Antisense Version Research Articles

LARP4 Is Regulated by Tumor Necrosis Factor Alpha in a Tristetraprolin-Dependent Manner.

LARP4 is a protein with unknown function that independently binds to poly(A) RNA, RACK1, and the poly(A)-binding protein (PABPC1). Here, we report on its regulation. We found a conserved AU-rich element (ARE) in the human LARP4 mRNA 3' untranslated region (UTR). This ARE, but not its antisense version or a point-mutated version, significantly decreased the stability of β-globin reporter mRNA. We found that overexpression of tristetraprolin (TTP), but not its RNA binding mutant or the other ARE-binding proteins tested, decreased cellular LARP4 levels. RNA coimmunoprecipitation showed that TTP specifically associated with LARP4 mRNA in vivo. Consistent with this, mouse LARP4 accumulated to higher levels in TTP gene knockout (KO) cells than in control cells. Stimulation of WT cells with tumor necrosis factor alpha (TNF-α), which rapidly induces TTP, robustly decreased LARP4 with a coincident time course but had no such effect on LARP4B or La protein or on LARP4 in the TTP KO cells. The TNF-α-induced TTP pulse was followed by a transient decrease in LARP4 mRNA that was quickly followed by a subsequent transient decrease in LARP4 protein. Involvement of LARP4 as a target of TNF-α-TTP regulation provides a clue as to how its functional activity may be used in a physiologic pathway.

BpGH3.5, an early auxin-response gene, regulates root elongation in Betula platyphylla × Betula pendula

The GH3 family is an important class of early auxin-response genes involved in the development of the hypocotyls and roots in Arabidopsis thaliana, but the role of this gene family in woody plants is poorly understood. In this study, we cloned a GH3-like gene from Betula platyphylla × Betula pendula (birch) named BpGH3.5 and produced transgenic birch lines that overexpressed either a sense or antisense version of the BpGH3.5 gene using Agrobacterium-mediated transformation. We found that both types of transgenic lines exhibited short primary and lateral roots in vitro, which was caused by a smaller sized root apical meristem with a fewer cells compared with the non-transgenic plant as observed in paraffin sections. The qRT-PCR results showed that the expression of genes associated with auxin and cytokinin metabolism and signaling changed in the transgenic lines. These results indicated that cytokinin and auxin crosstalk caused a small meristem, short-root phenotype in BpGH3.5 transgenic lines. In addition, transgenic sense and antisense BpGH3.5 lines showed reduced indole-3-acetic acid and N-1-napthylphthalamic-acid sensitivity. Taken as a whole, our results suggested that BpGH3.5 had a complex function and sophisticated mechanism of regulation in woody plants.

Improving peppermint essential oil yield and composition by metabolic engineering.

Peppermint (Mentha × piperita L.) was transformed with various gene constructs to evaluate the utility of metabolic engineering for improving essential oil yield and composition. Oil yield increases were achieved by overexpressing genes involved in the supply of precursors through the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. Two-gene combinations to enhance both oil yield and composition in a single transgenic line were assessed as well. The most promising results were obtained by transforming plants expressing an antisense version of (+)-menthofuran synthase, which is critical for adjusting the levels of specific undesirable oil constituents, with a construct for the overexpression of the MEP pathway gene 1-deoxy-D-xylulose 5-phosphate reductoisomerase (up to 61% oil yield increase over wild-type controls with low levels of the undesirable side-product (+)-menthofuran and its intermediate (+)-pulegone). Elite transgenic lines were advanced to multiyear field trials, which demonstrated consistent oil yield increases of up to 78% over wild-type controls and desirable effects on oil composition under commercial growth conditions. The transgenic expression of a gene encoding (+)-limonene synthase was used to accumulate elevated levels of (+)-limonene, which allows oil derived from transgenic plants to be recognized during the processing of commercial formulations containing peppermint oil. Our study illustrates the utility of metabolic engineering for the sustainable agricultural production of high quality essential oils at a competitive cost.

Transgenic resistance by N gene of a Peanut bud necrosis virus isolate of characteristic phylogeny

The nucleocapsid protein (N) gene of a Tospovirus devastating tomato crop in the south Indian state of Tamil Nadu was cloned and characterized. The high identity of the cloned sequence to a Peanut bud necrosis virus (PBNV) tomato isolate (97.8/99.6% nucleotide/amino acid) and a PBNV peanut isolate (94.4/96.3% nucleotide/amino acid) identified the Tospovirus as an isolate of PBNV, designated PBNV Coimbatore tomato (PBNV CT) isolate. Phylogenetic analysis of PBNV CT N gene provided useful insights into the movement and evolution of PBNV within Indian Territory. The characteristic phylogeny of PBNV CT N gene implied its potential to be an efficient transgene to confer effective PBNV resistance on crop plants. The efficacy of PBNV CT N gene in conferring PBNV resistance was studied by generating tobacco (Nicotiana tabacum L. cv Wisconsin) lines transgenic to the sense or antisense version of the gene. Several transgenic lines showed transgenic mRNA and/or protein accumulation, ranging from very high to undetectable levels, accompanied by different degrees of PBNV resistance. The undetectable or very low levels of transgene transcripts in certain PBNV-resistant sense or antisense N gene transgenic lines suggested RNA-mediated resistance by post-transcriptional gene silencing (PTGS) mechanism. However, PBNV resistance of certain transgenic lines with high levels of N gene transcripts was suggestive of possible operation of RNA-mediated non-PTGS mechanism(s) of resistance in those lines. Moreover, the high levels of N protein in certain PBNV-resistant sense N gene transgenic lines suggested protein-mediated resistance. The results predict the potential of PBNV CT N gene to confer effective PBNV resistance on tomato and other economically important crops.

Glucose-induced Remodeling of Intermediary and Energy Metabolism in Procyclic Trypanosoma brucei

The procyclic form of Trypanosoma brucei is a parasitic protozoan that normally dwells in the midgut of its insect vector. In vitro, this parasite prefers d-glucose to l -proline as a carbon source, although this amino acid is the main carbon source available in its natural habitat. Here, we investigated how l -proline is metabolized in glucose-rich and glucose-depleted conditions. Analysis of the excreted end products of (13)C-enriched l -proline metabolism showed that the amino acid is converted into succinate or l -alanine depending on the presence or absence of d-glucose, respectively. The fact that the pathway of l -proline metabolism was truncated in glucose-rich conditions was confirmed by the analysis of 13 separate RNA interference-harboring or knock-out cell lines affecting different steps of this pathway. For instance, RNA interference studies revealed the loss of succinate dehydrogenase activity to be conditionally lethal only in the absence of d-glucose, confirming that in glucose-depleted conditions, l -proline needs to be converted beyond succinate. In addition, depletion of the F(0)/F(1)-ATP synthase activity by RNA interference led to cell death in glucose-depleted medium, but not in glucose-rich medium. This implies that, in the presence of d-glucose, the importance of the F(0)/F(1)-ATP synthase is diminished and ATP is produced by substrate level phosphorylation. We conclude that trypanosomes develop an elaborate adaptation of their energy production pathways in response to carbon source availability.

Selection of non-branching lines induced by introducing Ls-like cDNA into Chrysanthemum ( Dendranthema × grandiflorum (Ramat.) Kitamura) “Shuho-no-chikara”

A partial Ls-like cDNA (525bp) was isolated from Chrysanthemum “95B1-45” line, which exhibited a non-branching characteristic. The fragments showed homology with lateral suppressor ( Ls) partial cDNA from tomato that stimulated the initiation of axillary meristems. After the fragments was constructed with sense and antisense versions, these constructs were employed in Agrobacterium-mediated transformation of Chrysanthemum ( Dendranthema × grandiflorum (Ramat.) Kitamura) “Shuho-no-chikara”. The leaf segments (5 mm × 5 mm) from in vitro shoots were inoculated with Agrobacterium cultures for 10 min and co-cultured with Agrobacterium tumefaciens for three days at 25 °C. Transgenic plants were obtained only in Agrobacterium C58C1 containing pCAMBIA2301 constructed with Ls-like partial cDNA in antisense version. Forty shoots (1 was in positive control and 39 in Ls-antisense version) in Agrobacterium C58C1 were confirmed as putative transformants in PCR analysis with nptII primer. In the Southern analysis, results showed that 35 of the 39 shoots integrated npt II gene were confirmed to contain Ls-like partial cDNA in antisense version. Therefore, the 35 transformants were multiplied and planted in green house for integrated Ls-like partial cDNA expression. One (2.9%) among the 36 transformed lines showed lateral bud inhibition of more than 40%, with another one (2.9%) in lateral bud inhibition of 20–30%, and the remaining ones in less than 20%. However, in the second investigation (in a lateral branch in October 29), nine lines belonged to the range of 20–30% lateral bud inhibition and the remaining ones were in the range of less than 20%. The expression of non-branching characteristics showed significant difference even in the same line according to season.

Transgenic tobacco plants overexpressing chloroplastic ferredoxin-NADP(H) reductase display normal rates of photosynthesis and increased tolerance to oxidative stress.

Ferredoxin-NADP(H) reductase (FNR) catalyzes the last step of photosynthetic electron transport in chloroplasts, driving electrons from reduced ferredoxin to NADP+. This reaction is rate limiting for photosynthesis under a wide range of illumination conditions, as revealed by analysis of plants transformed with an antisense version of the FNR gene. To investigate whether accumulation of this flavoprotein over wild-type levels could improve photosynthetic efficiency and growth, we generated transgenic tobacco (Nicotiana tabacum) plants expressing a pea (Pisum sativum) FNR targeted to chloroplasts. The alien product distributed between the thylakoid membranes and the chloroplast stroma. Transformants grown at 150 or 700 micromol quanta m(-2) s(-1) displayed wild-type phenotypes regardless of FNR content. Thylakoids isolated from plants with a 5-fold FNR increase over the wild type displayed only moderate stimulation (approximately 20%) in the rates of electron transport from water to NADP+. In contrast, when donors of photosystem I were used to drive NADP+ photoreduction, the activity was 3- to 4-fold higher than the wild-type controls. Plants expressing various levels of FNR (from 1- to 3.6-fold over the wild type) failed to show significant differences in CO2 assimilation rates when assayed over a range of light intensities and CO2 concentrations. Transgenic lines exhibited enhanced tolerance to photooxidative damage and redox-cycling herbicides that propagate reactive oxygen species. The results suggest that photosynthetic electron transport has several rate-limiting steps, with FNR catalyzing just one of them.

Fumarate Is an Essential Intermediary Metabolite Produced by the Procyclic Trypanosoma brucei

The procyclic stage of Trypanosoma brucei, a parasitic protist responsible for sleeping sickness in humans, converts most of the consumed glucose into excreted succinate, by succinic fermentation. Succinate is produced by the glycosomal and mitochondrial NADH-dependent fumarate reductases, which are not essential for parasite viability. To further explore the role of the succinic fermentation pathways, we studied the trypanosome fumarases, the enzymes providing fumarate to fumarate reductases. The T. brucei genome contains two class I fumarase genes encoding cytosolic (FHc) and mitochondrial (FHm) enzymes, which account for total cellular fumarase activity as shown by RNA interference. The growth arrest of a double RNA interference mutant cell line showing no fumarase activity indicates that fumarases are essential for the parasite. Interestingly, addition of fumarate to the medium rescues the growth phenotype, indicating that fumarate is an essential intermediary metabolite of the insect stage trypanosomes. We propose that trypanosomes use fumarate as an essential electron acceptor, as exemplified by the fumarate dependence previously reported for an enzyme of the essential de novo pyrimidine synthesis (Takashima, E., Inaoka, D. K., Osanai, A., Nara, T., Odaka, M., Aoki, T., Inaka, K., Harada, S., and Kita, K. (2002) Mol. Biochem. Parasitol. 122, 189-200).

Cross Talk between a Fungal Blue-Light Perception System and the Cyclic AMP Signaling Pathway

Blue light regulates many physiological and developmental processes in fungi. In Trichoderma atroviride the complex formed by the BLR-1 and BLR-2 proteins appears to play an essential role as a sensor and transcriptional regulator in photoconidiation. Here we demonstrate that the BLR proteins are necessary for carbon deprivation induced conidiation, even in the absence of light, pointing to the existence of an unprecedented cross talk between light and carbon sensing. Further, in contrast to what has been found in all other fungal systems, clear BLR-independent blue-light responses, including the activation of protein kinase A (PKA) and the regulation of gene expression, were found. Expression of an antisense version of the pkr-1 gene, encoding the regulatory subunit of PKA, resulted in a nonsporulating phenotype, whereas overexpression of the gene produced colonies that conidiate even in the dark. In addition, overexpression of pkr-1 blocked the induction of early light response genes. Thus, our data demonstrate that PKA plays an important role in the regulation of light responses in Trichoderma. Together, these observations suggest that the BLR complex plays a general role in sensing environmental cues that trigger conidiation and that such a role can be separated from its function as a transcription factor.

Light-Activated Phosphatase

Light influences many plant processes such as development, circadian responses including flowering, and growth. Plants are able to sense and to respond specifically to different wavelengths of light; red and far-red light are sensed by phytochromes, which are light-regulated serine and threonine kinases. Using a yeast two-hybrid screen, Kim et al. identified a protein phosphatase 2A catalytic subunit (from pea) that interacted with the Arabidopsis phytochrome A. The authors designated the phosphatase FyPP for flower-specific, phytochrome-associated protein phosphatase. In vitro, FyPP interacted with phytochrome A and B, with a preference for phytochrome B. When the phytochromes were pretreated with far-red light, they were designated Pfrs and FyPP had a preference for these. If Pfrs were not phosphorylated, they interacted more strongly with FyPP. Red light stimulates the translocation of phytochromes to the nucleus. Using a green-fluorescent protein fusion protein, the authors determined that FyPP was exclusively cytosolic. Thus, FyPP appears to interact specifically with the cytosolic Pfrs. In vitro phosphatase assays demonstrated that FyPP was able to dephosphorylate phytochrome A in the presence of Zn2+ or Fe2+ and that dephosphorylation occurred more quickly for the Pfr than for the red phytochrome (Pr). Overexpression of pea FyPP in Arabidopsis delayed flowering, whereas an antisense version produced plants that flowered earlier than wild-type plants. Thus, FyPP appears to be a negative regulator of the long-day flowering response.D.-H. Kim, J.-G. Kang, S.-S. Yang, K.-S. Chung, P.-S. Song, C.-M. Park, A phytochrome-associated protein phsophatase 2A modulates light signals in flowering time control in Arabidopsis. Plant Cell 14, 3043-3056 (2002). [Abstract] [Full Text]

Light-Activated Phosphatase

Light influences many plant processes such as development, circadian responses including flowering, and growth. Plants are able to sense and to respond specifically to different wavelengths of light; red and far-red light are sensed by phytochromes, which are light-regulated serine and threonine kinases. Using a yeast two-hybrid screen, Kim et al. identified a protein phosphatase 2A catalytic subunit (from pea) that interacted with the Arabidopsis phytochrome A. The authors designated the phosphatase FyPP for flower-specific, phytochrome-associated protein phosphatase. In vitro, FyPP interacted with phytochrome A and B, with a preference for phytochrome B. When the phytochromes were pretreated with far-red light, they were designated Pfrs and FyPP had a preference for these. If Pfrs were not phosphorylated, they interacted more strongly with FyPP. Red light stimulates the translocation of phytochromes to the nucleus. Using a green-fluorescent protein fusion protein, the authors determined that FyPP was exclusively cytosolic. Thus, FyPP appears to interact specifically with the cytosolic Pfrs. In vitro phosphatase assays demonstrated that FyPP was able to dephosphorylate phytochrome A in the presence of Zn 2+ or Fe 2+ and that dephosphorylation occurred more quickly for the Pfr than for the red phytochrome (Pr). Overexpression of pea FyPP in Arabidopsis delayed flowering, whereas an antisense version produced plants that flowered earlier than wild-type plants. Thus, FyPP appears to be a negative regulator of the long-day flowering response. D.-H. Kim, J.-G. Kang, S.-S. Yang, K.-S. Chung, P.-S. Song, C.-M. Park, A phytochrome-associated protein phsophatase 2A modulates light signals in flowering time control in Arabidopsis . Plant Cell 14 , 3043-3056 (2002). [Abstract] [Full Text]

Trichoderma atroviride G-protein alpha-subunit gene tga1 is involved in mycoparasitic coiling and conidiation.

The soil fungus Trichoderma atroviride, a mycoparasite, responds to a number of external stimuli. In the presence of a fungal host, T. atroviride produces hydrolytic enzymes and coils around the host hyphae. In response to light or nutrient depletion, asexual sporulation is induced. In a biomimetic assay, different lectins induce coiling around nylon fibers; coiling in the absence of lectins can be induced by applying cyclic AMP (cAMP) or the heterotrimeric G-protein activator mastoparan. We isolated a T. atroviride G-protein alpha-subunit (Galpha) gene (tgal) belonging to the fungal subfamily with the highest similarity to the Galpha1 class. Generated transgenic lines that overexpress Galpha show very delayed sporulation and coil at a higher frequency. Furthermore, transgenic lines that express an activated mutant protein with no GTPase activity do not sporulate and coil at a higher frequency. Lines that express an antisense version of the gene are hypersporulating and coil at a much lower frequency in the biomimetic assay. The loss of Tgal in these mutants correlates with the loss of GTPase activity stimulated by the peptide toxin Mas-7. The application of Mas-7 to growing mycelial colonies raises intracellular cAMP levels, suggesting that Tgal can activate adenylyl cyclase. In contrast, cAMP levels and cAMP-dependent protein kinase activity drop when diffusible host signals are encountered and the mycoparasitism-related genes ech42 and prb1 are highly expressed. Mycoparasitic signaling is unlikely to be a linear pathway from host signals to increased cAMP levels. Our results demonstrate that the product of the tga1 gene is involved in both coiling and conidiation.

Metabolic engineering of essential oil yield and composition in mint by altering expression of deoxyxylulose phosphate reductoisomerase and menthofuran synthase.

Peppermint (Mentha x piperita L.) was independently transformed with a homologous sense version of the 1-deoxy-d-xylulose-5-phosphate reductoisomerase cDNA and with a homologous antisense version of the menthofuran synthase cDNA, both driven by the CaMV 35S promoter. Two groups of transgenic plants were regenerated in the reductoisomerase experiments, one of which remained normal in appearance and development; another was deficient in chlorophyll production and grew slowly. Transgenic plants of normal appearance and growth habit expressed the reductoisomerase transgene strongly and constitutively, as determined by RNA blot analysis and direct enzyme assay, and these plants accumulated substantially more essential oil (about 50% yield increase) without change in monoterpene composition compared with wild-type. Chlorophyll-deficient plants did not afford detectable reductoisomerase mRNA or enzyme activity and yielded less essential oil than did wild-type plants, indicating cosuppression of the reductoisomerase gene. Plants transformed with the antisense version of the menthofuran synthase cDNA were normal in appearance but produced less than half of this undesirable monoterpene oil component than did wild-type mint grown under unstressed or stressed conditions. These experiments demonstrate that essential oil quantity and quality can be regulated by metabolic engineering. Thus, alteration of the committed step of the mevalonate-independent pathway for supply of terpenoid precursors improves flux through the pathway that leads to increased monoterpene production, and antisense manipulation of a selected downstream monoterpene biosynthetic step leads to improved oil composition.

BDNF gene transfer to the mammalian brain using CNS-derived neural precursors.

Neural stem cell lines represent a homogeneous source of cells for genetic, developmental, and gene transfer and repair studies in the nervous system. Since both gene transfer of neurotrophic factors and cell replacement strategies are of immediate interest for therapeutical purposes, we have generated BDNF-secreting neural stem cell lines and investigated to what extent different endogenous levels of BDNF expression affect in vitro survival, proliferation and differentiation of these cells. Also, we have investigated the in vivo effects of such BDNF gene transfer procedure in the rat neostriatum. Hippocampus- and cerebellum-derived cell lines reacted differently to manipulations aimed at varying their levels of BDNF production. Over-expression of BDNF enhanced survival of both cell types, in a serum-deprivation assay. Conversely, and ruling out unspecific effects, expression of an antisense version of BDNF resulted in compromised survival of cerebellum-derived cells, and in a lethal phenotype in hippocampal progenitors. These data indicate that endogenous BDNF level strongly influences the in vitro survival of these cells. These effects are more pronounced for hippocampus- than for cerebellum-derived progenitors. Hippocampus-derived BDNF overproducers showed no major change in their capacity to differentiate towards a neuronal phenotype in vitro. In contrast, cerebellar progenitors overproducing BDNF did not differentiate into neurons, whereas cells expressing the antisense BDNF construct generated cells with morphological features of neurons and expressing immunological neuronal markers. Taken together, these results provide evidence that BDNF controls both the in vitro survival and differentiation of neural stem cells. After in vivo transplantation of BDNF-overproducing cells to the rat neostriatum, these survived better than the control ones, and induced the expected neurotrophic effects on cholinergic neurons. However, long-term (3 months) administration of BDNF resulted in detrimental effects, at this location. These findings may be of importance for the understanding of brain development, for the design of therapeutic neuro-regenerative strategies, and for cell replacement and gene therapy studies.

Ribozyme-mediated decrease in mumps virus nucleocapsid mRNA level and progeny in infected vero cells.

The effects of endogenously expressed ribozymes directed to the mumps virus nucleocapsid (NP) mRNA were studied during viral infection. To this end, eukaryotic expression vectors encoding ribozymes or controls of passive hybridization effects were constructed and used to transfect mumps permissive Vero cells. Transcripts spanning trans-acting ribozymes of the hammerhead and hairpin types were designed to hydrolyze the first 5'GUC-3' sequence downstream from the initiation site and to hybridize to a 16 base sequence containing the putative cleavage site. Control vectors encoded mutated and catalytically inactive forms of the ribozymes or a 16 base antisense version of the target sequence. When stably expressed in cells, both ribozymes and passive control RNAs reduced viral yields. A ribozyme-mediated effect on viral growth was, however, observed, as both ribozyme types reduced viral titers by approximately 80%, well above the highest inhibition level of approximately 35% found when noncatalytic RNAs were expressed. In addition, levels of NP mRNA were generally lower in cells expressing catalytic RNAs, supporting the observed inhibition of viral growth. Although cleavage in vitro of a synthetic analog of the NP mRNA was demonstrated using RNAs isolated from ribozyme-expressing cells, in vivo cleavage products were not detectable despite the use of sensitive methods, possibly because of degradation phenomena. We also suggest here that additional controls should be conducted when semicompetitive RT-PCR methods are used to evaluate intracellular cleavage by ribozymes, as the results may depend on the initial target RNA concentration.

Tomato Spotted Wilt Virus Resistance in Chrysanthemum Expressing the Viral Nucleocapsid Gene.

Three tomato spotted wilt virus (TSWV) nucleocapsid (N) gene constructs were employed for Agrobacterium-mediated transformation of chrysanthemum (Dendranthema grandiflora) cv. Polaris. These constructs contained either a full-length N gene (pTSWVN+), a full-length N gene encoding a truncated N protein (pTSWVNt), or an antisense version of the full-length N gene (pTSWVN-), all derived from a dahlia isolate of TSWV (TSWV-D). Initial resistance screens were conducted on cuttings made from 152 pTSWVN+, 37 pTSWVNt, and 47 pTSWVN- transformed plants employing a highly virulent, heterologous strain of TSWV (TSWV-GB) isolated from chrysanthemum and vectored by thrips. This screening served to eliminate the majority of TSWV-susceptible transgenic lines. More rigorous resistance tests with three rounds of mechanical inoculation with TSWV-GB identified one pTSWVNt and two pTSWVN- transformed lines that exhibited a total lack of systemic symptoms and no virus accumulation. Six other lines, including some pTSWVN+, exhibited a lack of one or more of the destructive necrotic TSWV symptoms (stem canker and apical bud death) and a delay in symptom expression. Both sense and antisense constructs, therefore, were found to be effective at yielding TSWV resistance in chrysanthemum. Molecular analysis revealed that the highly TSWV-resistant pTSWVNt line had no detectable levels of N protein. All three resistant lines had low levels of N gene transcript and at least three transgene insertion sites within their genomes, although susceptible lines often had a similar number of insertion sites. The generation of Polaris lines resistant to TSWV transmitted either mechanically or by thrips represents the first time a major ornamental crop has been genetically engineered for disease resistance.

Differential expression of antisense in regenerated tobacco plants transformed with an antisense version of a tomato ACC oxidase gene

Ethylene production was measured during vegetative and reproductive development in normal tobacco plants and in transgenic tobacco plants carrying antisense genes for tomato ACC oxidase driven by the 35S CaMV promoter (Hamilton et al., 1990). When expressed in three independently derived transgenic plants, the antisense ethylene gene failed to affect ethylene production in young/mature leaves or in stems but it did inhibit ethylene production in roots by 37–58%. Ethylene production in developing flowers (i.e. from small unopened flower buds up until open flowers at anthesis) was not affected in transgenic plants but ethylene production in fruits was inhibited by 35%. The most dramatic effect on ethylene production in transgenic plants was seen immediately after wounding leaf tissue, in which case the antisense gene inhibited wound ethylene production by 72%. Thus, the antisense gene composed of a 35S CaMV promoter driving a heterologous ACC oxidase sequence had differential effects on ethylene production in tobacco plants.

1010 DELAYED PETAL SENESCENCE IN TRANSGENIC CARNATION USING ANTISENSE ACC OXIDASE

Ethylene is essential for the senescence process in many fruit and flowers. In the last two steps in the biosynthesis of ethylene in plants ACC synthase converts S-adenosyl methionine to 1-aminocyclopropane-1-carboxylic acid(ACC). ACC oxidase (ACO) then degrades ACC to ethylene. Inhibitors of ethylene synthesis, such as amino-oxyacetic acid, and of the response to ethylene, such as silver thiosulphate, delay or prevent senescence. By expression of an antisense version of ACO RNA, we have generated two varieties of transgenic carnation which produce flowers with an extended vase life. These were produced using the cultivars Red Sim and White Sim. Flowers from these plants produce very little ethylene and normally fail to display the inrolling phenotype typical of senescence in this species. At the time after harvest when inrolling would normally lake place (5 days), the antisense ACO flowers produce only barely detectable levels of endogenous ACO mRNA or ACS (ACC Synthase) mRNA. Exposure to exogenous ethylene(100ppm) induces inrolling and production of ACS and ACO mRNA species. Such carnations will be valuable both as a commercial product and as a tool for further exploring the role of ethylene in carnation flower senescence and leaf wound response.

Genetic engineering of potyvirus resistance using constructs derived from the zucchini yellow mosaic virus coat protein gene.

Three versions of the zucchini yellow mosaic virus (ZYMV) coat protein gene were engineered for expression in plants: the full-length coat protein sequence, the conserved core portion of the gene, and an antisense version. These constructs were introduced into muskmelon (Cucumis melo) and tobacco plants (Nicotiana tabacum) via Agrobacterium tumefaciens-mediated transformation; gene expression was verified by Northern and Western analysis. Transgenic R0 and R1 muskmelon plants expressing the full-length coat protein gene exhibited apparent immunity to ZYMV infection: There was a lack of symptom development during a 3-mo observation period and no measurable virus accumulation as determined by ELISA. Melon plants expressing the core or antisense constructs showed a several-day delay of systemic symptom development and reduction in virus titer. Furthermore, transgenic R1 tobacco plants expressing the full-length coat protein, core, or antisense constructs of ZYMV, a nonpathogen of tobacco, showed a short delay in symptom development and reduced virus titer when inoculated with the heterologous potyviruses, potato virus Y, and tobacco etch virus. The transgenic tobacco plants were not protected against the non-potyvirus, tobacco mosaic virus.

Antisense RNA inhibition of ?-glucuronidase gene expression in transgenic tobacco plants

Antisense RNA was used to specifically inhibit the expression of a GUS gene introduced in a transgenic plant. A tobacco transformant containing a single intact copy of the GUS gene and showing relatively high constitutive levels of GUS activity (GUS +) was re-transformed with an Agrobacterium Ti-derived binary vector containing an antisense version of this reporter gene. The sense and antisense GUS genes were each under the regulation of the CaMV 35S promoter. Re-transformed plants contained 1-5 copies of the antisense construct and all showed a greater than 90% reduction in GUS activity relative to the original GUS + plant. This reduction in GUS activity correlated closely with the levels of GUS enzyme and steady state GUS mRNA observed in these plants. The relatively low levels of sense and antisense GUS transcripts found in the re-transformed plants may indicate a rapid degradation of the RNA:RNA duplex in the cell.