No single way to understand singlet oxygen signalling in plants

When plant cells are under environmental stress, several chemically distinct reactive oxygen species (ROS) are generated simultaneously in various intracellular compartments and these can cause oxidative damage or act as signals. The conditional flu mutant of Arabidopsis, which generates singlet oxygen in plastids during a dark-to-light transition, has allowed the biological activity of singlet oxygen to be determined, and the criteria to distinguish between cytotoxicity and signalling of this particular ROS to be defined. The genetic basis of singlet-oxygen-mediated signalling has been revealed by the mutation of two nuclear genes encoding the plastid proteins EXECUTER (EX)1 and EX2, which are sufficient to abrogate singlet-oxygen-dependent stress responses. Conversely, responses due to higher cytotoxic levels of singlet oxygen are not suppressed in the ex1/ex2 background. Whether singlet oxygen levels lower than those that trigger genetically controlled cell death activate acclimation is now under investigation.     

Role of singlet oxygen in chloroplast to nucleus retrograde signaling in Chlamydomonas reinhardtii

High light illumination of photosynthetic organisms stimulates the production of singlet oxygen by photosystem II and causes photooxidative stress. In Chlamydomonas reinhardtii, singlet oxygen also induces the expression of the nuclear-encoded glutathione peroxidase homologous gene GPXH. We provide evidence that singlet oxygen stimulates GPXH expression by activating a signaling mechanism outside the thylakoid membrane. Singlet oxygen from photosystem II could be detected with specific probes in the aqueous phase of isolated thylakoid suspensions and the cytoplasm of high light stressed cells. This indicates that singlet oxygen can stimulate a response farther from its production site than generally believed.     

Ultraviolet-B-induced oxidative stress and antioxidant defense system responses in ascorbate-deficient vtc1 mutants of Arabidopsis thaliana

Ultraviolet-B (UV-B) radiation has a negative impact on plant cells, and results in the generation of reactive oxygen species (ROS). In order to increase our understanding of the effects of UV-B on antioxidant processes, we investigated the response of an ascorbate-deficient Arabidopsis thaliana mutant vtc1 to short-term increased UV-B exposure. After UV-B supplementation, vtc1 mutants exhibited oxidative damage. Evidence for damage included an increase in H(2)O(2) content and the production of thiobarbituric acid reactive substances (TBARS); a decrease in chlorophyll content and chlorophyll fluorescence parameters were also reported. The vtc1 mutants had higher total glutathione than the wild type (WT) during the first day of UV-B treatment. We found reduced ratio of glutathione/total glutathione and increased ratio of dehydroascorbate/total ascorbate in the vtc1 mutants, compared to the WT plants. In addition, the enzymes responsible for ROS scavenging, including superoxide dismutase, catalase, and ascorbate peroxidase, had insufficient activity in the vtc1 mutants, compared to the WT plants. The same reduced activity in the vtc1 mutants was reported for the enzymes responsible for the regeneration of ascorbate and glutathione (including monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase). These results suggest that the ascorbate-deficient mutant vtc1 is more sensitive to supplementary UV-B treatment than WT plants and ascorbate can be considered an important antioxidant for UV-B radiation.     

Carotenoids protectPhaffia rhodozyma against singlet oxygen damage

Summary The only known habitat of the astaxanthin-containingPhaffia rhodozyma is in slime fluxes of deciduous trees at high altitudes. In this habitat, the function of carotenoids inP. rhodozyma is probably to provide protection against photogenerated antifungal substances in the tree flux such as singlet oxygen (¹O₂). To investigate the role of carotenoids inP. rhodozyma, genetic selections were employed to determine if carotenogenic yeast strains ofP. rhodozyma have enhanced ability to quench¹O₂. Singlet oxygen was generated in liquid culture by the interaction of visible light (-550 nm) with the photosensitizer rose bengal or by the activation of -terthienyl with ultraviolet light (=366 nm). In each case the treatments selected for growth of pigmented strains ofP. rhodozyma. Albino (carotenoid-less) or yellow (-carotene producing) strains grew less well in media containing¹O₂. Addition of the¹O₂ quencher sodium azide to the medium with -terthienyl allowed growth of non-pigmented strains. Since the ecological niche ofP. rhodozyma is highly specific, we investigated whether extracts of birch trees (Betula), the original source ofP. rhodozyma, contained a compound that would select for pigmented populations of the yeast. WhenP. rhodozyma strains were exposed to ethyl acetate extracts ofBetula papyrifera excited with 366 nm ultraviolet light, only pigmented cells were able to grow. These results suggest that carotenogenesis developed inP. rhodozyma in response to the presence of photoactivatable antifungal compounds produced by the host tree.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues.     

Photomorphogenic mutants of Arabidopsis thaliana reveal activities of multiple photosensory systems during light-stimulated apical-hook opening

Fluence rate-response curves were generated for red-, far-red-, and blue-light-stimulated apical-hook opening in seedlings of several photomorphogenic mutants of Arabidopsis thaliana (L.) Heynh. Compared to wild-type plants, hook opening was reduced in the phytochrome-deficient hy1, hy2, and hy6 mutants in red and far-red light at all fluence rates tested, and in low-fluence blue light, but was normal under high-irradiance blue light. In contrast, the blue-light-response mutants (blu1, blu2, and blu3) lacked the high-irradiance-dependent hook-opening response in blue light while hook opening was normal in low-fluence blue light and in red and farred light at all fluence rates tested. Hook opening in the phytochrome-B-deficient hy3 mutant was similar to wild type in all light conditions tested. The effects of the different mutations on light-induced hook opening indicate that a phytochrome(s) other than phytochrome B mediates hook opening stimulated by red, far-red and lowfluence blue light, while a blue-light-absorbing photoreceptor mediates the blue-light-sensitive high-irradiance response. Although the phytochrome and blue-light photosensory systems appear to work independently for the most part, some of their signal-transduction components may interact since the hy4, and hy5 mutants showed reduced hook-opening responses under conditions dependent on the phytochrome and blue-light-photosensory systems.We thank Jeff Young and Brian Parks for their many helpful suggestions during the progress of this research. This work was supported by National Science Foundation Grant No. DCB-9106697.     

Overexpression of phytoene synthase gene from Salicornia europaea alters response to reactive oxygen species under salt stress in transgenic Arabidopsis

A phytoene synthase gene SePSY was isolated from euhalophyte Salicornia europaea L. The 1655 bp full-length SePSY has an open reading frame of 1257 bp and encodes a 419-amino acid protein. The overexpression of SePSY enhanced the growth of transgenic Arabidopsis. When the plants were exposed to 100 mM NaCl, the photosynthesis rate and photosystem II activity (Fv/Fm) increased from 92% to 132% and from 9.3% to 16.6% in the transgenic lines than in the wild-type, respectively. The transgenics displayed higher activities of SOD and POD and lower contents of H(2)O(2) and MDA than the WT. In conclusion, the transgenic lines showed higher tolerance to salt stress than WT plants by increased photosynthesis efficiency and antioxidative capacity. This is the first report about improving the salt tolerance by genetic manipulation of carotenoid biosynthesis.     

Arabidopsis mutants deregulated in RCI2A expression reveal new signaling pathways in abiotic stress responses

To uncover new pathways involved in low-temperature signal transduction, we screened for mutants altered in cold-induced expression of RCI2A, an Arabidopsis gene that is not a member of the CBF/DREB1 regulon and is induced not only by low temperature but also by abscisic acid (ABA), dehydration (DH) and NaCl. This was accomplished by generating a line of Arabidopsis carrying a transgene consisting of the RCI2A promoter fused to the firefly luciferase coding sequence. A number of mutants showing low or high RCI2A expression in response to low temperature were identified. These mutants also displayed deregulated RCI2A expression in response to ABA, DH or NaCl. Interestingly, however, they were not altered in stress-induced expression of RD29A, a CBF/DREB1-target gene, suggesting that the mutations affect signaling intermediates of CBF/DREB1-independent regulatory pathways. Several mutants showed alterations in their tolerance to freezing, DH or salt stress, as well as in their ABA sensitivity, which indicates that the signaling intermediates defined by the corresponding mutations play an important role in Arabidopsis tolerance to abiotic stresses. Based on the mutants identified, we discuss the involvement of CBF/DREB1-independent pathways in modulating stress signaling.     

Concurrent activation of cell death-regulating signaling pathways by singlet oxygen in Arabidopsis thaliana

Upon a dark/light shift the conditional flu mutant of Arabidopsis starts to generate singlet oxygen ((1)O(2)), a non-radical reactive oxygen species that is restricted to the plastid compartment. Immediately after the shift, plants stop growing and develop necrotic lesions. We have established a protoplast system, which allows detection and characterization of the death response in flu induced by the release of (1)O(2). Vitamin B6 that quenches (1)O(2) in fungi was able to protect flu protoplasts from cell death. Blocking ethylene production was sufficient to partially inhibit the death reaction. Similarly, flu mutant seedlings expressing transgenic NahG were partially protected from the death provoked by the release of (1)O(2), indicating a requirement for salicylic acid (SA) in this process, whereas in cells depleted of both, ethylene and SA, the extent of cell death was reduced to the wild-type level. The flu mutant was also crossed with the jasmonic acid (JA)-depleted mutant opr3, and with the JA, OPDA and dinor OPDA (dnOPDA)-depleted dde2-2 mutant. Analysis of the resulting double mutants revealed that in contrast to the JA-induced suppression of H(2)O(2)/superoxide-dependent cell death reported earlier, JA promotes singlet oxygen-mediated cell death in flu, whereas other oxylipins such as OPDA and dnOPDA antagonize this death-inducing activity of JA.     

Sugar-induced tolerance to the herbicide atrazine in Arabidopsis seedlings involves activation of oxidative and xenobiotic stress responses

Exogenous sucrose confers to Arabidopsis seedlings a very high level of tolerance to the herbicide atrazine that cannot be ascribed to photoheterotrophic growth. Important differences of atrazine tolerance between sucrose and glucose treatments showed that activation of chloroplast biogenesis per se could not account for induced tolerance. Sucrose-induced acquisition of defence mechanisms was shown by the gene expression pattern of a chloroplastic iron superoxide dismutase and by enhancement of whole-cell glucose-6-phosphate dehydrogenase activity. Activation of these defence mechanisms depended on both soluble sugar and atrazine. Moreover, acquisition of sucrose protection was shown to unmask atrazine-induced gene expression, such as that of a cytosolic glutathione-S-transferase, which remained otherwise cryptic because of the lethal effects of atrazine in the absence of soluble sugars.     

DNA methylation increases throughout Arabidopsis development

We used amplified fragment length polymorphisms (AFLP) to analyze the stability of DNA methylation throughout Arabidopsis development. AFLP can detect genome-wide changes in cytosine methylation produced by DNA demethylation agents, such as 5-azacytidine, or specific mutations at the DDM1 locus. In both cases, cytosine demethylation is associated with a general increase in the presence of amplified fragments. Using this approach, we followed DNA methylation at methylation sensitive restriction sites throughout Arabidopsis development. The results show a progressive DNA methylation trend from cotyledons to vegetative organs to reproductive organs.     

Tocopherol as singlet oxygen scavenger in photosystem II

Singlet oxygen is formed in the photosystem II reaction center in the quench of P680 triplets, and the yield is dependent on light intensity and the reduction level of plastoquinone. Singlet oxygen in PS II triggers the degradation of the D1 protein. We investigated the participation of tocopherol as a singlet oxygen scavenger in this system. For this purpose, we inhibited tocopherol biosynthesis at the level of the HPP-dioxygenase in the alga Chlamydomonas reinhardtii under conditions in which plastoquinone did not limit the photosynthesis rate. In the presence of the inhibitor and in high light for 2 h, photosynthesis in vivo and photosystem II was inactivated, the D1 protein was degraded, and the tocopherol pool was depleted and fell below its turnover rate/h. The inhibited system could be fully resuscitated upon the addition of a chemical singlet oxygen quencher (diphenylamine), and partly by synthetic cell wall permeable short chain alpha- and gamma-tocopherol derivatives. We conclude that under conditions of photoinhibition and extensive D1 protein turnover tocopherol has a protective function as a singlet oxygen scavenger.     

Blue-light promotion of flowering is absent in hy4 mutants of Arabidopsis

Loss of a blue-light photoreceptor in the hy4 mutants of Arabidopsis thaliana (L.) Heynh substantially delayed flowering (>100 d to flower vs. 40–50 d), especially with blue light exposure from lamps lacking much red (R) and/or far-red (FR) light. Red night breaks were promotory but flowering was still later for the hy4-101 mutant. However, with exposure to light from FR-rich lamps, flowering of all mutants was early and no different from the wild type. Thus, flowering of Arabidopsis involves a blue-light photoreceptor and other, often more effective photoreceptors. The latter may involve phytochrome photoresponses to R and FR, but with little or no phytochrome response to blue wavelengths.Abbreviations HIR high irradiance response - FR far-red - R red - WT wild type     

The Role of GIGANTEA Gene in Mediating the Oxidative Stress Response and in Arabidopsis

The Arabidopsis GIGANTEA (GI) gene has been shown to be involved in the regulation of the oxidative stress response; however, little is known about the mechanism by which GI gene regulates the oxidative stress response. We show here that enhanced tolerance of the gi-3 mutant to oxidative stress is associated, at least in part, with constitutive activation of superoxide dismutase (SOD) and ascorbate peroxidase (APX) genes. The gi-3 plants were more tolerant to parquart (PQ) or hydrogen peroxide (H₂O₂)-mediated oxidative stress than wild-type plants. Analyses of concentrations of endogenous H₂O₂ and superoxide anion radicals as well as lipid peroxidation revealed that enhanced tolerance of gi-3 plants to oxidative stress was not due to defects in the uptake of PQ or the sequestration of PQ from its site of action, and that the gi-3 mutation alleviated oxidative damage of plant cells from PQ stress. Moreover, the gi-3 mutant showed constitutive activation of cytosolic Cu/ZnSOD and plastidic FeSOD as well as cytosolic APX1 and stromal APX genes, which at least in part contributed to constitutive increases in activities of anti-oxidative enzymes SOD and APX, respectively. To our knowledge, we demonstrate, for the first time, that GI gene regulates the oxidative stress response, at least in part, through modulation of SOD and APX genes.     

Analysis of storage proteins in normal and aborted seeds from embryo-lethal mutants of Arabidopsis thaliana

The major storage proteins isolated from wild-type seeds of Arabidopsis thaliana (L.) Heynh., strain Columbia, were studied by sucrose gradient centrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Both the hypocotyl and cotyledons of mature embryos contained abundant 12 S (cruciferin) and 2 S (arabin) proteins that appeared similar in size and subunit composition to the cruciferin (12 S) and napin (1.7 S) seed-storage proteins of Brassica napus. The 12 S protein from Arabidopsis was resolved by SDS-PAGE into two groups of subunits with approximate relative molecular weights of 22–23 kDa (kilodalton) and 30–34 kDa. These polypeptides accumulated late in embryo development, disappeared early in germination, and were not detected in other vegetative or reproductive tissues. Accumulation of the 12 S proteins in aborted seeds from nine embryo-lethal mutants with different patterns of abnormal development was studied to determine the extent of cellular differentiation in arrested embryos from each mutant line. Abundant 12 S proteins were found in arrested embryos from two mutants with late lethal phases, but not in seven other mutants with lethal phases ranging from the globular to the cotyledon stages of embryo development. These results indicate that the accumulation of seed-storage proteins in wild-type embryos of Arabidopsis is closely tied to morphogenetic changes that occur during embryo development. Embryo-lethal mutants may therefore be useful in future studies on the developmental regulation of storage-protein synthesis.Abbreviations kDa kilodalton - M_r relative molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Two translesion synthesis DNA polymerase genes, AtPOLH and AtREV1, are involved in development and UV light resistance in Arabidopsis

Plants are continually exposed to external and internal DNA-damaging agents. Although lesions can be removed by different repair processes, damages often remain in the DNA during replication. Synthesis of template damages requires the replacement of replicative enzymes by translesion synthesis polymerases, which are able to perform DNA synthesis opposite specific lesions. These proteins, in contrast to replicative polymerases, operate at low processivity and fidelity. DNA polymerase η and Rev 1 are two proteins found in eukaryotes that are involved in translesion DNA synthesis. In Arabidopsis, DNA polymerase η and Rev 1 are encoded by AtPOLH and AtREV1 genes, respectively. Transgenic plants over-expressing AtPOLH showed increased resistance to ultraviolet light. Only plants with moderate AtREV1 over-expression were obtained, indicating that this enzyme could be toxic at high levels. Transgenic plants that over-expressed or disrupted AtREV1 showed reduced germination percentage, but the former exhibited a higher stem growth rate than the wild type during development.     

拟南芥活性氧不敏感型突变体的筛选与特性分析

采用 EMS化学诱变方法与 H2 O2 氧化胁迫选择 ,以根在重力作用下的弯曲生长为指标 ,筛选得到拟南芥活性氧不敏感型突变体。对突变体杂交后代遗传分析表明 ,突变株对活性氧不敏感性状为隐性单基因突变所致 ;生理生化分析表明突变体对 H2 O2 有很强的抗性 ,表现为气孔开度对 H2 O2 不敏感和 H2 O2 胁迫时较低的膜脂过氧化水平。运用 L SCM技术并结合 H2 O2 荧光探针 H2 DCFDA检测外源 ABA诱导保卫细胞内产生 H2 O2 的情况 ,结果显示突变体体内荧光强度比对照低 ,暗示了突变体体内消除 H2 O2 的能力可能有所提高 ,增强了植株对氧化胁迫的抗性。拟南芥活性氧不敏感突变体的筛选 ,不仅为人们深入研究活性氧在细胞内的作用提供良好的实验材料 ,而且还将大大加深人们对信号转导途径的再认识