Independent Transgenic Tobacco Research Articles

Overexpression of the Tectona grandis TgNAC01 regulates growth, leaf senescence and confer salt stress tolerance in transgenic tobacco plants.

NAC transcription factors play critical roles in xylem secondary development and in regulation of stress response in plants. NAC proteins related to secondary cell wall development were recently identified and characterized in Tectona grandis (teak), one of the hardwood trees of highest economic importance in the world. In this work, we characterized the novel TgNAC01 gene, which is involved in signaling pathways that mediate teak response to stress. Abscisic acid (ABA) increases TgNAC01 expression in teak plants. Therefore, this gene may have a role in signaling events that mediate ABA-dependent osmotic stress responsive in this plant species. Stable expression in tobacco plants showed that the TgNAC01 protein is localized in the cell nucleus. Overexpression of TgNAC01 in two out three independent transgenic tobacco lines resulted in increased growth, leaf senescence and salt tolerance compared to wild type (WT) plants. Moreover, the stress tolerance of transgenic plants was affected by levels of TgNAC01 gene expression. Water potential, gas exchange and chlorophyll fluorescence were used to determine salt stress tolerance. The 35S:TgNAC01-6 line under 300 mM NaCl stress responded with a significant increase in photosynthesis rate, stomatal conductance, transpiration and carboxylation efficiency, but lower water potential compared to WT plants. The data indicate that the TgNAC01 transcription factor acts as a transcriptional activator of the ABA-mediated regulation and induces leaf senescence.

Reaction to Hydrogen-Peroxide Action in Nicotiana tabacum Plants Transformed by the Cholinoxidase Gene (codA)

Genetic engineering of glycine betaine (GB) synthesis offers a new approach to improvements in the productivity and stress resistance of cultivated plants. The possible effect of the bacterial heterologous sequence on the reaction of plants under oxidative stress (OS) induced by 3 mM of hydrogen peroxide (H2O2) was determined. The independent transgenic tobacco lines (Nicotiana tabacum L.) Cod 16 and Cod 45 with the choline oxidase (codA) gene from Arthrobacter gloiformis, which is responsible for GB synthesis, were used. The transgenic lines differed from wild-type plants with lower resistance to H2O2, which led to a more significant decrease in the pool of photosynthetic pigments and a greater intensity of lipid peroxidation and dynamics of superoxide dismutase (SOD) and peroxidase (PO) activity. In terms of OS manifestation, transgenic lines with initially similar values of GB accumulation (0.825–0.910 mmol/g) had significant differences among themselves. In the Cod 16 line, the reaction to OS induction was earlier (1 h after H2O2 treatment), and there was a more significant increase in PO activity and proline content than in the Cod 45 line and the original variety. In the Cod 45 line, there is a significant increase in SOD activity and proline content in response to OS induction 24 h after treatment, and, in contrast to the wild-type plants and the Cod 16 line, it is accompanied by a significantly higher content of carotenoids in the leaves. When genetic-engineering methods are used to increase the productivity and resistance of plants to stress, it is necessary to take into account the functional interaction of antioxidants and osmoprotectors.

A PeMYB-like gene induced by brassinosteroids regulates cellulose synthesis in tobacco

MYB is a well-known key transcriptional regulator involved in plant development and metabolism. In this study, a new MYB gene was isolated from Phyllostachys edulis and named PeMYB-like. A close link between brassinosteroids (BRs) and PeMYB-like gene expression was suggested in this study. Functional characterization of PeMYB-like was elucidated through overexpression of this gene in tobacco. Expression of PeMYB-like in ten independent transgenic tobacco lines was quantified by semi-quantitative RT-PCR. The expression levels NtCESA1 and NtCESA2 in transgenic plants were higher than that in control plants. The transgenic lines also showed a significant increase in cellulose content, suggesting that PeMYB-like may enhance cellulose biosynthesis through the upregulation of genes encoding enzymes involved in cellulose biosynthesis in plant stems. Our study indicated that there may be a close link between PeMYB-like gene expression and cellulose biosynthesis; moreover, BR signaling pathway might be involved in cellulose biosynthesis processes through the regulation of the MYB gene transcription.

Salt and osmotic stress response of tobacco plants overexpressing Lepidium latifolium L. Ran GTPase gene

Our earlier study has identified Lepidium latifolium Ran (LlaRan) as a cold stress downregulated gene from L. latifolium, a cold desert plant. Further, independent transgenic tobacco (Nicotiana tabacum L. var Xanthi) lines overexpressing LlaRan were developed for functional characterization of the gene. We analysed the tolerance of tobacco plants over expressing LlaRan gene to salt and PEG stresses at germination which represents a critical stage in life cycle of the plants. Germination performance of the four independent transgenic lines (T1 generation) were assessed for germination under distilled water, NaCl (150 mM) and osmotic (15% PEG 4000) stresses. The emergence of radical, plumule and cotyledonary leaf were recorded for 10 days on daily basis and its rate assessed in terms of Timson’s index. It was observed that the germination of wild type tobacco seeds was better than the assessed transgenic lines. It may be inferred that the transgenic lines seeds under study were more sensitive to salt and drought stress elucidating their hypersensitivity at germination level.

An Intergenic Region Shared by At4g35985 and At4g35987 in Arabidopsis thaliana Is a Tissue Specific and Stress Inducible Bidirectional Promoter Analyzed in Transgenic Arabidopsis and Tobacco Plants

On chromosome 4 in the Arabidopsis genome, two neighboring genes (calmodulin methyl transferase At4g35987 and senescence associated gene At4g35985) are located in a head-to-head divergent orientation sharing a putative bidirectional promoter. This 1258 bp intergenic region contains a number of environmental stress responsive and tissue specific cis-regulatory elements. Transcript analysis of At4g35985 and At4g35987 genes by quantitative real time PCR showed tissue specific and stress inducible expression profiles. We tested the bidirectional promoter-function of the intergenic region shared by the divergent genes At4g35985 and At4g35987 using two reporter genes (GFP and GUS) in both orientations in transient tobacco protoplast and Agro-infiltration assays, as well as in stably transformed transgenic Arabidopsis and tobacco plants. In transient assays with GFP and GUS reporter genes the At4g35985 promoter (P85) showed stronger expression (about 3.5 fold) compared to the At4g35987 promoter (P87). The tissue specific as well as stress responsive functional nature of the bidirectional promoter was evaluated in independent transgenic Arabidopsis and tobacco lines. Expression of P85 activity was detected in the midrib of leaves, leaf trichomes, apical meristemic regions, throughout the root, lateral roots and flowers. The expression of P87 was observed in leaf-tip, hydathodes, apical meristem, root tips, emerging lateral root tips, root stele region and in floral tissues. The bidirectional promoter in both orientations shows differential up-regulation (2.5 to 3 fold) under salt stress. Use of such regulatory elements of bidirectional promoters showing spatial and stress inducible promoter-functions in heterologous system might be an important tool for plant biotechnology and gene stacking applications.

Cloning and characterization of the new multiple stress responsible gene I (MuSI) from sweet potato

New multiple-stress related gene isolated from sweet potato and designated it as MusI (multiple stress responsible gene I). Sequence analysis revealed that its full length cDNA was 998 bp long and included a 717 bp open reading frame encoding for 238 amino acids. Comparison of its cDNA and genomic DNA sequence showed that 3 exons were divided by 2 introns in its ORF region. Its deduced amino acid sequence contained a conserved rubber elongation factor (REF) domain and showed high homology with many stress-related proteins. Therefore, it was named MuSI (multiple stress responsible gene I). Southern hybridization analysis indicated that the MuSI gene may belong to a multi-gene family. Expression pattern of the MuSI gene showed that it was differently expressed among roots, stems, leaves, and flowers of a sweet potato, and its expression level was especially high in flowers and white fibrous roots. Its expression was also highly induced by various stress signals including dehydration, high salt, heavy metal, oxidation, and plant hormones. Stress tolerance experiment using transgenic plants overexpressing the MuSI gene showed that all independent transgenic tobacco lines have enhanced tolerance to high temperature stress. Among them, transgenic line 6 particularly showed tolerance to salt, heavy metal, and osmotic stress as well. These results suggest that the MuSI gene functions as a positive regulator of various stress responses and may be useful in improving stress tolerance of transgenic plants.

Inhibition of biphasic ethylene production enhances tolerance to abiotic stress by reducing the accumulation of reactive oxygen species in Nicotiana tabacum.

Reactive oxygen species (ROS), such as H(2)O(2), are important plant cell signaling molecules involved in responses to biotic and abiotic stresses and in developmental and physiological processes. Despite the well-known physiological functions of ethylene production and stress signaling via ROS during stresses, whether ethylene acts alone or in conjunction with ROS has not yet been fully elucidated. Therefore, we investigated the relationship between ethylene production and ROS accumulation during the response to abiotic stress. We used three independent transgenic tobacco lines, CAS-AS-2, -3 and -4, in which an antisense transcript of the senescence-related ACC synthase (ACS) gene from carnation flower (CARACC, Gen-Bank accession No. M66619) was expressed heterologously. Biphasic ethylene biosynthesis was reduced significantly in these transgenic plants, with or without H(2)O(2) treatment. These plants exhibited significantly reduced H(2)O(2)-induced gene-specific expression of ACS members, which were regulated in a time-dependent manner. The higher levels of NtACS1 expression in wild-type plants led to a second peak in ethylene production, which resulted in a more severe level of necrosis and cell death, as determined by trypan blue staining. In the transgenic lines, upregulated transcription of CAB, POR1 and RbcS resulted in increased photosynthetic performance following salt stress. This stress tolerance of H(2)O(2)-treated transgenic plants resulted from reduced ethylene biosynthesis, which decreased ROS accumulation via increased gene expression and activity of ROS-detoxifying enzymes, including MnSOD, CuZnSOD, and catalase. Therefore, it is suggested that ethylene plays a potentially critical role as an amplifier for ROS accumulation, implying a synergistic effect between biosynthesis of ROS and ethylene.

Inheritance of marker and target genes in seed and vegetative progenies of transgenic tobacco plants carrying the buckwheat serine protease inhibitor gene

The buckwheat serine protease inhibitor (BWI-1) target gene (ISP) was expressed under the control of the constitutive 35S promoter of the cauliflower mosaic virus was expressed in transgenic tobacco plants and conferred antibacterial resistance. A stable and linked inheritance and expression of the marker nptII and target genes were observed in a random sample of independent transgenic tobacco plants after longterm propagation by nodal segments or multiple (for 1.5 years) regenerations under nonselective conditions; the transgene insert was preserved in the T1 seed progeny. Transgenic plants displayed numerous alterations in microsporogenesis. A loss of kanamycin (Km) resistance was accompanied by a loss of antibacterial activity in two lines. Segregation was observed for Km resistance in line C7 and for seedling size in line C22.

Functional analysis reveals effects of tobacco alternative oxidase gene (NtAOX1a) on regulation of defence responses against abiotic and biotic stresses

Mitochondrial AOX (alternative oxidase) is the terminal oxidase of the CN (cyanide)-resistant alternative respiratory pathway in plants. To investigate the role of the tobacco AOX gene (NtAOX1a) (where Nt is Nicotiana tabacum) under deleterious conditions which could induce ROS (reactive oxygen species) accumulation, we generated and characterized a number of independent transgenic tobacco (N. tabacum) lines with altered NtAOX1a gene expression and AP (alternative pathway) capacity. AOX efficiently inhibited the production of low-temperature-induced H2O2 and might be a major enzyme for scavenging H2O2 at low temperature. Furthermore, NtAOX1a may act as a regulator of KCN-induced resistance to TMV (tobacco mosaic virus) through the regulation of H2O2. Notably, a moderate accumulation of H2O2 under the control of NtAOX1a was crucial in viral resistance. Analysis of seed germination indicated an important role for NtAOX1a in germination under H2O2-induced oxidative stress when the CP (cytochrome pathway) was inhibited. These results demonstrate that NtAOX1a is necessary for plants to survive low temperature, pathogen attack and oxidative stress by scavenging ROS under these adverse conditions when the CP is restricted.

Expression of Rhizobium pydA-pydB fusion gene in Nicotiana tabacum confers resistance to the toxic aromatic compound 3-hydroxy-4-pyridone

The toxic aromatic compound 3-hydroxy-4-pyridone (HP) is an intermediate in both synthesis and degradation of mimosine, which is produced by the tree legume Leucaena leucocephala. The L. leucocephala root-nodule symbiont Rhizobium TAL1145 contains a dioxygenase (pydA) and a hydrolase (pydB) gene that produce enzymes necessary for the degradation of HP. In order to coordinately express both genes in plant tissues under a single promoter, three different pydA-pydB fusion constructs (G0, G3, and G7) with varying glycine linkers between the two genes were developed. Prior to transferring the fusion constructs into L. leucocephala, which is highly recalcitrant to genetic transformation, we tested the expression and activity of the hybrid proteins in Nicotiana tabacum, a model plant system that can be easily transformed and analyzed. Seven independent transgenic tobacco lines were generated by Agrobacterium-mediated transformation, and stable integration and expression of pydA-pydB in these transgenic lines were confirmed by polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR) and Western analysis. Only one of the fusion constructs, G3, containing a 9-nucleotide linker between pydA and pydB, provided significant levels of resistance to 3 mM HP, indicating that the hybrid protein produced by this fusion construct could degrade HP.

One Novel Mitochondrial Citrate Synthase from Oryza sativa L. can Enhance Aluminum Tolerance in Transgenic Tobacco

Rice exhibits the greatest aluminum (Al) tolerance compared with other cereals such as wheat, barley, maize, etc. A full-length gene, OsCS1, encoding citrate synthase, which is highly induced by aluminum toxicity in rice (Oryza sativa L.), was isolated. Sequence analysis and the sub-cellular localization of OsCS1 in yeast revealed that it is a mitochondrial citrate synthase. OsCS1 was induced by Al toxicity. Several independent transgenic tobacco lines expressing OsCS 1 exhibited increased citrate efflux and extraordinary Al tolerance. Possible outlook for OsCS1 to be applied to enhance plant tolerance to Al toxicity was also discussed.

Construction vascular-specific expression bi-directional promoters in plants

Promoters that have been widely used for both basic research and biotechnological application in plants are generally unidirectional. Here we describe a strategy to bi-directionalize the vascular-specific expression grp1.8 promoter (here named GRPp) and 4CL1 promoter (here named 4CL1p) so that one promoter can direct the vascular-specific expression of two genes, one on each end of the promoter. The minimal promoter ( 35Smini or GRP mini), when fused at the 5′ end of the specific expression promoter ( GRPp or 4CL1p) to form bi-directional promoter ( 35Smini-GRPp, 35Smini-4CL1p or GRPmini-GRPp), was able to direct expression of the glucuronidase ( gus) and green fluorescent protein ( gfp) gene in all independent transgenic tobacco lines. Stable expression of gusA and gfp genes in transgenic plants was analyzed by histochemical staining for GUS and fluorescence microscopic observation under UV for GFP in transgenic plants. The remarkable transcript levels of GFP and GUS were detected by real-time PCR in independent transgenic tobacco lines. Their vascular-specific bi-directional promoters should be used to vascular-specific expression several functional genes in transgenic plants simultaneously.

Anchorage to the cytosolic face of the endoplasmic reticulum membrane: a new strategy to stabilize a cytosolic recombinant antigen in plants

The levels of accumulation of recombinant vaccines in transgenic plants are protein specific and strongly influenced by the subcellular compartment of destination. The human immunodeficiency virus protein Nef (negative factor), a promising target for the development of an antiviral vaccine, is a cytosolic protein that accumulates to low levels in transgenic tobacco and is even more unstable when introduced into the secretory pathway, probably because of folding defects in the non-cytosolic environment. To improve Nef accumulation, a new strategy was developed to anchor the molecule to the cytosolic face of the endoplasmic reticulum (ER) membrane. For this purpose, the Nef sequence was fused to the C-terminal domain of mammalian ER cytochrome b5, a long-lived, tail-anchored (TA) protein. This consistently increased Nef accumulation by more than threefold in many independent transgenic tobacco plants. Real-time polymerase chain reaction of mRNA levels and protein pulse-chase analysis indicated that the increase was not caused by higher transcript levels but by enhanced protein stability. Subcellular fractionation and immunocytochemistry indicated that Nef-TA accumulated on the ER membrane. Over-expression of mammalian or plant ER cytochrome b5 caused the formation of stacked membrane structures, as observed previously in similar experiments performed in mammalian cells; however, Nef-TA did not alter membrane organization in tobacco cells. Finally, Nef could be removed in vitro by its tail-anchor, taking advantage of an engineered thrombin cleavage site. These results open up the way to use tail-anchors to improve foreign protein stability in the plant cytosol without perturbing cellular functions.

Tobacco Isoenzyme 1 of NAD(H)-Dependent Glutamate Dehydrogenase Catabolizes Glutamate in Vivo

Glutamate (Glu) dehydrogenase (GDH, EC 1.4.1.2-1.4.1.4) catalyzes in vitro the reversible amination of 2-oxoglutarate to Glu. The in vivo direction(s) of the GDH reaction in higher plants and hence the role(s) of this enzyme is unclear, a situation confounded by the existence of isoenzymes comprised totally of either GDH beta- (isoenzyme 1) or alpha- (isoenzyme 7) subunits, as well as another five alpha-beta isoenzyme permutations. To clarify the in vivo direction of the reaction catalyzed by GDH isoenzyme 1, [(15)N]Glu was supplied to roots of two independent transgenic tobacco (Nicotiana tabacum) lines with increased isoenzyme 1 levels (S4-H and S49-H). The [(15)N]ammonium (NH(4)(+)) accumulation rate in these lines was elevated approximately 65% compared with a null segregant control line, indicating that isoenzyme 1 catabolizes Glu in roots. Leaf glutamine synthetase (GS) was inhibited with a GS-specific herbicide to quantify any contribution by GDH toward photorespiratory NH(4)(+) reassimilation. Transgenic line S49-H did not show enhanced resistance to the herbicide, indicating that the large pool of isoenzyme 1 in S49-H leaves was unable to compensate for GS and suggesting that isoenzyme 1 does not assimilate NH(4)(+) in vivo.

Engineering expression of bacterial polyphosphate kinase in tobacco for mercury remediation

To develop the potential of plants to sequester and accumulate mercurials from the contaminated sites, we engineered a tobacco (Nicotiana tabacum) plant to express a bacterial ppk gene, encoding polyphosphate kinase (PPK), under control of a plant promoter. The designated plant expression plasmid pPKT116 that contains the entire coding region of ppk was used for Agrobacterium-mediated gene transfer into tobacco plants. A large number of independent transgenic tobacco plants were obtained, in some of which the ppk gene was stably integrated in the plant genome and substantially translated to the expected PPK protein in the transgenic tobacco. The presence of Hg2+ did not cause considerable morphological abnormalities in the transgenic tobacco, which grew, flowered, and set seed similarly to the wild-type tobacco on the medium containing normally toxic levels of Hg2+. The ppk-transgenic tobacco showed more resistance to Hg2+ and accumulated more mercury than its wild-type progenitors. These results suggest that ppk-specified polyphosphate has abilities to reduce mercury toxicity, probably via chelation mechanism, and also to accumulate mercury in the transgenic tobacco. Based on the results obtained in the present study, the expression of ppk gene in transgenic tobacco plants might provide a means for phytoremediation of mercury pollution.

Overexpression of the feedback-insensitive anthranilate synthase gene in tobacco causes tryptophan accumulation.

A total of 35 independent transgenic tobacco plants were produced using the Agrobacterium tumefaciens-leaf segment co-cultivation method followed by selection with kanamycin for the nptII gene. The vector also carried the tobacco feedback-insensitive anthranilate synthase gene (ASA2). Many of the lines showed increased ASA2 mRNA levels but only three contained increased free tryptophan (Trp) and many lines contained lower Trp than the untransformed control. The line with the highest Trp level (threefold that of the untransformed control) contained increased anthranilate synthase activity (AS) both in leaves and a cell suspension culture derived from the plant while the feedback insensitivity was most evident in the suspension culture. Other kinetic data also indicated that the ASA2 encoded AS alpha-subunit was more abundant in the tissue culture than in leaves. Progeny seedlings from this line were resistant to certain toxic Trp analogs, especially alpha-methyltryptophan (alphaMT) and less so to the most commonly used analog, 5-methyltryptophan. Shoots formed more readily from leaves of two of the transgenic lines than from leaves of the untransformed control on alphaMT, indicating that it might be possible to use ASA2 as a selectable marker gene and alphaMT as the selection agent.

Transgenic tobacco expressing an Arisaema heterophyllum agglutinin gene displays enhanced resistance to aphids

Tobacco leaf discs were transformed with a plasmid, pBIAHA, containing the selectable marker neomycin phosphotransferase gene (nptII) and an Arisaema heterophyllum agglutinin gene (aha) via Agrobacterium tumefaciens-mediated transformation. Thirty-two independent transgenic tobacco plants were regenerated. PCR and Southern blot analyses confirmed that multiple copies of the aha gene had integrated into the plant genome. Northern blot analysis revealed that the aha gene was expressed at various levels in the transgenic plants. Insect bioassay test showed that transgenic plants expressing multiple copies of the aha gene reduced the rate of population increase of the peach potato aphid (Myzus persicae Sulzer). This is the first report that transgenic tobacco plants expressing the aha gene display enhanced resistance to aphids. Key words: Insect bioassay, Arisaema heterophyllum agglutinin, transformation, transgenic tobacco, peach potato aphid (Myzus persicae Sulzer)

Transgenic tobacco expressing Pinellia ternata agglutinin confers enhanced resistance to aphids.

Tobacco leaf discs were transformed with a plasmid, pBIPTA, containing the selectable marker neomycin phosphotransferase gene (nptII) and Pinellia ternata agglutinin gene (pta) via Agrobacterium tumefaciens-mediated transformation. Thirty-two independent transgenic tobacco plants were regenerated. PCR and Southern blot analyses confirmed that the pta gene had integrated into the plant genome and northern blot analysis revealed transgene expression at various levels in transgenic plants. Genetic analysis confirmed Mendelian segregation of the transgene in T1 progeny. Insect bioassays showed that transgenic plants expressing PTA inhibited significantly the growth of peach potato aphid (Myzus persicae Sulzer). This is the first report that transgenic plants expressing pta confer enhanced resistance to aphids. Our study indicates that the pta gene can be used as a supplement to the snowdrop (Galanthus nivalis) lectin gene (gna) in the control of aphids, a sap-sucking insect pest causing significant yield losses of crops.

Complete sequence of the binary vector pBI121 and its application in cloning T-DNA insertion from transgenic plants

The widely used expression vector pBI121 for plant transformation was reconstructed, and the complete sequence of 14758 bp is now available (accession number AF485783). The T-DNA region (6193 bp) contains the right border, expression cassettes for a neomycin phosphotransferase II (NPTII) selection marker and a β-glucuronidase (GUS) reporter gene, and the left border. The non-T-DNA region (8565 bp) was constructed according to the Bin 19 vector. We applied the vector information to clone the plant/T-DNA junction region from three independent transgenic tobacco plants. Knowledge of the complete sequence of this vector will be useful for an accurate description of vector size, determination of the integrity of T-DNA, identification of independent lines, the locus where it is inserted, the T-DNA copy number in those stable transformants, or construction of a smaller vector. In addition, the complete sequence (5667 bp) of the transient expression vector pBI221 (accession number AF502128) carrying the ampicillin resistance and gus reporter genes is also reported.

Inhibition of tobacco NADH-hydroxypyruvate reductase by expression of a heterologous antisense RNA derived from a cucumber cDNA: implications for the mechanism of action of antisense RNAs.

Tobacco plants were genetically transformed to generate antisense RNA from a gene construct comprised of a full-length cucumber NADH-dependent hydroxypyruvate reductase (HPR) cDNA placed in reverse orientation between the cauliflower mosaic virus 35S promoter and a nopaline synthase termination/polyadenylation signal sequence. In vivo accumulation of antisense HPR RNA within eight independent transgenic tobacco plants resulted in reductions of up to 50% in both native HPR activity and protein accumulation relative to untransformed tobacco plants (mean transgenote HPR activity = 67% wild type, mean transgenote HPR protein = 63% wild type). However, in contrast to previous reports describing antisense RNA effects in plants, production of the heterologous HPR antisense RNA did not systematically reduce levels of native tobacco HPR mRNA (mean transgenote HPR mRNA level = 135% wild type). Simple regression comparison of the steady-state levels of tobacco HPR mRNA to those of HPR antisense RNA showed a weak positive correlation (r value of 0.548, n = 9; n is wild type control plus eight independent transformants; significant at 85% confidence level), supporting the conclusion that native mRNA levels were not reduced within antisense plants. Although all transgenic antisense plants examined displayed an apparent reduction in both tobacco HPR protein and enzyme activity, there is no clear correlation between HPR activity and the amount of either sense (r = 0.267, n = 9) or antisense RNA (r = 0.175, n = 9). This compares to a weak positive correlation between HPR mRNA levels and the amount of HPR activity observed in wild-type SR1 tobacco plants (r = 0.603, n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)