35S and e35S-4ocs were found to drive high constitutive expression in the transgenic non-nodulated plants. In contrast, the UBQ1 promoter was very poorly expressed and appeared unsuitable for A. verticillata. We also showed that none of the promoters studied were active in the nodule infected cells, whatever the developmental stage studied.     

An improved transformation protocol for studying gene expression in hairy roots of sugar beet (Beta vulgaris L.)

A transformation protocol, based on co-inoculation with two strains of Agrobacterium, Agrobacterium tumefaciens LBA4404 and A. rhizogenes 15834 containing a binary vector with the GUS gene, was established for the induction of transgenic hairy roots from sugar beet (Beta vulgaris L.) explants. It resulted in marked improvement in the formation of hairy roots and the integration of the binary vector T-DNA into the host genome. Of 250 inoculated sugar beet hypocotyls, 84% yielded hairy roots 5–7 days after inoculation, of which 70% were co-transformed with the binary vector T-DNA. To determine stable expression of alien genes in hairy roots, the nematode resistance gene Hs1 ^pro-1 was used as a reporter gene. In addition, molecular marker analysis was applied to monitor stable incorporation of a translocation from the wild beet B. procumbens. The molecular analysis and the nematode (Heterodera schachtii) resistance test in vitro demonstrated that the genomic structure and the expression of the Hs1 ^pro-1 -mediated nematode resistance were well-maintained in all hairy root cultures even after repeated sub-culture. Received: 25 November 1997 / Revision received: 26 May 1998 / Accepted: 15 June 1998     

Structure and regulation of OPR1 and OPR2, two closely related genes encoding 12-oxophytodienoic acid-10,11-reductases from Arabidopsis thaliana

The genes of two closely related 12-oxophytodienoic acid reductases (EC 1.3.1.42), OPR1 and OPR2, were identified on a 7079-bp-long genomic fragment from Arabidopsis thaliana (L.) Heynh. The organization of these two genes was determined and putative cis elements were identified. Promoter-β-glucuronidase (GUS) fusions expressed in transgenic Arabidopsis thaliana and Nicotiana tabacum L. plants revealed differences in OPR-promoter-driven GUS expression in flowers. While the OPR1 promoter directed GUS expression in young seeds, the OPR2 promoter directed pollen-specific expression. Both OPR1 and OPR2, were predominantly expressed in roots. Stress treatments, like local and systemic wounding, UV-C illumination and coldness, resulted in transient changes in steady-state OPR mRNA levels, but no concurrent changes in polypeptide level or enzyme activity were detected. Received: 2 October 1998 / Accepted: 22 December 1998     

Inhibition of CAT enzyme activity in Arabidopsis thaliana

Previously it was shown that transient chloramphenicol acetyltransferase (CAT) marker gene expression in Arabidopsis thaliana and Nicotiana tabacum resulted in significant differences in the accumulation of the CAT reaction products in radioactive CAT assays. Compared to Nicotiana tabacum, conversion of chloramphenicol to the acetylated products in Arabidopsis thaliana extracts was rather low. Here we report that the low CAT enzyme activity can be attributed in part to a heat sensitive CAT inhibitory effect in extracts of Arabidopsis thaliana. CAT enzyme activity in transgenic tobacco is inhibited by extracts from Arabidopsis. This inhibitory effect diminishes when Arabidopsis extracts were heat incubated. CAT activity in transgenic Arabidopsis lines was very low and was only detected in heat incubated extracts. Alternatively, enzyme-linked immunosorbent assays (ELISAs) can be used to detect the CAT protein in transgenic Arabidopsis.Abbreviations CAT chloramphenicol acetyltransferase - CAM chloramphenicol - ELISA enzyme linked immunosorbent assay     

Genetic transformation through the use of hyperhydric tobacco meristems

Exposed shoot meristems from normal and hyperhydric (vitrified) tobacco, Nicotiana tabacum, were bombarded with gold particles either coated with plasmid DNA containing neomycin phosphotransferase (NPTII), rolC and -glucuronidase (GUS) genes (plasmid pGA-GUSGFrolC) or left uncoated. Meristems bombarded with uncoated particles were co-cultivated with Agrobacterium tumefaciens strain EHA 101 harboring the binary vector pGA-GUSGFrolC. Whole-plant transformants were produced from 4 of 40 hyperhydric meristems bombarded with uncoated particles followed by co-cultivation with A. tumefaciens. One transgenic plant was obtained from 40 normal, non-hyperhydric meristems treated. Transformation was verified by growth on kanamycin-containing medium, GUS assays, PCR, and Southern analysis. The plants tested through Southern analysis appeared to have 2 or more copies of the transgene insert. Seeds obtained from self-pollination of these transgenic plants segregated 3:1 or 15:1 (kanamycin resistant:sensitive) when germinated on medium containing 100 mg/l kanamycin, indicating transfer of foreign genes through the sexual cycle. Whole-plant transformants were not produced from 50 normal tobacco meristems bombarded with plasmid-coated gold particles and not exposed to engineered A. tumefaciens, but 1 plant of 60 bombarded hyperhydric meristems produced transgenic roots, the result of a chimera. We suggest that hyperhydric meristems are more readily transformed.     

Indole acetonitrile-sensitivity of transgenic tobacco containing Arabidopsis thaliana nitrilase genes

 The Arabidopsis thaliana genome has four nitrilase (nitrile aminohydrolase, EC 3.5.5.1) genes (NIT1 to NIT4). These nitrilases catalyze hydrolysis of indole-3-acetonitrile (IAN) to indole-3-acetic acid (IAA). Growth of A. thaliana is inhibited by IAN probably due to hydrolysis of IAN to IAA, while the tobacco (Nicotiana tabacum) genome has only NIT4 homologs and is resistant to IAN. In this study, we introduced A. thaliana NIT1 to NIT4 into tobacco. Introduction of NIT1, NIT2 or NIT3 into tobacco conferred growth inhibition by IAN. NIT2 transgenic plants were highly sensitive to IAN, and NIT1 and NIT3 transgenic plants were moderately sensitive. On the other hand, NIT4 transgenic plants were less sensitive to IAN, although some morphological changes in the roots were observed as the wild-type tobacco. These findings suggest that the ability of transgenic tobacco to convert IAN to IAA in vivo is markedly different among transgenes of NIT1 to NIT4. Received: 22 November 1999 / Revision received: 28 January 2000 / Accepted: 4 February 2000     

High-efficiency <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-mediated transformation of heat inducible sHSP18.2-GUS in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis>

The chimerical gene, Arabidopsis thaliana sHSP18.2 promoter fused to E. coli gusA gene, was Agrobacterium rhizogenes-mediated transformed into Nicotiana tabacum as a heat-regulatable model, and the thermo-inducible expression of GUS activity in N. tabacum transgenic hairy roots was profiled. An activation of A. rhizogenes with acetosyringone (AS) before cocultured with tobacco's leaf disc strongly promoted transgenic hairy roots formation. Transgenic hairy roots formation efficiency of A. rhizogenes precultured with 200 μM AS supplementation was 3.1-fold and 7.5-fold, respectively, compared to the formation efficiency obtained with and without AS supplementation in coculture. Transgenic hairy roots transformed with different AS concentration exhibited a similar pattern of thermo-inducibility after 10 min to 3 h heat treatments detected by GUS expression. The peak of expressed GUS specific activity, 399,530 pmol MUG per mg total protein per min, of the transgenic hairy roots was observed at 48 h after 3 h of 42°C heat treatment, and the expressed GUS specific activity was 7–26 times more than that reported in A. thaliana, tobacco BY-2 cells and Nicotiana plumbaginifolia. Interference caused by AS supplementation on the growth of transgenic hairy roots, time-course of GUS expression and its expression level were not observed.     

An S-RNase promoter from Nicotiana alata functions in transgenic N. alata plants but not Nicotiana tabacum

Nicotiana tabacum and Nicotiana alata plants were transformed with genomic clones of two S-RNase alleles from N. alata. Neither the S ₂ clone, with 1.6 kb of 5 sequence, nor the S ₆ clone, with 2.8 kb of 5 sequence, were expressed at detectable levels in transgenic N. tabacum plants. In N. alata, expression of the S ₂ clone was not detected, however the S ₆ clone was expressed (at low levels) in three out of four transgenic plants. An S ₆-promoter-GUS fusion gene was also expressed in transgenic N. alata but not N. tabacum. Although endogenous S-RNase genes are expressed exclusively in floral pistils, the GUS fusion was expressed in both styles and leaves.     

Hairy root production in Arabidopsis thaliana: cotransformation with a promoter-trap vector results in complex T-DNA integration patterns

 In comparison with the production of transgenic plants, the generation of hairy roots has the advantage that more independent transgenic lines can be produced in a shorter period of time. Therefore, we wanted to combine this approach with the promoter-trapping strategy to identify nematode-induced plant promoters. For the efficient production and culture of transgenic hairy root lines of Arabidopsis thaliana, the standard Agrobacterium rhizogenes transformation procedure was modified to avoid rapid callusing of the hairy roots. An average of 0.72 independent kanamycin-resistant (Km^R) roots were obtained per leaf piece. However, a much lower frequency of reporter gene activation was obtained than expected from experiments with the same vectors in Agrobacterium tumefaciens: of more than 700 independent Km^R hairy roots tested, only 8 were β-glucuronidase (GUS) positive. DNA hybridization was done on ten hairy root lines, of which one had a single truncated T-DNA and the others multiple copies of T-DNA that led to complex hybridization patterns. In a parallel analysis of A. thaliana plants transformed with the same vectors using A. tumefaciens, relatively simple T-DNA integration patterns were obtained. The low occurrence of GUS-positive hairy root lines in our experiments could be explained by the multiple T-DNA copies, especially in inverted array, that result in high frequencies of gene inactivation. Received: 11 August 1998 / Revision received: 17 February 1999 / Accepted: 18 March 1999     

The mannopine synthase promoter contains vectorial cis-regulatory elements that act as enhancers and silencers

A 479-bp bi-directional promoter controls the expression of two genes (mas1′ and mas2′) that encode enzymes for the synthesis of the opine mannopine in plant tissues infected with Agrobacterium tumefaciens. This 5′ regulatory region (mas promoter) contains all the cis-acting elements involved in mediating the complex regulatory properties of these genes in plants. Using different mas promoter regions fused to a minimal 35S promoter (35SΔ108), we found that the regulatory properties of these divergent promoters result from the presence of orientation-dependent negative and positive regulatory regions. Some of these elements have the unusual property of acting as enhancers in one orientation and as silencers in the other. Using electrophoretic mobility shift analysis (EMSA), we showed that the functional mas promoter regions identified by fluorometric and histochemical assays for reporter gene activity in transgenic plants have the ability specifically to bind nuclear protein factors from Nicotiana tabacum, Phaseolus vulgaris, Solanum tuberosum, and Arabidopsis thaliana. Received: 7 May 1999 / Accepted: 5 August 1999     

Production of the Escherichia coli betaine-aldehyde dehydrogenase, an enzyme required for the synthesis of the osmoprotectant glycine betaine, in transgenic plants

In several organisms osmotic stress tolerance is mediated by the accumulation of the osmoprotective compound glycine betaine. With the ambition to transfer the betaine biosynthetic pathway into plants not capable of synthesizing this osmoprotectant, the Escherichia coli gene betB encoding the second enzyme in the pathway, betaine-aldehyde dehydrogenase was introduced into Nicotiana tabacum. The betB structural gene was fused to the promoter of ats1a, a gene coding for the small subunit of Rubisco in Arabidopsis thaliana. Two types of constructs were made, either encoding the N-terminal transit peptide for chloroplast targeting or without the targeting signal for cytoplasmic localization of the BetB polypeptide. Analysis of transgenic N. tabacum plants harboring these constructs showed that in both cases the transgenes were expressed. Northern analysis of the plants demonstrated the accumulation of betB-related mRNA of the correct size. The production and processing of the corresponding polypeptides could be demonstrated by immunoblotting using polyclonal antisera raised against the BetB polypeptide. The transit peptide encoded by ats1a was able to direct BetB to the chloroplast, as suggested by the presence of the correctly processed BetB polypeptide in the chloroplast fraction. High betaine-aldehyde dehydrogenase activity was detected in transgenic plants, both in those where the chimeric gene product was targeted to the chloroplast and those where it remained in the cytoplasm. The transgenic tobacco acquired resistance to the toxic intermediate, betaine aldehyde, in the betaine biosynthetic pathway indicating that the bacterial enzyme is biologically active in its new host. Furthermore, these transgenic plants were able to convert exogenously supplied betaine aldehyde efficiently to glycine betaine.     

A Brassica S-locus related gene promoter directs expression in both pollen and pistil of tobacco

A portion (1.5 kb) of the promoter region of an S63 S-locus related (SLR) glycoprotein gene from the sporophytically self-incompatible species Brassica oleracea was inserted upstream of the β-glucuronidase (GUS) gene in binary vector pBI101.1. The resulting construct was then introduced into Nicotiana tabacum through Agrobacterium-mediated transformation. The expression pattern of GUS under the control of the S63 SLR promoter fragment was found to be similar to that already reported for expression of the GUS gene directed by an S-locus specific gene promoter in transgenic N. tabacum. Furthermore, this pattern of expression resembled more closely that reported for S-genes of the self-incompatible species Nicotiana alata, which has a gametophytic self-incompatibility system, than the predicted pattern of expression of S-genes in B. oleracea.     

Morphological changes in transgenic poplar induced by expression of the rice homeobox gene OSH1

Genetically transformed lombardy poplar (Po-pulus nigra L. var. italica Koehne) plants were regenerated after co-cultivation of stem segments with Agrobacterium tumefaciens strain LBA4404 that harbored a binary vector which included the rice gene for a homeodomain protein (OSH1) and a gene for neomycin phosphotransferase. The expression of the OSH1 gene under control of the cauliflower mosaic virus 35S promoter induced morphological abnormalities in the leaves and stems of the newly generated transgenic poplar plants. This result suggests that OSH1 can function as a regulator of morphogenesis in transgenic poplar, as it does in transgenic rice, Arabidopsis, and tobacco plants. Received: 16 October 1998 / Revision received: 27 November 1998 / Accepted: 12 December 1998     

A geminivirus-induced gene silencing system for gene function validation in cassava

We have constructed an African cassava mosaic virus (ACMV) based gene-silencing vector as a reverse genetics tool for gene function analysis in cassava. The vector carrying a fragment from the Nicotiana tabacumsulfur gene (su), encoding one unit of the chloroplast enzyme magnesium chelatase, was used to induce the silencing of the cassava orthologous gene resulting in yellow–white spots characteristic of the inhibition of su expression. This result suggests that well developed sequence databases from model plants like Arabidopsis thaliana, Nicotiana benthamiana, N. tabacum, Lycopersicon esculentum and others could be used as a major source of information and sequences for functional genomics in cassava. Furthermore, a fragment of the cassava CYP79D2endogenous gene, sharing 89% homology with CYP79D1endogenous gene was inserted into the ACMV vector. The resultant vector was inducing the down regulation of the expression of these two genes which catalyze the first-dedicated step in the synthesis of linamarin, the major cyanogenic glycoside in cassava. At 21 days post-inoculation (dpi), a 76% reduction of linamarin content was observed in silenced leaves. Using transgenic plants expressing antisense RNA of CYP79D1and CYP79D2, Siritunga and Sayre (2003) obtained several lines with a reduction level varying from 60% to 94%. This result provides the first example of direct comparison of the efficiency of a virus-induced gene silencing (VIGS) system and the transgenic approach for suppression of a biosynthetic pathway. The ACMV VIGS system will certainly be a complement and in some cases an alternative to the transgenic approach, for gene discovery and gene function analysis in cassava.