Sunyo Jung

Porphyrin Biosynthesis Control under Water Stress: Sustained Porphyrin Status Correlates with Drought Tolerance in Transgenic Rice

A controlled flow of porphyrin metabolites is critical for organisms, but little is known about the control of porphyrin biosynthesis under environmental stress. We monitored transgenic rice (Oryza sativa) plants expressing Myxococcus xanthus protoporphyrinogen oxidase (PPO) for their response to drought stress. Transgenic plants showed significantly improved drought tolerance, as indicated by a higher shoot water potential, less oxidative damage, and a more favorable redox balance compared with wild-type plants. Both transgenic and wild-type plants responded to the onset of drought stress, even prior to changes in shoot water potential and oxidative metabolism, by drastically scavenging porphyrin intermediates in leaves, which was crucial for alleviating reactive oxygen species-induced stress. Protoporphyrin IX, protochlorophyllide, magnesium-protoporphyrin IX, and its methyl ester were absent or hardly detected with the intensification of water stress (–3.1 MPa) in the wild type, whereas transgenic plants retained these intermediates to some extent. Additionally, the expression and activity of most enzymes involved in porphyrin biosynthesis, particularly in the chlorophyll branch, were primarily down-regulated under dehydrating conditions, with stronger repression in the wild type than in transgenic plants. There was up-regulation of Glutamate 1-Semialdehyde Aminotransferase, PPO1, and Fe Chelatase2 transcripts in drought-stressed transgenic plants, enabling the transgenic plants to make larger pools of 5-aminolevulinic acid and protoporphyrin IX available for subsequent steps in the heme branch. Overexpression of PPO ultimately protected the transgenic plants from drought-induced cytotoxicity, demonstrating clearly that manipulation of porphyrin biosynthesis can produce drought-tolerant plants. Our results support a possible role for tetrapyrroles in signaling their metabolic state and in plant protection under drought stress conditions.

Antioxidant responses of cucumber (Cucumis sativus) to photoinhibition and oxidative stress induced by norflurazon under high and low PPFDs.

Photooxidative damage is exacerbated by norflurazon (NF), which blocks carotenoid biosynthesis. This study examined the influence of photosynthetic photon flux density (PPFD) on the overall responses of both non-enzymatic and enzymatic antioxidants to NF-caused oxidative damage in leaves of cucumber (Cucumis sativus). Seven-day-old cucumber plants were exposed to NF under either low PPFD (30 µmol m(-2) s(-1)) or high PPFD (300 µmol m(-2) s(-1)) for 3 days. The NF plants exposed at high PPFD had lower levels of F(v)/F(m) ratio, quantum yield of electron transport, and 33-kDa protein of photosystem II as compared with the NF plants at low PPFD. In the NF plants, there was a reduction in total chlorophylls and carotenoids except newly formed zeaxanthin in either PPFD. The NF plants at high PPFD resulted in less level of photochemical quenching, q(P), and Stern-Volmer quenching, NPQ, than those of the plants at low PPFD, whereas both plants had similar level of non-photochemical quenching coefficient, q(N). However, the level of PPFD did not significantly affect the NF-caused induction of antioxidant enzymes including peroxidase, superoxide dismutase, glutathione reductase, and ascorbate peroxidase.

A comparative study of the cytotoxicity of skin irritants on cultured human oral and skin keratinocytes

Summary The cytotoxicity of irritants on human oral and skin keratinocyte culture models was compared. Keratinocytes were exposed to sodium lauryl sulphate and benzalkonium chloride at concentrations of 10–4–10–7 M for 24 h. Cytotoxicity was evaluated by changes in niitochondrial metabolic activity (MIT assay) and plasma membrane integrity (LDH leakage). Our results showed that oral and skin keratinocytes are equally sensitive to the irritants tested. There were marked similarities in susceptibility between each cell type cultured from six individuals. The immunohistochemical staining pattern of both cell types resembled that of the basal cell.

Mechanism of paraquat tolerance in cucumber leaves of various ages

Abstract Differential sensitivity to paraquat was observed between cucumber cultivars and leaf age. Physiological responses to paraquat, including antioxidative enzyme activity, were investigated in leaf age classes of cucumber to identify mechanisms of paraquat tolerance. Leaf injury for ‘Naeseosamcheok’, ‘Daehandadagi’, ‘Baekgwangdadagi’, ‘Sangrokheukjinju’, and ‘Eunseongbaekdadagi’ cultivars was less than that of six other cultivars tested, averaged over leaf age and herbicide rate. The level of foliar injury caused by paraquat was Leaf 1 > 2 > 3 > 4 in seven of 11 cultivars used, where 4 was the youngest leaf. There was a positive correlation between leaf age and its relative susceptibility to paraquat, regardless of growth stage. Lipid peroxidation was less in the youngest leaf (Leaf 4) than in the older leaves at all herbicide concentrations. The youngest leaf had higher values for apparent photosynthesis than the oldest leaf. Differential leaf response to paraquat was partially correlated with the change in superoxide dismutase, ascorbate peroxidant, and glutathione reductase activities in treated leaves. Enzyme activity of ascorbate peroxidase (APX) was higher in Leaf 4 than in Leaves 1, 2, or 3 in untreated plants and after exposure to paraquat. APX isozymes were more abundant in treated than in untreated leaves and produced in higher amounts in younger than in older leaves. Application of ascorbate and glutathione before paraquat treatment protected cucumber leaves from paraquat injury. Nomenclature: Paraquat; cucumber, Cucumis sativus L.

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.

Differential Antioxidant Responses and Perturbed Porphyrin Biosynthesis after Exposure to Oxyfluorfen and Methyl Viologen in Oryza sativa.

We compared antioxidant responses and regulation of porphyrin metabolism in rice plants treated with oxyfluorfen (OF) or methyl viologen (MV). Plants treated with MV exhibited not only greater increases in conductivity and malondialdehyde but also a greater decline in Fv/Fm, compared to plants treated with OF. MV-treated plants had greater increases in activities of superoxide dismutase (SOD) and catalase (CAT) as well as transcript levels of SODA and CATA than OF-treated plants after 28 h of the treatments, whereas increases in ascorbate peroxidase (APX) activity and transcript levels of APXA and APXB were greater in OF-treated plants. Both OF- and MV-treated plants resulted in not only down-regulation of most genes involved in porphyrin biosynthesis but also disappearance of Mg-porphyrins during the late stage of photooxidative stress. By contrast, up-regulation of heme oxygenase 2 (HO2) is possibly part of an efficient antioxidant response to compensate photooxidative damage in both treatments. Our data show that down-regulated biosynthesis and degradation dynamics of porphyrin intermediates have important roles in photoprotection of plants from perturbed porphyrin biosynthesis and photosynthetic electron transport. This study suggests that porphyrin scavenging as well as strong antioxidative activities are required for mitigating reactive oxygen species (ROS) production under photooxidative stress caused by OF and MV.

Differential antioxidant responses in catalase-deficient maize mutants exposed to norflurazon

Maize catalase (CAT) mutants lacking in CAT isozymes were used to investigate the response of CAT and superoxide dismutase (SOD) to norflurazon (NF) caused oxidative stress in 18-d post-pollination scutella. NF treatment caused an increase in malonyldialdehyde content, which is an indicator of oxidative stress, and a decrease in a content of photoprotectant carotenoids in the scutella. CAT activity decreased greatly with increasing NF concentration in standard maize line (W64A) and CAT-2 null mutant (WA10C), however, it remained relatively constant in CAT-2/CAT-3 double null mutant (WDN10). Cat1 transcript increased in the NF-treated scutella of W64A and WDN10, with a greater Cat1 transcript in WDN10, whereas Cat2 transcript decreased in the NF-treated scutella of all lines examined. In all lines, SOD activities were not changed noticeably at various NF concentrations. In the scutella of W64A, Sod4A decreased in response to NF but the other Sod transcript contents were unchanged. The CAT-deficient mutants reacted with raised transcripts of Sod1, Sod3, Sod4 or Sod4A.

Expression of recombinant protoporphyrinogen oxidase influences growth and morphological characteristics in transgenic rice

Transgenic rice plants expressing a Bacillus subtilis protoporphyrinogen oxidase (Protox), the last shared enzyme of the porphyrin pathway, in the cytoplasm (C89) or the plastids (P72) were compared with wild-type rice plants in their growth characteristics. Production of tiller buds 18 d after seeding was more profuse in transgenic plants than in wild-type plants, especially in plastid-targeted plants. Transgenic plants had 12–27% increase in tiller number and 17–33% increase in above-ground biomass compared with wild-type plants 4 and 8 weeks after transplanting of 2-week-old rice seedlings, demonstrating that tiller production and above-ground biomass correlate with each other. Cytoplasm-expressed and plastid-targeted transgenic plants also had a distinct phenotypic characteristic of narrower and more horizontal leaves than wild-type plants. Phenotypic and anatomical characteristics of the transgenic plants were clearly different from wild-type plants, indicating that regulation of porphyrin biosynthesis by expression of B. subtilis Protox in rice influences morphological characteristics of plant growth as well as biomass.

Effects of Light-Emitting Diode Irradiation on Growth Characteristics and Regulation of Porphyrin Biosynthesis in Rice Seedlings

We examined the effects of light quality on growth characteristics and porphyrin biosynthesis of rice seedlings grown under different wavelengths from light emitting diodes (LEDs). After 10 days of exposure to various wavelengths of LEDs, leaf area and shoot biomass were greater in seedlings grown under white and blue LEDs than those of green and red LEDs. Both green and red LED treatments drastically decreased levels of protoporphyrin IX (Proto IX) and Mg-porphyrins compared to those of white LED, while levels of Mg-Proto IX monomethyl ester and protochlorophyllide under blue LED were decreased by 21% and 49%, respectively. Transcript levels of PPO1 were greatly upregulated in seedlings grown under red LED compared to white LED, whereas transcript levels of HO2 and CHLD were upregulated under blue LED. Overall, most porphyrin biosynthetic genes in the Fe-porphyrin branch remained almost constant or upregulated, while most genes in the Mg-porphyrin branch were downregulated. Expression levels of nuclear-encoded photosynthetic genes Lhcb and RbcS noticeably decreased after exposure to blue and red LEDs, compared to white LED. Our study suggests that specific wavelengths of LED greatly influence characteristics of growth in plants partly through altering the metabolic regulation of the porphyrin biosynthetic pathway, and possibly contribute to affect retrograde signaling.

Characterization of Transgenic Rice Plants Expressing an Arabidopsis FAD7

Fatty acid ω-3 desaturase (FAD) is the key enzyme catalyzing the formation of trienoic fatty acids. We utilized an Arabidopsis FAD7 gene and the seven independent transgenic rice plants harbouring 1 to 3 copies of this gene were generated. The expression of FAD7 mRNA was different among independent transgenic lines regardless of the copy number. The total linolenic acid (18:3) contents reduced by about 7 – 32 % in transgenic rice plants but the linoleic acid (18:2) content increased accordingly. With or without wounding treatments, the jasmonate content was higher in transgenic lines than in wild-type rice plant. The transgenic lines overproducing jasmonate also showed increased expression of PR1b mRNA and allene oxide synthase inresponse to wounding.