Responses of MxPPO overexpressing transgenic tall fescue plants to two diphenyl-ether herbicides, oxyfluorfen and acifluorfen

We generated transgenic tall fescue (Festuca arundinacea Schreb. cv. Kentucky-31) plants harboring a synthetic Myxococcus xanthus protoporphyrinogen oxidase (MxPPO) gene through Agrobacterium-mediated gene transfer. Successful integration of the transgene into the genome of transgenic plants confirmed by polymerase chain reaction (PCR) and Southern blot analysis, and the functional expression of the MxPPO gene at the mRNA level in transgenic lines was validated by Northern blot analysis. Responses of transgenic and non-transgenic tall fescue plants to diphenyl-ether herbicides such as oxyfluorfen and acifluorfen have been evaluated in respect of various physiological and biochemical parameters. Differential responses were observed in chlorophyll content, in vivo H₂O₂ deposition and lipid peroxidation in both transgenic and non-transgenic plants exposed to oxyfluorfen or acifluorfen. Isozyme profiles of four antioxidant-enzymes, including peroxidase (POD), catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX), were also investigated in transgenic and non-transgenic plants using native PAGE analysis. Compared to the transgenic lines, higher staining activities of the examined antioxidant-enzymes observed in non-transgenic plants subjected to 100 μM of oxyfluorfen or acifluorfen suggests that non-transgenic plants are unable to prevent the photodynamic induced oxidative stress caused by herbicides. In addition, both transgenic and non-transgenic plants exposed to oxyfluorfen exhibited proportionally increased band-staining patterns in contrast to acifluorfen, which suggests that oxyfluorfen has relatively greater or more rapid effects on leaves than acifluorfen. Both Ki-Won Lee and Nagib Ahsan have contributed equally to this work.     

Dual targeting of Myxococcus xanthus protoporphyrinogen oxidase into chloroplasts and mitochondria and high level oxyfluorfen resistance

Much attention has been paid to the signal sequences of eukaryotic protoporphyrinogen oxidases (protoxes); both the organelles targeted by protoxes and the role of protoxes in conferring resistance against protox‐inhibiting herbicides, such as oxyfluorfen, have been examined. However, there have been no reports on the translocation of prokaryotic protoxes. This study investigated the targeting ability of Myxococcus xanthus protox in vitro and in vivo. In an in vitro translocation assay using a dual import system, M. xanthus protein was detected in chloroplasts and mitochondria, suggesting that the M. xanthus protox protein was targeted into both organelles. In order to confirm the in vitro dual targeting ability of M. xanthus, we used a stable transgenic strategy to investigate dual targeting in vivo. In transgenic rice plants overexpressing M. xanthus protox, M. xanthus protox antibody cross‐reacted with proteins with predicted molecular masses of 50 kDa from both chloroplasts and mitochondria, and this in vivo transgene expression corresponded to a prominent increase in chloroplastic and mitochondrial protox activity. Seeds from the transgenic lines M4 and M7 germinated in solid Murashige and Skoog media of up to 500 µm of oxyfluorfen, whereas wild‐type seeds did not germinate in 1 µm . After 4‐week‐old‐rice plants were treated with oxyfluorfen for 3 d, lines M4 and M7 exhibited normal growth, whereas the wild‐type line was severely bleached and necrotized. The herbicidal resistance is attributed to the insignificant accumulation of photodynamic protoporphyrin IX in cytosol because the high chloroplastic and mitochondrial protox activity in oxyfluorfen‐treated transgenic lines, compared with that in oxyfluorfen‐treated and untreated wild‐type plants, metabolizes protoporphyrinogen IX to chlorophyll and heme. A practical application of the dual targeting of M. xanthus protox for obtaining outstanding resistance to peroxidizing herbicides is discussed.     

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.     

Alterations in the antioxidative system of suspension-cultured soybean cells (Glycine max) induced by oxidative stress

The objectives of this study were the changes of antioxidative key enzyme activities under stress conditions induced by a peroxidizing herbicide using photoheterotrophi-cally grown, suspension-cultured soybean celts ( Glycine max L.). Within two days, 50 to 500 n M oxyfluorfen. a p-nitrodiphenyl ether herbicide, caused up to 100% inhibition of growth, while simultaneously, the chlorophyll was 25% to completely bleached. The major cellular antioxidants ascorbate and glutathione showed different responses. Under stress conditions with more than 250 n M oxyfluorfen, the cellular ascorbate- concentration was halved, whereas dehydroascorbate remained roughly constant. The glutathione content (approximately one-fifth of that of ascorbate in untreated control cells) increased nearly 3-fold in the presence of 250 n M oxyfluorfen. Under this condition, oxidized glutathione was 5 times above the control level. The specific activities of selected enzymes participating in cellular defence, namely ascor-bate peroxidase, glutathione reductase, rnonodehydroascorbate reductase. peroxidase and catalase increased by 40 to 70% with oxyfluorfen concentrations between 50 and 500 n M , while dehydroascorbate reductase showed a significant decrease. Glutathione transferase activity even increased 6-fold under oxyfluorfen stress.     

杨树HDA902基因在低温胁迫应答反应中的功能

组蛋白去乙酰化酶在植物非生物胁迫应答反应中具有重要的调控作用。利用RT-PCR的方法从毛果杨中克隆了组蛋白去乙酰化酶基因HDA902。利用农杆菌介导法将其遗传转化到烟草中,并对转基因植株进行低温耐受性分析。研究结果表明,HDA902在烟草中的表达显著提高了转基因株系对低温的耐受性。叶片NBT和DAB染色结果表明,在低温处理后转基因烟草比野生型烟草产生较少的活性氧。丙二醛和脯氨酸含量测定结果表明,在低温条件下,转基因烟草叶片的脯氨酸含量显著高于野生型烟草,而丙二醛含量显著低于野生型烟草。这些研究结果表明,HDA902参与低温胁迫应答反应,其过量表达提高了植株耐低温的能力。     

Resistance mechanisms in protoporphyrinogen oxidase (PROTOX) inhibitor-resistant transgenic rice

We investigated the mechanism for conferring herbicide resistance in transgenic rice. Plants from Line M4 were resistant to PROTOX inhibitors and had yields similar to those from wild-type (WT) rice.Myxococcus xanthus PROTOX mRNA was abundantly expressed in the transgenic leaf tissues, and theM. xanthus PROTOX gene was stably transmitted into the T₄ generation. We detected a protein with a predicted molecular mass of 50 kD, equal to the weight ofM. xanthus PROTOX, in M4 but not WT plants. Furthermore, several PROTOX-inhibitor herbicides — acifluorfen, oxyfluorfen, carfentrazone-ethyl, and oxadiazon — caused significant cellular leakage and lipid peroxidation in the WT, but not in the transgenics. Total PROTOX activity in untreated transformed rice was 17-fold higher than in the WT, with activity being inhibited in the latter genotype by 55%, 59%, 53%, or 60% as a result of treatment with acifluorfen, oxyfluorfen, carfentrazone-ethyl, or oxadiazon, respectively. However, PROTOX activities in transgenic rice were similar to their corresponding, untreated controls. The accumulation of Proto IX was 15-to 21-fold higher in the WT than in M4 when plants were treated with PROTOX inhibitors. In the former, their epicuticular wax and chloroplasts were severely damaged after oxyfluorfen treatment The lack of damage in transformed plants suggests that M4 does not accumulate Proto IX, probably due to the production of herbicide-resistant chloroplastic and mitochondria PROTOX.     

Accumulation of overproduced ferritin in the chloroplast provides protection against photoinhibition induced by low temperature in tobacco plants

Wild-type tobacco plants (Nicotiana tabacum L. cv. Petit Havanna line SR1) and plants transformed with full-length alfalfa ferritin cDNA with the chloroplast transit peptide under the control of a Rubisco small subunit gene promoter (C3 and C8) were cold-treated at 0 degrees C with continuous light (250mumolm(-2)s(-1)). These transgenic plants had higher chlorophyll content and higher F(v)/F(m) chlorophyll-a fluorescence induction parameters than wild-type plants after 2 or 3d of cold treatment in C3 and C8 transgenic plants, respectively. Thermoluminescence studies on the high-temperature bands suggest that these plants suffered less oxidative damage in comparison to the wild-type genotype. The present experiments provide evidence that transgenic tobacco lines overexpressing alfalfa ferritin, which is accumulated in the chloroplasts, may show higher tolerance to various stress factors, generating ROS including low temperature-induced photoinhibition.     

Response to oxidative stress induced by cadmium and copper in tobacco plants (Nicotiana tabacum) engineered with the trehalose-6-phosphate synthase gene (AtTPS1)

The response of tobacco plants genetically engineered with the AtTPS1 gene to stress induced by excess Cu and Cd was evaluated in hydroponic solution (100 and 400 μM Cu and 50 and 200 μM Cd) after a 48 h exposure. Two transgenic lines, transformed with the AtTPS1 (trehalose-6-phosphate synthase) gene from Arabidopsis, with different levels of trehalose-6-phosphate synthase expression (B5H, higher and B1F, lower), and a wild type (WT) were investigated. Protein content, antioxidative enzymes (CAT, POD, SOD, and APX), glucose, fructose, lipid peroxidation, hydrogen peroxide and Cd and Cu contents were determined in leaves. The two transgenic lines were differently influenced by Cd and Cu exposure as they induced a different antioxidant enzymatic defense response. B1F and B5H plants showed a better acclimation to Cd and excess Cu compared to WT. Furthermore B1F was more tolerant than B5H to Cd and excess Cu. B1F accumulated less Cd and Cu in leaves, probably due to a more efficient exclusion mechanism. Catalase was shown to be the most important enzyme in the antioxidative system of these plants.     

Overexpression of Rice Ferrochelatase I and II Leads to Increased Susceptibility to Oxyfluorfen Herbicide in Transgenic Rice

Protoporphyrin IX is a photosensitizer and a causative agent of rice membrane lipid peroxidation in plant cells. Protoporphyrinogen IX oxidase (PPO) is the molecular target of PPO-inhibiting herbicides, which trigger a massive increase in protoporphyrin IX. Thus, any possible method to decrease the levels of protoporphyrin IX upon challenge with PPO-inhibiting herbicides could be employed to generate plants resistant to such herbicides. We generated transgenic rice plants overexpressing rice ferrochelatase isogenes encoding ferrochelatase enzymes, which convert protoporphyrin IX into protoheme, to see whether the transgenic plants have phenotypes resistant to PPO-inhibiting herbicides. The resulting transgenic rice plants were all susceptible to oxyfluorfen (a diphenyl-ether-type PPO-inhibiting herbicide), as judged by cellular damage with respect to cellular leakage, chlorophyll loss, and lipid peroxidation. In particular, the transgenic plants expressing rice ferrochelatase II without its plastid targeting sequence showed higher transgene expression and oxyfluorfen susceptibility than lines expressing the intact ferrochelatase II. Possible susceptibility mechanisms to oxyfluorfen herbicide in the transgenic rice plants are discussed.     

Increase of glycinebetaine synthesis improves drought tolerance in cotton

The tolerance to drought stress of the homozygous transgenic cotton (Gossypium hirsutum L.) plants with enhanced glycinebetaine (GB) accumulation was investigated at three development stages. Among the five transgenic lines investigated, lines 1, 3, 4, and 5 accumulated significantly higher levels of GB than the wild-type (WT) plants either before or after drought stress, and the transgenic plants were more tolerant to drought stress than the wild-type counterparts from young seedlings to flowering plants. Under drought stress conditions, transgenic lines 1, 3, 4, and 5 had higher relative water content, increased photosynthesis, better osmotic adjustment (OA), a lower percentage of ion leakage, and less lipid membrane peroxidation than WT plants. The GB levels in transgenic plants were positively correlated with drought tolerance under water stress. The results suggested that GB may not only protect the integrity of the cell membrane from drought stress damage, but also be involved in OA in transgenic cotton plants. Most importantly, the seedcotton yield of transgenic line 4 was significantly greater than that of WT plants after drought stress, which is of great value in cotton production.     

Transgenic tomato plants alter quorum sensing in plant growth-promoting rhizobacteria

Two Gram-negative, plant growth-promoting rhizobacteria (PGPRs), denominated as M12 and M14, were classified by 16S rDNA sequencing as Burkholderia graminis species. Both strains were shown to produce a variety of N-acyl-homoserine lactone (AHL) quorum sensing (QS) signalling molecules. The involvement of these molecules in plant growth promotion and the induction of protection against salt stress was examined. AHL production was evaluated in vitro by thin-layer chromatography using AHL biosensors, and the identity of the AHLs produced was determined by liquid chromatography-tandem mass spectrometry. The in situ production of AHLs by M12 and M14 in the rhizosphere of Arabidopsis thaliana plants was detected by co-inoculation with green fluorescent protein-based biosensor strains and confocal laser scanning microscopy. To determine whether plant growth promotion and protection against salt stress were mediated by QS, these PGPRs were assayed on wild-type tomato plants, as well as their corresponding transgenics expressing YenI (short-chain AHL producers) and LasI (long-chain AHL producers). In wild-type tomato plants, only M12 promoted plant growth, and this effect disappeared in both transgenic lines. In contrast, M14 did not promote growth in wild-type tomatoes, but did so in the LasI transgenic line. Resistance to salt stress was induced by M14 in wild-type tomato, but this effect disappeared in both transgenic lines. The strain M12, however, did not induce salt resistance in wild-type tomato, but did so in LasI tomato plants. These results reveal that AHL QS signalling molecules mediate the ability of both PGPR strains M12 and M14 to promote plant growth and to induce protection against salt stress.     

Defence response produced during photodynamic damage in transgenic rice overexpressing 5-aminolevulinic acid synthase

Photodynamic and photoprotective responses at different irradiances were investigated in transgenic rice (Oryza sativa) expressing Bradyrhizobium japonicum 5-aminolevulinic acid synthase (ALA-S). With high irradiance (HI) of 350 µmol m^?2 s^?1, transgenic lines P5 and P14 showed a decrease in contents of chlorophyll (Chl) and the chloroplast-encoded gene psbA mRNA, whereas a decrease in light-harvesting Chl-binding proteins was observed only in P14. These effects were not observed in the wild-type (WT) line treated with HI or all of the lines treated with low irradiance (LI) of 150 µmol m^?2 s^?1. HI resulted in a greater decrease in the quantum yield of photosystem 2 and a greater increase in non-photochemical quenching (NPQ) in the transgenic lines, particularly in P14, compared to WT. Photoprotective zeaxanthin contents increased at HI, even though carotenoid contents were lower in the transgenic lines compared to WT. When exposed to HI, superoxide dismutase greatly increased in transgenic lines P5 and P14, but peroxidase and glutathione reductase increased only in P14, in which more photodynamic damage occurred. Thus the greater expression of ALA-S in the transgenic plants developed the stronger protective functions, i.e. the increased values of NPQ and zeaxanthin, as well as more photodynamic reactions, i.e. decreased photosynthetic component and efficiency, in the photosynthetic complexes. However, the photodynamic reactions indicate that the antioxidant capacity was insufficient to cope with the severe stress triggered by photoactive porphyrins in the transgenic rice expressing ALA-S.     

Effect of chlorophyll reduction in Arabidopsis thaliana by methyl jasmonate or norflurazon on antioxidant systems.

Methyl jasmonate (MeJA) and norflurazon (NF) treatments resulted in a substantial decrease in photosynthetic activities and chlorophylls (Chls) in Arabidopsis thaliana plants, causing a senescence-like yellowing and a bleaching in MeJA- and NF-treated plants, respectively. Non-radiative energy dissipation through q(N) and non-photochemical quenching increased greatly in NF-treated plants in concomitance with an increase in photoprotectants antheraxanthin and zeaxanthin from interconversion of violaxanthin, although they were not changed in MeJA-treated plants. A significant accumulation of anthocyanin was observed only in MeJA-treated plants, not in NF-treated plants. Total activities of catalase (CAT, EC 1.11.1.6), peroxidase (POD, EC 1.11.1.7), superoxide dismutase (EC 1.15.1.1) and glutathione reductase (EC 1.6.4.2) increased greatly in response to MeJA, particularly a 100-fold increase in POD activity 7 days after MeJA treatment. NF application to plants exhibited less increase in antioxidant enzymes than MeJA-treated plants. NF-treated young leaves had a greater decline in Chls and CAT activity, and less zeaxanthin accumulation compared to NF-treated mature leaves, indicating that NF-treated young leaves are more susceptible to excess light exposure and a possible photooxidative stress. Both MeJA- and NF-treated Arabidopsis plants suffered destruction of Chls, however, they developed differential antioxidant responses during the stress, in large part by an increased anthocyanin level in the epidermis and enzymatic antioxidants in MeJA-treated plants and by accumulation of antheraxanthin and zeaxanthin, and enhanced energy dissipation in NF-treated plants.     

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.     

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.