Either soluble or plastidic expression of recombinant protoporphyrinogen oxidase modulates tetrapyrrole biosynthesis and photosynthetic efficiency in transgenic rice

Protoporphyrinogen oxidase (Protox) is the last shared enzyme of the porphyrin pathway. As a continuation of our previous work in which the transgenic rice plants expressing the Bacillus subtilis Protox in the cytoplasm or the plastid showed resistance to diphenyl ether herbicide, this study was undertaken to identify the effects of tertapyrrole biosynthesis in these transgenic rice plants. The transgenic plants either targeted into plastids or expressed in cytoplasm showed higher Protox activity than wild-type plants did. Photosynthetic activity, measured as a quantum yield of photosystem II, was slightly higher in transgenic plants than in wild-type plants, but chlorophyll contents were not significantly different between transgenic and wild-type plants. As for porphyrin biosynthesis, both cytoplasm-expressed and plastid-targeted transgenic plants showed increased synthesis of aminolevulinic acid, Mg-Proto IX, and protoheme in comparison to wild-type plants whereas synthesis of protoporphyrin IX was similar for wild-type and transgenic plants. These results indicate that either cytoplasm or plastid expression of B. subtilis Protox in rice can upregulate the porphyrin pathway leading to increase in photosynthetic efficiency in plants.     

Expression of a <Emphasis Type="Italic">Bacillus subtilis</Emphasis> protoporphyrinogen oxidase gene in rice plants reduces sensitivity to peroxidizing herbicides

Protoporphyrinogen oxidase (Protox) in the porphyrin pathway is the target site of the peroxidizing herbicides such as carfentrazone-ethyl and oxyfluorfen. In an attempt to develop herbicide-resistant plants, transgenic rice plants were generated via expression of herbicide-insensitive Bacillus subtilis Protox gene fused to the transit sequence for targeting to the plastid using Agrobacterium-mediated gene transformation. Homozygous transgenic rice lines of T₃ generation selected by hygromycin resistance test were examined if they are resistant to the herbicides carfentrazone-ethyl and oxyfluorfen. The homozygous transgenic lines had single copy insertion of B. subtilis Protox gene into their genomes and express its mRNA. Compared to wild-type rice, the transgenic lines were less susceptible to the herbicides when examined with respect to growth, electrolyte leakage, chlorophyll loss and lipid peroxidation. The in vitro Protox activities in transgenic lines were about 56 % higher than those in wild-type rice. With 10 µM concentration of the herbicides in the enzyme assays, Protox activities in transgenic lines were similar to those in non-inhibited wild-type rice. Less amount of protoporphyrin IX was accumulated in transgenic lines than in wild-type rice upon the treatment of the herbicides at 10 µM concentration. Our results indicated that expression of B. subtilis Protox gene was stably transmitted into T₃ rice plants and reduced their sensitivity to carfentrazone-ethyl and oxyfluorfen.This work was supported by Ministry of Agriculture and Forestry of Korea and Agricultural Plant Stress Research Center (grant No. R11-2001-09203000-0) funded by Korea Science and Engineering Foundation.     

Transgenic Rice Plants Expressing Bacillus subtilis Protoporphyrinogen Oxidase Gene Show Low Herbicide Oxyfluorfen Resistance

Transgenic rice plants harbouring Bacillus subtilis protoporphyrinogen oxidase (Protox) gene, which is targeted into plastid, were generated by Agrobacterium-mediated transformation using a rice (Oryza sativa L. cv. Dongjin) and their gene integration at T₁ generation by Southern and mRNA expression in T₂ generation by Northern blotting were analyzed. Their herbicide-resistant trait was further confirmed by in vitro leaf segment assay and in planta bioassays such as seed germination assay and measurement of growth inhibition. The herbicide oxyfluorfen resistance in transgenic rice plants was not very high. The results showed that the Protox from B. subtilis can not be applicable as a gene source to generate a high level oxyfluorfen tolerance in plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

Overexpression of a cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase in tobacco enhances photosynthesis and growth

Transgenic tobacco plants expressing a cyanobacterial fructose-1,6/sedoheptulose-1,7-bisphosphatase targeted to chloroplasts show enhanced photosynthetic efficiency and growth characteristics under atmospheric conditions (360 p.p.m. CO2). Compared with wild-type tobacco, final dry matter and photosynthetic CO2 fixation of the transgenic plants were 1.5-fold and 1.24-fold higher, respectively. Transgenic tobacco also showed a 1.2-fold increase in initial activity of ribulose 1,5 bisphosphate carboxylase/oxygenase (Rubisco) compared with wild-type plants. Levels of intermediates in the Calvin cycle and the accumulation of carbohydrates were also higher than those in wild-type plants. This is the first report in which expression of a single plastid-targeted enzyme has been shown to improve carbon fixation and growth in transgenic plants.     

Characterization of the potato <Emphasis Type="Italic">upreg1</Emphasis>gene,encoding a mutated ADP-glucose pyrophosphorylase large subunit,in transformed rice

The potato upreg1, which encodes a mutated ADP-glucose pyrophosphorylase (AGPase) large subunit, was introduced into rice to evaluate its potential to enhance sink-driven yield productivity in this crop. We also wished to elucidate the activities of the up-regulated allosteric variants of potato AGPase large subunit gene in rice. A T-DNA vector containing the upreg1 gene under the control of the rice glutelin promoter was constructed with a MAR sequence and transformed into rice using Agrobacterium-mediated transformation. Transgenic plants were selected on medium supplemented with phosphinothricin and confirmed by the application of herbicide. A total of 38 transgenic plants were subsequently obtained in which the integration upreg1 into the rice genome was confirmed by Southern blotting. The exogenous AGPase in transgenic rice plants showed a high affinity for 3-phosphoglycerate activator and a low affinity for the orthophosphate inhibitor, as observed in lettuce. The transgenic rice also showed increases in the number of grains per particle, the number of panicles per plant, and also in the fresh weight of the above-ground mass of plant which was about 15% higher than non-transgenic ‘Nak-dong’. The number of seeds per tiller was also found to be about 10% higher in the transgenic plants. However, the net photosynthesis rate showed very little difference in the transgenic rice, and we could not therefore confirm any linkage with the deregulation of allosteric effects. Based on these results, upreg1 mutant genes can be used for the genetic improvement of plant AGPases other than potato and to effectively increase crop yield productivity.     

Response of transgenic rape plants bearing the <Emphasis Type="Italic">Osmyb4</Emphasis> gene from rice encoding a trans-factor to low above-zero temperature

Accumulation of soluble sugars (sucrose, fructose, and glucose), proline, phenols (total phenols and flavonoids), and antocyanins during adaptation to low-temperature stress (4°C) of two lines of spring rape (Brassica napus L., cv. Westar) characterized by weak (Bn-1) and strong (Bn-3) expression of the Osmyb4 transgene was studied. Vegetatively propagated transgenic and wild-type plants were grown in the hydroponic culture at 24°C; at the stage of 5–6 leaves, plants were exposed to 4°C for 5 days and then returned to the optimum temperature of 24°C for recovery. Transgenic plants were established to manifest improved cold and frost tolerance, which was evident from more active biomass accumulation at 4°C as compared with wild-type plants and from sustaining their viability after 2-day-long exposure to −6°C. Determination of MDA content showed that one of the reasons of their improved cold tolerance was their capability of maintaining oxidative homeostasis under low-temperature stress. This suggestion is supported by intense accumulation of phenols and antocyanins, manifesting pronounced antioxidant effects, by transgenic plants during their cold adaptation. Thus, during 2–5 days of plant exposure to 4°C, in transgenic plants the total content of phenols increased by 2.6–3.7 times, flavonoids — by 3.7–4.7 times, and antocyanins — by 3.5–5.3 times as compared with control plants growing at 24°C. Transgenic Bn-3 plants with strong expression of the Osmyb4 gene accumulated phenols and antocyanins at 4°C more actively than Bn-1 plants characterized by weak expression of this gene. Transgenic rape plants subjected to cold stress accumulated more proline, manifesting stress-protection effects, and lesser accumulation of soluble sugars. Before the beginning of experiment, the content of soluble sugars was approximately similar in wild-type plants and transgenic lines; at 4°C their level in transgenic plants was substantially lower than in control plants. As distinct from the process of cold adaptation, during recovery, the content of all tested stress-protection compounds dropped sharply. The results obtained indicate that active expression of the Osmyb4 gene from rice in the rape plants was accompanied not only by accumulation of compatible osmolytes but also by biosynthesis of antioxidants of phenolic nature.     

Characterization of sunlight-grown transgenic rice plants expressing Arabidopsis phytochrome A

Transgenic rice (Oryza sativa) overexpressing Arabidopsis phytochrome A (phyA) was cultivated up to the T₃ generation in paddy to elucidate the role of phyA in determining the plant architecture and the productivity of sunlight-grown rice plants. PhyA is light-labile and controls plant growth in response to the far-red light-dependent high-irradiance response as well as the very low fluence response. The Arabidopsis phyA gene linked to the rice rbcS promoter was transformed into embryogenic rice calli, and the calli were regenerated to whole plants. Compared to wild-type seedlings, the rbcS::PHYA transgenic seedlings contained more phyA when grown in the dark, and at least 10-fold more phyA when exposed to white light. When grown in paddy, the phyA transgenic plants in general exhibited reduced plant height (dwarfing), larger grain size, higher chlorophyll content, smaller tiller number, and low grain fertility compared to wild-type plants. The heading stage was not significantly changed. However, it is likely that a certain level of phyA is a prerequisite for induction of such changes. It is suggested that phyA overproduction in rice could be a useful tool to improve rice grain productivity by the larger grain size that increases grain yield and the dwarfing that tolerates lodging-associated damage.     

Expression of the rice cytoplasmic cysteine synthase gene in tobacco reduces ozone-induced damage

Cysteine serves as a precursor for the synthesis of various sulfur-containing metabolites, and the cysteine synthase (CS) gene plays a central role in the sulfur cycle in nature. In the present study, rcs1, a cytosolic CS gene of rice, was introduced into the genome of tobacco (Nicotiana tabacum). The tolerance of wild-type tobacco plants as well as of the resulting transgenic tobacco plants overexpressing the rcs1 gene to toxic levels of ozone (O₃, 0.15 μ mol⁻¹) was measured after various lengths of exposure. Leaf lesions in plants exposed for 2 weeks to O₃ were more prevalent in the leaves of the wild-type plants than in those of the transgenic tobacco plants. Transgenic tobacco plants showed a higher growth rate and a higher chlorophyll content than the wild-type plants. Cysteine synthase activity and cysteine and glutathione contents were higher in transgenic plants than in wild-type plants irrespective of the length of the O₃ treatment. Our results indicate that the CS gene plays a role in the protection of the plant against toxic O₃ gas, probably through the mechanism of an over-accumulation of such sulfur-rich antioxidants as cysteine and glutathione.     

Transgenic Agrostis mongolica Roshev. with enhanced tolerance to drought and heat stresses obtained from Agrobacterium-mediated transformation

Efficient procedures for regeneration and Agrobacterium-mediated transformation were established for Agrostis mongolica Roshev. and generated transgenic plants tolerant to drought and heat stresses using a regulatory gene from Arabidopsis, ABF3, which controls the ABA-dependent adaptive responses. The identification and selection of regenerable and reproducible callus type was a key factor for successful transformation. The transformation efficiency was 49.2% and gfp expression was detected in hygromycin-resistant calli and stem of putative transgenic plants. The result of Southern blot analysis showed that the ABF3 transgene was stably integrated into the genome of transgenic plants. Of the five transgenic lines analyzed, single transgene integration was observed in two lines and two copy integration was observed in three transgenic lines. Northern blot analysis confirmed that ubi::ABF3 was expressed in all transgenic lines. Transgenic plants exhibited neither growth inhibition nor visible vegetative phenotypic alternations. However, both transgenic and wild-type plants were highly sterile and did not flower during 3 years of growth period in the open field under subtropical Jeju Island climate. The stomata of the transgenic plants opened less than did stomata of the wild-type plants, and water content of the transgenic leaves remained about 3–4 fold higher than observed for wild-type leaves under drought stress. The transgenic plants showed about 2 fold higher survival rates under drought stress and about 3 fold higher survival rates under heat stress when compared to wild-type plants. Thus, overexpression of the Arabidopsis ABF3 gene results in enhancement of both drought and heat stress tolerance in Agrostis mongolica Roshev.     

Over-expressing GsGST14 from Glycine soja enhances alkaline tolerance of transgenic Medicago sativa

Glutathione-S-transferases (GSTs) are ubiquitous enzymes that play a key role in stress tolerance and cellular detoxification. The GST gene GsGST14 selected from the gene expression profiles of Glycine soja under alkaline stress was transformed into alfalfa (Medicago sativa L.). Transgenic alfalfa plants showed 1.73–1.99 times higher GST activity than wild-type plants. Transgenic alfalfa grew well in the presence of 100 mM NaHCO₃, while wild-type plants exhibited chlorosis and stunted growth, even death. There were marked changes in malondialdehyde content and relative membrane permeability caused by alkaline stress in non-transgenic lines compared to transgenic lines. The results indicate that the gene GsGST14 could enhance alkaline resistance in transgenic alfalfa.     

Overexpressing <Emphasis Type="Italic">SgNCED1</Emphasis> in Tobacco Increases ABA Level,Antioxidant Enzyme Activities,and Stress Tolerance

Abscisic acid (ABA) regulates plant adaptive responses to various environmental stresses. 9-cis-epoxycarotenoid dioxygenase (NCED) is the key enzyme of ABA biosynthesis in higher plants. A NCED gene, SgNCED1, was overexpressed in transgenic tobacco plants which resulted in 51–77% more accumulation of ABA in leaves. Transgenic tobacco plants decreased stomatal conductance, transpiration rate, and photosynthetic rate but induced activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate-peroxidase (APX). Hydrogen peroxide (H₂O₂) and nitric oxide (NO) in leaves were also induced in the transgenic plants. Compared to the wild-type control, the transgenic plants improved growth under 0.1 M mannitol-induced drought stress and 0.1 M NaCl-induced salinity stress. It is suggested that the ABA-induced H₂O₂ and NO generation upregulates the stomatal closure and antioxidant enzymes, and therefore increases drought and salinity tolerance in the transgenic plants.     

A cytosolic NADP-malic enzyme gene from rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) confers salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

NADP-malic enzyme (NADP-ME, EC 1.1.1.40) functions in many different pathways in plant and may be involved in plant defense such as wound and UV-B radiation. Here, expression of the gene encoding cytosolic NADP-ME (cytoNADP-ME, GenBank Accession No. AY444338) in rice (Oryza sativa L.) seedlings was induced by salt stress (NaCl). NADP-ME activities in leaves and roots of rice also increased in response to NaCl. Transgenic Arabidopsis plants over-expressing rice cytoNADP-ME had a greater salt tolerance at the seedling stage than wild-type plants in MS medium-supplemented with different levels of NaCl. Cytosolic NADPH/NADP⁺ concentration ratio of transgenic plants was higher than those of wild-type plants. These results suggest that rice cytoNADP-ME confers salt tolerance in transgenic Arabidopsis seedlings.     

Improved salt tolerance in tobacco plants by co-transformation of a betaine synthesis gene <Emphasis Type="Italic">BADH</Emphasis> and a vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene <Emphasis Type="Italic">SeNHX1</Emphasis>

Three types of transgenic tobacco plants were acquired by separate transformation or co-transformation of a vacuolar Na⁺/H⁺ antiporter gene, SeNHX1, and a betaine synthesis gene, BADH. When exposed to 200 mM NaCl, the dual gene-transformed plants displayed greater accumulation of betaine and Na⁺ than their wild-type counterparts. Photosynthetic rate and photosystem II activity in the transgenic plants were less affected by salt stress than wild-type plants. Transgenic plants exhibited a greater increase in osmotic pressure than wild-type plants when exposed to NaCl. More importantly, the dual gene transformed plants accumulated higher biomass than either of the single transgenic plants under salt stress. Taken together, these findings indicate that simultaneous transformation of BADH and SeNHX1 genes into tobacco plants can enable plants to accumulate betaine and Na⁺, thus conferring them more tolerance to salinity than either of the single gene transformed plants or wild-type tobacco plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Increasing maize seed weight by enhancing the cytoplasmic ADP-glucose pyrophosphorylase activity in transgenic maize plants

ADP-glucose pyrophosphorylase (AGPase) plays a key role in regulating starch biosynthesis in cereal seeds and is likely the most important determinant of seed strength. The Escherichia coli mutant glgC gene (glgC16), which encodes a highly active and allosterically insensitive AGPase, was introduced into maize (Zea mays L.) under the control of an endosperm-specific promoter. Developing seeds from transgenic maize plants showed up to 2–4-fold higher levels of AGPase activity in the presence of 5 mM inorganic phosphate (Pi). Transgenic plants with higher cytoplasmic AGPase activity under Pi-inhibitory conditions showed increases (13–25%) in seed weight over the untransformed control. In addition, in all transgenic maize plants, the seeds were fully filled, and the seed number of transgenic plants had no significant difference compared with that of untransformed control. These results indicate that increasing cytoplasmic AGPase activity has a marked effect on sink activity and, in turn, seed weight in transgenic maize plants.     

Spontaneous plant regeneration in transformed roots and calli from Tylophora indica: changes in morphological phenotype and tylophorine accumulation associated with transformation by Agrobacterium rhizogenes

We examined the effects of genetic transformation by Agrobacterium rhizogenes on the production of tylophorine, a phenanthroindolizidine alkaloid, in the Indian medicinal plant, Tylophora indica. Transformed roots induced by the bacterium grew in axenic culture and produced shoots or embryogenic calli in the absence of hormone treatments. However, hormonal treatment was required to regenerate shoots in root explants of wild type control plants. Transformed plants showed morphological features typically seen in transgenic plants produced by A. rhizogenes, which include, short internodes, small and wrinkled leaves, more branches and numerous plagiotropic roots. Plants regenerated from transformed roots showed increased biomass accumulation (350–510% in the roots and 200–320% in the whole plants) and augmented tylophorine content (20–60%) in the shoots, resulting in a 160–280% increase in tylophorine production in different clones grown in vitro.     

Transgenic rice tolerant to high temperature with elevated contents of dienoic fatty acids

Transgenic rice plants in which the content of dienoic fatty acids was increased as a result of co-suppression of fatty acid desaturase were more tolerant to high temperatures than untransformed wild-type plants, as judged by growth rate and chlorophyll content. When untransformed wild-type and transgenic rice seedlings were incubated at 35 °C, seedlings of the transgenic rice lines showed approximately 1.6 and 2.1 times the growth of untransformed wild-type seedlings, as assayed by shoot and root mass, respectively. The chlorophyll content of the transgenic leaves after 9 d at 35 °C was also higher than that of wild-type rice. The maximum photochemical efficiency of photosystem 2 was also higher in transgenic plants than in wild-type plants upon high temperature stress.     

Enhanced tolerance to drought stress in transgenic rice plants overexpressing a small heat-shock protein,sHSP17.7

Exposure of rice (Oryza sativa L.) seedlings to a high temperature (42°C) for 24 h resulted in a significant increase in tolerance to drought stress. To try to determine the mechanisms of acquisition of tolerance to drought stress by heat shock, the rice small heat-shock protein gene, sHSP17.7, the product of which was shown to act as molecular chaperones in vitro and in vivo in our previous study, was overexpressed in the rice cultivar “Hoshinoyume”. Western and Northern blot analyses showed higher expression levels of sHSP17.7 protein in three transgenic lines than in one transgenic line. Drought tolerance was assessed in these transgenic lines and wild-type plants by withholding water for 6 days for evaluation of the ability of plants to continue growth after water-stress treatments. Although no significant difference was found in water potential of seedlings between transgenic lines and wild-type plants at the end of drought treatments, only transgenic seedlings with higher expression levels of sHSP17.7 protein could regrow after rewatering. Similar results were observed in survival rates after treatments with 30% polyethylene glycol (PEG) 3640 for 3 days. These results suggest that overproduction of sHSP17.7 could increase drought tolerance in transgenic rice seedlings.     

Photosynthetic characteristics and tolerance to photo-oxidation of transgenic rice expressing C4 photosynthesis enzymes

The photosynthetic characteristics of four transgenic rice lines over-expressing rice NADP-malic enzyme (ME), and maize phosphoenolpyruvate carboxylase (PC), pyruvate,orthophosphate dikinase (PK), and PC+PK (CK) were investigated using outdoor-grown plants. Relative to untransformed wild-type (WT) rice, PC transgenic rice exhibited high PC activity (25-fold increase) and enhanced activity of carbonic anhydrase (more than two-fold increase), while the activity of ribulose-bisphosphate carboxylase/oxygenase (Rubisco) and its kinetic property were not significantly altered. The PC transgenic plants also showed a higher light intensity for saturation of photosynthesis, higher photosynthetic CO₂ uptake rate and carboxylation efficiency, and slightly reduced CO₂ compensation point. In addition, chlorophyll a fluorescence analysis indicates that PC transgenic plants are more tolerant to photo-oxidative stress, due to a higher capacity to quench excess light energy via photochemical and non-photochemical means. Furthermore, PC and CK transgenic rice produced 22–24% more grains than WT plants. Taken together, these results suggest that expression of maize C₄ photosynthesis enzymes in rice, a C₃ plant, can improve its photosynthetic capacity with enhanced tolerance to photo-oxidation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Transgenic grapefruit plants expressing the PAPETALA3-IPT gp gene exhibit altered expression of PR genes

Transgenic grapefruit plants (Citrus paradisi cv. ‘Duncan’) with the isopentenyltransferase (ipt) gene under the control of APETALA3 promoter have been produced using Agrobacterium-mediated transformation. The relative expression level of the ipt gene was between 2.3 and 7 times higher in transformed plants than in the wild-type but despite the presence of a tissue-specific promoter, the expression was not limited only to flower tissue. Increased levels of trans-zeatin riboside between 9.4 and 32-fold found in transgenic grapefruit were considered the consequence of ectopic expression of the ipt gene. Chlorophyll levels in fully expanded uppermost leaves were also about 30% higher in transgenic than in wild-type plants. Involvement of cytokinins in control of expression of three pathogenesis-related protein genes: β-1,3-glucanase, a stress related PR gene 24P220, and an acidic chitinase, 24P262 was examined. Expression of β-1,3-glucanase, and 24P220 gene were significantly enhanced in transgenic plants while the expression of chitinase was reduced to low levels. Our results confirm the effect of cytokinins on expression of genes implicated in the response of grapefruit plants to pathogen attack and suggest a possible role of cytokinins in pathogen resistance.