Non-transformed Research Articles

Regulation of astaxanthin and its intermediates through cloning and genetic transformation of β-carotene ketolase in Haematococcus pluvialis

Astaxanthin, a high-value ketocarotenoid used in the pharmaceutical and nutraceutical industries is mainly produced from green alga, Haematococcus pluvialis. It is biosynthesized by the action of key enzyme, β-carotene ketolase (BKT) on β-carotene through intermediates echinenone and canthaxanthin. In this study, the β-carotene ketolase (bkt) gene was isolated from H. pluvialis and cloned in a vector pRT100 and further mobilized to a binary vector pCAMBIA 1304. The T-DNA of pCAMBIA 1304, which consists of cloned bkt, was successfully transformed to H. pluvialis through Agrobacterium mediation. The cloning and transformation of bkt in H. pluvialis was confirmed by Southern blotting and also by PCR analysis. Total carotenoids and astaxanthin content in the transformed cells were found to be 2–3-fold higher, while the intermediates like echinenone and canthaxanthin were found to be 8–10-fold higher than in the control cells. The expression level of carotenogenic genes like phytoene synthase (psy), phytoene desaturase (pds), lycopene cyclase (lcy), bkt, and β-carotene hydroxylase (bkh) were found to be higher in transformed cells compared to the non-transformed (NT) H. pluvialis.

Peroxisomal APX knockdown triggers antioxidant mechanisms favourable for coping with high photorespiratory H2 O2 induced by CAT deficiency in rice.

The physiological role of peroxisomal ascorbate peroxidases (pAPX) is unknown; therefore, we utilized pAPX4 knockdown rice and catalase (CAT) inhibition to assess its role in CAT compensation under high photorespiration. pAPX4 knockdown induced co-suppression in the expression of pAPX3. The rice mutants exhibited metabolic changes such as lower CAT and glycolate oxidase (GO) activities and reduced glyoxylate content; however, APX activity was not altered. CAT inhibition triggered different changes in the expression of CAT, APX and glutathione peroxidase (GPX) isoforms between non-transformed (NT) and silenced plants. These responses were associated with alterations in APX, GPX and GO activities, suggesting redox homeostasis differences. The glutathione oxidation-reduction states were modulated differently in mutants, and the ascorbate redox state was greatly affected in both genotypes. The pAPX suffered less oxidative stress and photosystem II (PSII) damage and displayed higher photosynthesis than the NT plants. The improved acclimation exhibited by the pAPX plants was indicated by lower H2 O2 accumulation, which was associated with lower GO activity and glyoxylate content. The suppression of both pAPXs and/or its downstream metabolic and molecular effects may trigger favourable antioxidant and compensatory mechanisms to cope with CAT deficiency. This physiological acclimation may involve signalling by peroxisomal H2 O2 , which minimized the photorespiration.

Overexpression of the rice phosphate transporter gene OsPT6 enhances tolerance to low phosphorus stress in vegetable soybean

Low phosphorus (P) availability is a major constraint in the growth and production of vegetable soybean. To improve P efficiency in vegetable soybean, a rice (Oryza sativa L.) phosphate transporter gene, OsPT6, was transferred into the vegetable soybean cultivar ‘NY-1001’ using an Agrobacterium-mediated cotyledonary-node transformation system. OsPT6 was driven by the constitutive cauliflower mosaic virus (CaMV) 35S promoter in the binary vector pCAMBIA3301-OsPT6. Two independent fertile T0 transgenic plants (12PT6-1 and 12PT6-2) were obtained and confirmed by PCR and Southern blotting analyses. The overexpression of the OsPT6 gene of the T2 transgenic plants was detected by quantitative RT-PCR analysis. The tolerance of the T2 transgenic lines overexpressing OsPT6 to low concentrations of phosphate (low-Pi; 10μMPi) was evaluated by hydroponic culture using a modified Hoagland's nutrient solution. The P contents in leaves, stems, and roots of the transgenic plants were significantly higher than those of the non-transformed (NT) plants under the concentrations of both low- and normal-Pi of standard Hoagland's nutrient solution. Under low-Pi stress, the transgenic plants grew better and exhibited significant increases in plant height, root length, root weight, number of pods and seeds, and seed weight per plant compared with the NT plants. Our data indicate that the overexpression of OsPT6 in transgenic vegetable soybean lines improves P accumulation and growth performance and thereby represents a promising approach for the improvement of P efficiency in vegetable soybean.

Impact of Enhanced Capacity to Scavenge Reactive Oxygen Species on Cold Tolerance of Tobacco

Premise of research. Reactive oxygen species (ROS) accumulate in plant tissues when the plant is exposed to stress, causing damage to cell structure and metabolism. Plants have a variety of mechanisms to prevent such damage, including a number of enzymes that scavenge ROS. The impact of specific enzymes, however, is as yet poorly understood.Methodology. The impact on plant tolerance to cold stress of enzymes involved in scavenging ROS was explored by comparing transplastomic tobacco lines overexpressing genes encoding glutathione reductase (GR), dehydroascorbate reductase (DHAR), glutathione-S-transferase (GST), and manganese superoxide dismutase, singly or in pairwise combinations, with nontransformed (NT) tobacco.Pivotal results. Chilling to 10°C resulted in less photoinhibition in lines overexpressing GR alone or in combination with GST, compared to NT tobacco, but more photoinhibition in lines overexpressing DHAR alone or in combination with GR. These differences were not, however, reflected in other indicators of stress. When chilled at 4°C, all lines showed similar damage to NT, except under relatively high photosynthetically active radiation, in which case photoinhibition was reduced in all transplastomic lines compared to NT. Growth over several weeks in a greenhouse was inhibited in the transplastomic lines when the duration included transient cool air temperatures. When optimal air temperature was maintained, growth over a similar duration was equal in transplastomics and NT tobacco.Conclusions. These experiments suggest a subtle interaction of enhanced ROS scavenging and tolerance of chilling stress.

The knockdown of chloroplastic ascorbate peroxidases reveals its regulatory role in the photosynthesis and protection under photo-oxidative stress in rice

The inactivation of the chloroplast ascorbate peroxidases (chlAPXs) has been thought to limit the efficiency of the water–water cycle and photo-oxidative protection under stress conditions. In this study, we have generated double knockdown rice (Oryza sativa L.) plants in both OsAPX7 (sAPX) and OsAPX8 (tAPX) genes, which encode chloroplastic APXs (chlAPXs). By employing an integrated approach involving gene expression, proteomics, biochemical and physiological analyses of photosynthesis, we have assessed the role of chlAPXs in the regulation of the protection of the photosystem II (PSII) activity and CO2 assimilation in rice plants exposed to high light (HL) and methyl violagen (MV). The chlAPX knockdown plants were affected more severely than the non-transformed (NT) plants in the activity and structure of PSII and CO2 assimilation in the presence of MV. Although MV induced significant increases in pigment content in the knockdown plants, the increases were apparently not sufficient for protection. Treatment with HL also caused generalized damage in PSII in both types of plants. The knockdown and NT plants exhibited differences in photosynthetic parameters related to efficiency of utilization of light and CO2. The knockdown plants overexpressed other antioxidant enzymes in response to the stresses and increased the GPX activity in the chloroplast-enriched fraction. Our data suggest that a partial deficiency of chlAPX expression modulate the PSII activity and integrity, reflecting the overall photosynthesis when rice plants are subjected to acute oxidative stress. However, under normal growth conditions, the knockdown plants exhibit normal phenotype, biochemical and physiological performance.

The mitochondrial glutathione peroxidase GPX3 is essential for H2O2 homeostasis and root and shoot development in rice

Glutathione (GSH) peroxidases (GPXs: EC 1.11.1.9 and EC1.11.1.12) are non-heme thiol peroxidases that catalyze the reduction of H2O2 or organic hydroperoxides to water, and they have been identified in almost all kingdoms of life. The rice glutathione peroxidase (OsGPX) gene family is comprised of 5 members spread throughout a range of sub cellular compartments. The OsGPX gene family is induced in response to exogenous H2O2 and cold stress. In contrast, they are down regulated in response to drought and UV-B light treatments. Transgenic rice plants have been generated that lack mitochondrial OsGPX3. These GPX3s plants showed shorter roots and shoots compared to non-transformed (NT) plants, and higher amounts of H2O2 mitochondrial release were observed in the roots of these plants cultivated under normal conditions. This accumulation of H2O2 is positively associated with shorter root length in GPX3s plants compared to NT ones. Moreover, GPX3 promoter analysis indicated that it is mainly expressed in root tissue. These results suggest that silencing the mitochondrial OsGPX3 gene impairs normal plant development and leads to a stress-induced morphogenic response via H2O2 accumulation.

Overexpression of tobacco osmotin (Tbosm) in soybean conferred resistance to salinity stress and fungal infections

Salinity and fungal diseases are the two significant constraints limiting soybean productivity. In order to address these problems, we have transformed soybean cv. Pusa 16 via somatic embryogenesis with salinity induced and apoplastically secreted pathogenesis-related tobacco osmotin (Tbosm) gene using Agrobacterium-mediated genetic transformation. Integration of Tbosm in randomly selected five GUS assay-positive independently transformed soybean plants was confirmed by polymerase chain reaction (PCR) and Southern hybridization. Reverse transcriptase-PCR (RT-PCR) and Western blotting confirmed that the Tbosm was expressed in three of the five transformed soybean plants. Further the Western blotting revealed that the truncated osmotin protein accumulated more in apoplastic fluid. The transformed (T(1)) soybean plants survived up to 200 mM NaCl, whereas non-transformed (NT) plants could withstand till 100 mM and perished at 150 mM NaCl. The biochemical analysis revealed the T(1) soybean plants accumulated higher amount of proline, chlorophyll, APX, CAT, SOD, DHAR, MDHAR, and RWC than NT plants. Leaf gas exchange measurements revealed that T(1) soybean plants maintained higher net photosynthetic rate, CO(2) assimilation, and stomatal conductance than NT plants. The three T(1) soybean plants expressing the osmotin gene also showed resistance against three important fungal pathogens of soybean--Microsphaera diffusa, Septoria glycines and Phakopsora pachyrhizi. The T(1) soybean plants produced 32-35 soybean pods/plant containing 10.3-12.0 g of seeds at 200 mM NaCl, whereas NT plant produced 28.6 soybean pods containing 9.6 g of seeds at 100 mM NaCl. The present investigation clearly shows that expression of Tbosm enhances salinity tolerance and fungal disease resistance in transformed soybean plants.

Transgenic Potato Overexpressing the Amaranthus caudatus Agglutinin Gene to Confer Aphid Resistance

Potato (Solanum tuberosum L.) was transformed with a pBCACAc vector harboring the Amaranthus caudatus agglutinin (ACA) gene mediated by Agrobacterium tumefaciens (Smith and Townsend 1907) Conn. One hundred and eighteen of a total 320 regenerated plants were confirmed to possess the ACA gene integrated into the potato genome as detected by polymerase chain reaction (PCR) and Southern blot analysis. Three transgenic events (referred to as PA1, PA2, and PA3) with one copy of the ACA gene insertion and no obvious phenotypic change were selected and further evaluated for expression level of ACA protein and performance in insect bioassays. Based on western blot assays, the content of ACA protein in PA1, PA2, and PA3 events was high at 0.11, 0.32, and 0.29%, respectively, of total soluble leaf protein. The propagation of peach‐potato aphids (Myzus persicae Sulzer) in the three events was significantly inhibited, with 49, 18.5, and 25 aphids per plant, respectively, compared to 102.5 aphids per plant in nontransformed (NT) controls based on insect bioassays in greenhouse trials. The results of a field trial (in Beijing in 2009) showed that the number of aphids was significantly lower in transgenic plants than in NT. Tuber weight and plant yield of the three transgenic events were significantly higher than those of NT. Our results indicated that PA2 and PA3 can be regarded as potential germplasm to breed new cultivars that may decrease production loss from resistance to aphids and reduced indirect virus damage.

Role of peroxidases in the compensation of cytosolic ascorbate peroxidase knockdown in rice plants under abiotic stress

Current studies, particularly in Arabidopsis, have demonstrated that mutants deficient in cytosolic ascorbate peroxidases (APXs) are susceptible to the oxidative damage induced by abiotic stress. In contrast, we demonstrate here that rice mutants double silenced for cytosolic APXs (APx1/2s) up-regulated other peroxidases, making the mutants able to cope with abiotic stress, such as salt, heat, high light and methyl viologen, similar to non-transformed (NT) plants. The APx1/2s mutants exhibited an altered redox homeostasis, as indicated by increased levels of H₂O₂ and ascorbate and glutathione redox states. Both mutant and NT plants exhibited similar photosynthesis (CO₂) assimilation and photochemical efficiency) under both normal and stress conditions. Overall, the antioxidative compensatory mechanism displayed by the mutants was associated with increased expression of OsGpx genes, which resulted in higher glutathione peroxidase (GPX) activity in the cytosolic and chloroplastic fractions. The transcript levels of OsCatA and OsCatB and the activities of catalase (CAT) and guaiacol peroxidase (GPOD; type III peroxidases) were also up-regulated. None of the six studied isoforms of OsApx were up-regulated under normal growth conditions. Therefore, the deficiency in cytosolic APXs was effectively compensated for by up-regulation of other peroxidases. We propose that signalling mechanisms triggered in rice mutants could be distinct from those proposed for Arabidopsis.

Overexpression of 2-cysteine peroxiredoxin enhances tolerance to methyl viologen-mediated oxidative stress and high temperature in potato plants

Oxidative stress is one of the major causative factors for injury to plants exposed to environmental stresses. Plants have developed diverse defense mechanisms for scavenging oxidative stress-inducing molecules. The antioxidative enzyme 2-cysteine peroxiredoxin (2-Cys Prx) removes peroxides and protects the photosynthetic membrane from oxidative damage. In this study, transgenic potato (Solanum tuberosum L. cv. Atlantic) expressing At2-Cys Prx under control of the oxidative stress-inducible SWPA2 promoter or enhanced CaMV 35S promoter (referred to as SP and EP plants, respectively) was generated using Agrobacterium-mediated transformation. The transgenic plants were tested for tolerance to stress. Following treatment with 3 μM methyl viologen (MV), leaf discs from SP and EP plants showed approximately 33 and 15% less damage than non-transformed (NT) plants. When 300 μM MV was sprayed onto whole plants, the photosynthetic activity of SP plants decreased by 25%, whereas that of NT plants decreased by 60%. In addition, SP plants showed enhanced tolerance to high temperature at 42 °C. After treatment at high temperature, the photosynthetic activity of SP plants decreased by about 7% compared to plants grown at 25 °C, whereas it declined by 31% in NT plants. These results indicate that transgenic potato can efficiently regulate oxidative stress from various environmental stresses via overexpression of At2-Cys Prx under control of the stress-inducible SWPA2 promoter.

Defensive Role of Tomato Polyphenol Oxidases against Cotton Bollworm (Helicoverpa armigera) and Beet Armyworm (Spodoptera exigua)

Tomato (Solanum lycopersicum) polyphenol oxidases (PPOs), enzymes that oxidize phenolics to quinones, have been implicated in plant resistance to insects. The role of PPO in resistance to cotton bollworm [Helicoverpa armigera (Hübner)] and beet armyworm [Spodoptera exigua (Hübner)] (Lepidoptera: Noctuidae) was evaluated. Consumption, weight gains, and mortality of larvae feeding on foliage of transgenic tomato lines overexpressing PPO (OP lines) and of larvae feeding on foliage of transgenic tomato lines with suppressed PPO (SP lines) were compared with consumption, weight gains, and mortality of larvae feeding on non-transformed (NT) plants. Increases in foliage consumption and weight gains were observed for cotton bollworms feeding on leaves of SP plants compared to NT and OP plants. PPO activity was negatively correlated with both weight gains and foliar consumption of cotton bollworm, substantiating the defensive role of PPO against this insect. Similarly, beet armyworm consumed less foliage (both young and old leaves) from OP plants than SP plants. Larvae feeding on OP leaves generally exhibited lower weight gains than those feeding on SP leaves. These results indicate that tomato PPO plays a role in resistance to both cotton bollworm and beet armyworm.

Enhanced tolerance of transgenic sweetpotato plants that express both CuZnSOD and APX in chloroplasts to methyl viologen-mediated oxidative stress and chilling

Oxidative stress is one of the major factors causing injury to plants exposed to environmental stress. Transgenic sweetpotato [Ipomoea batatas (L.) Lam. cv. Yulmi] plants with an enhanced tolerance to multiple environmental stresses were developed by expressing the genes of both CuZn superoxide dismutase (CuZnSOD) and ascorbate peroxidase (APX) under the control of an oxidative stress-inducible SWPA2 promoter in the chloroplasts of sweetpotato plants (referred to as SSA plants). SSA plants were successfully generated by the particle bombardment method and confirmed by PCR analysis. When leaf discs of SSA plants were subjected to 5 μM methyl viologen (MV), they showed approximately 45% less damage than non-transformed (NT) plants. When 200 μM MV was sprayed onto the whole plants, SSA plants showed a significant reduction in visible damage compared to leaves of NT plants, which were almost destroyed. The expression of the introduced CuZnSOD and APX genes in leaves of SSA plants following MV treatment was significantly induced, thereby reflecting increased levels of SOD and APX in the chloroplasts. APX activity in chloroplast fractions isolated from SSA plants was approximately 15-fold higher than that in their counterparts from NT plants. SSA plants treated with a chilling stress consisting of 4°C for 24 h exhibited an attenuated decrease in photosynthetic activity (Fv/Fm) relative to NT plants; furthermore, after 12 h of recovery following chilling, the Fv/Fm of SSA plants almost fully recovered to the initial levels, whereas NT plants remained at a lower level of Fv/Fm activity. These results suggest that SSA plants would be a useful plant crop for commercial cultivation under unfavorable growth conditions. In addition, the manipulation of the antioxidative mechanism in chloroplasts can be applied to the development of various other transgenic crops with an increased tolerance to multiple environmental stresses.

Effects of potato plants expressing a barley cystatin on the predatory bug Podisus maculiventris via herbivorous prey feeding on the plant

The aim of this study was to assess the effects of potato plants expressing a barley cystatin on a potentially cystatin-susceptible natural enemy by predation on susceptible and non-susceptible preys feeding on the plant. We have focussed on the impact of the variant HvCPI-1 C68 --> G, in which the only cysteine residue was changed by a glycine, on the growth and digestive physiology of the Colorado potato beetle (CPB), Leptinotarsa decemlineata, and the Egyptian cotton leafworm (ECW), Spodoptera littoralis. Moreover, we have studied the prey-mediated effects of the barley cystatin at the third trophic level, using the predatory spined soldier bug (SSB), Podisus maculiventris, as a model. Feeding trials conducted with CPB larvae reared on transgenic potato plants expressing the C68 --> G variant resulted in significantly lower weight gains compared to those fed on non-transformed (NT) plants. On the contrary, larger weight gains were obtained when ECW larvae, that lack digestive cysteine proteases, were reared on transgenic potato expressing the cystatin, as compared to larvae fed on NT plants. No negative effects on survival and growth were observed when SSB nymphs were exposed to HvCPI-1 C68 --> G by predation on either CPB or ECW larvae reared on transgenic potato plants expressing the barley cystatin, despite the fact that the inhibitor suppressed in vitro gut proteolysis of the predatory bug. To investigate the physiological background, biochemical analysis were carried out on guts of insects dissected at the end of the feeding assays.

Comparison of the Biotransformation of the14C-Labelled Insecticide Carbaryl by Non-Transformed and Human CYP1A1-, CYP1A2-, and CYP3A4-Transgenic Cell Cultures ofNicotiana tabacum

Transgenic tobacco-cell-suspension cultures expressing separately the human cytochrome P450 monooxygenases CYP1A1, CYP1A2, and CYP3A4 were utilized to study the biotransformation of the 14C-labelled insecticide carbaryl (=naphthalen-1-yl methylcarbamate). The resulting data were compared to similar data from the corresponding non-transformed (NT) tobacco-cell culture and commercially available membrane preparations (Bactosomes) of genetically modified bacteria separately containing the same human P450s. A rapid conversion rate of carbaryl was observed with the CYP1A1 and CYP1A2 cells, where only 49.7 and 0.2% of applied carbaryl (1 mg/l), respectively, remained after 24 h, as compared to 77.7% in the non-transformed culture. Unexpectedly, the corresponding results obtained from the CYP3A4 cultures were not definite. With 25 mg/l of carbaryl and 96 h of incubation, it was proven that the insecticide is also substrate of CYP3A4. This finding was supported by GC/EI-MS analysis of the primary metabolite pattern produced by the isozyme. This consisted of naphthalene-1-ol, N-(hydroxymethyl)carbaryl, 4-hydroxycarbaryl, and 5-hydroxycarbaryl, whereas the main product in non-transformed cells was N-(hydroxymethyl)carbaryl. Data obtained from the CYP1A1, CYP1A2, or CYP3A4 Bactosomes agreed with those of the P450-transgenic tobacco cells. Problems with GC/EI-MS analysis of carbaryl and its metabolites are discussed.

Ozone-induced ethylene and foliar injury responses are altered in 1-aminocyclopropane-1-carboxylate synthase antisense potato plants.

• In potato (Solanum tuberosum), two 1-aminocyclopropane-1-carboxylate synthase (ACS) genes are induced by ozone (O3 ). Antisense inhibition of these ethylene (C2 H4 ) biosynthetic genes allowed us to examine the relationship between O3 -induced C2 H4 emission and foliar injury. • Thirty-two lines (antisense for ST-ACS4 or ST-ACS5) were screened in the glasshouse for acute O3 -induced C2 H4 and lesions. Stomatal conductance and ACS transcripts were quantified for selected C2 H4 -altered lines. Six lines were field-tested for chronic O3 effects. • Ten lines produced less, and four lines produced more, acute-O3 -induced C2 H4 than nontransformed (NT) plants. Ethylene levels did not appear to be correlated with stomatal conductance. ST-ACS4 and -5 transcript were reduced in transgenic plants, except in two C2 H4 over-producing lines. In the field, these C2 H4 over-producing lines displayed stunting and leaf rolling in charcoal-filtered (CF) air and chronic O3 , and they sustained the most severe O3 injury. • When C2 H4 production was strongly suppressed or enhanced, corresponding reductions or increases in lesion severity were observed, suggesting a critical role for C2 H4 in the lesion formation process during O3 stress.

Root and hypocotyl growth in transgenic tomatoes that express the bacterial enzyme ACC deaminase

The seeds of tomatoes transformed with a bacterial ACC deaminase gene under the transcriptional control of either the cauliflower mosiac virus 355 promoter, the ro/D (root specific) promoter or the prb-1b (pathogenesis related) promoter, dand the seeds of nontransformed (NT) tomato plants were germinated in the presence or absence of ACC in the dark, in the light, or in the presence of UV-B light for 12 days. Roots grew to a greater length under UV-B than in the light or dark, in the absence of ACC for all of the plant lines tested. In contrast, root growth was inhibited by ACC under all conditions tested. The 35S/ACC deaminase transgenic seedlings had the longest hypocotyl, the ro/D/ACC deaminase had intermediate sized hypocotyls and the NT and the prb-1b/ACC deaminase had the shortest hypocotyl in the presence of ACC in the dark. Hypocotyl growth was inhibited by both light and UV-B in both the absence and presence of ACC in NT and transformed plants. Taken together these results indicate that root growth is more sensitive to applied ACC (the precursor of ethylene) than is hypocotyl growth under all of the light regimes tested.

Differential Resistance to Methyl Viologen in Transgenic Tobacco Plants that Express Sweet Potato Peroxidases

To analyze the physiological role of each peroxidase (POD) isoenzyme in environmental stress adaptation, transgenic tobacco ( Nicotiana tabacum ) plants expressing either a sweet potato ( Ipomoea batatas ) anionic POD ( swpa 1) or neutral POD ( swpn 1) were tested for responses to oxidant methyl viologen (MV). Fully expanded leaves of both swpa 1 - and swpn 1 -transgenic plants had about two times higher POD activity than non transformed (NT) plants. When tobacco leaf discs were subjected to MV at 10 and 100 µmol/L, swpa 1 -transgenic plants showed about a 25 % reduction in membrane damage relative to swpn 1 -transgenic or NT plants. Leaves of swpn 1 -transgenic and NT plants were bleached more than those of swpa 1 -transgenics by 1 µmol/L MV treatment, whereas all plants were severely damaged at 3 µmol/L MV. These results indicate that the increased H 2 O 2 -scavenging capacity provided by the swpa 1 POD (a guaiacol-type POD) contributes to increased protection against MV-mediated oxidative damage. Furthermore, differences in response to oxidant treatments indicate unique functions for the two isoenzymes encoded by swpa 1 and swpn 1 .

SOD2: a new type of tumor-suppressor gene?

The activity of superoxide dismutases (SOD) 1 and 2 was analysed in correlation with mRNA and chromosome content in 6 SV40-transformed (TF) and in non-transformed (NF) human fibroblast cell lines. Total SOD activity was fairly constant, whereas the ratio SOD2/SOD1 was much lower in TF than in NF. The decrease in SOD2 activity was correlated with a low mRNA content, and with the presence of various chromosomal rearrangements leading to deletions of the long arm of chromosome 6 where the gene is mapped. In contrast, chromosome 21, carrying the gene for SOD1, was not found to be deficient and the SOD1 activity was high. This shows that in TF, the activity of SOD2 is largely determined by gene dosage. It has been proposed that SOD activity could be inversely correlated with cell proliferation, and that SOD2 activity, in particular, was related to cell differentiation. Thus, there is a cascade of events occurring in cell transformation, involving gene deregulation, chromosome (gene) deletion, low mRNA and protein content, low enzyme activity, and acquisition of growth advantage which makes the SOD2 gene a possible new type of tumor-suppressor gene.

Colorado Potato Beetle Control with Genetically Engineered (Transgenic) Potato Plants, 1991

Abstract Test plots using 'Russet Burbank' potatoes that had been genetically engineered to contain a transformed Bacillus thuringiensis var. tenebrionis (Bt) gene were established at the UI Research and Extension Center, Aberdeen, ID. Two 'Russet Burbank' check plots and five lines, including a non-transformed (NT) line, of minitubers obtained from Monsanto, were hand planted on 20 May. All treatment plots were replicated six times. The transgenic potato lines planted ranged from pea to golf ball size mini-tubers. Due to the small average size, these tubers were planted at a shallow depth of not more than four inches. Individual plots were 1 row of ten hills (1 ft spacing between hills) with treatment rows bordered by a blank row. Between the blank rows was an untreated row of 'Russet Burbank' potatoes. Irrigation of plots was by sprinklers. No chemical controls for CPB were used other than to one of the 'Russet Burbank' check treatments when an application of Asana (0.02 lb Al/acre) was made on 11 Jul. On a weekly basis, all stages of beetles were counted and percent defoliation estimates were made using whole plant inspections of all plants within the single plot rows. Larval stages were separated into small (1-2 instar) and large (3-4 instar). Data were analyzed using ANOVA and Newman-Keuls studentized range test.

2-Deoxy-D-Glucose Uptake by Chick Embryo Cells: a Biochemical Indicator of Genetic Susceptibility to RNA Tumour Viruses

The enhanced glucose uptake by chick embryo cells as early as 72 h after infection with Rous sarcoma virus (RSV) was confirmed in this study to be an early indicator of cellular transformation. The glucose uptake of C/E BrL cells infected by various doses of BS-RSV of subgroup A showed that the relationship between the log-dose of virus and log-uptake was linear (slope, b1 = 1.30 +/- 0.14) when the ratio of the number of infectious virus particles to the number of cells in the culture was above 1:200. But infection of cultures with a relatively high dose of virus, for instance 10(3.5) focus forming units (f.f.u.) was ineffective for the measurement of cellular transformation using the criterion of glucose uptake, whereas a much lower dose such as 10(1.7) f.f.u. was sufficient to induce foci of transformed cells. We concluded therefore that the statistic of glucose uptake assay (GUA) measured at 72 p.i. is less sensitive than that of the focus count assay (FCA) measured after 10 days as a measure of assessing cellular transformation by RSV. Nevertheless, when the cultures were infected with a higher dose of virus (10(4.3) f.f.u. or more), the GUA could discriminate between the transformed (T) and non-transformed (NT) cultures. This was demonstrated in the two genetic crosses, line 7-2 X WC(F1) and line 7-2 C line. Embryo cultures of these two test-crosses were infected with viruses of subgroups A, B, C and D, and the T and NT phenotypes were ascertained. Also, on the basis of focus counts in the cultures the genetically resistant (R) and susceptible (S) phenotypes in response to various infections were determined. The T and NT phenotypes based on the GUA were compared with the S and R phenotypes, respectively, based on the FCA. It was found that in 47 of the 51 cultures, the phenotypic agreement was perfect, suggesting that glucose uptake by cells of embryo cultures exposed to RSV is a biochemical indicator of genetic susceptibility. The discordant results in 4 cultures are discussed in the light of present knowledge of cellular transformation by RSV.