活性氧不敏感型拟南芥的突变体对H2O2的响应

检测拟南芥ros突变株对H2O2响应的结果表明,此种突变体对H2O2有较强的耐受性,表现为气孔开度对H2O2不敏感和H2O2胁迫时的膜脂过氧化水平较低。采用激光扫描共聚焦显微术(LSCM)并结合H2O2荧光探针H2DCFDA检测外源ABA诱导保卫细胞的结果显示,突变体内荧光强度比野生型拟南芥低,暗示此种突变体消除H2O2的能力可能有提高,从而可增强植株抗氧化胁迫的能力。     

拟南芥根生长缺陷突变体rei1的分离和鉴定

植物体根发育是一个复杂的过程,尽管对其研究颇多,但对其中的分子机制尚缺乏足够认识。以模式植物拟南芥（Arabidopsis thaliana）为研究材料,在T-DNA突变体库中分离到一个拟南芥根生长缺陷突变体rei1（root elongationinhi-bited1）。通过表型分析发现,rei1在生长发育方面与野生型存在明显的差异,突变体的根较野生型短,且角果较小,花出现部分的败育。对突变体进行显微结构分析,发现突变体的根在内部结构上表现为表皮及皮层细胞形态不规则,排列疏松且横向膨大。遗传学分析表明,rei1是单基因隐性突变且与一个T-DNA插入共分离,通过图位克隆的方法成功分离了缺失的候选基因。以上研究结果表明,REI1对植物的根发育具有非常重要的调节作用。     

Weak Light-Induced Oxygen Consumption Observed during Photoreactivation Is Coupled to the Recovery of Oxygen Evolving Activity

During photoreactivation of the O₂-evolving center in Tris-inactivated/Mn-depletedthylakoids, a slow O₂-consumption occurred. This O₂-consumptionbecame detectable when the O₂-evolving activity of thylakoidswas inactivated by Tris-treatment and decreased as photoreactivationproceeded. The O₂-consumption and photoreactivation similarlyrequired Mn²⁺ at µM levels in addition to PSII electrondonors and shared severa common characteristics. Stimulationof O₂-consumption and photoreactivation by these cofactors werealways accompanied by enhancement in chlorophyll fluorescenceinduction, suggesting the involvement of a Mehler-type reactionin photoreactivation. Although the electron transport due tothis O₂-consumption was rapid enough to oxidize 4 Mn²⁺ ionsto reconstitute the tetranuclear Mn-cluster in each O₂-evolvingcenter in a few seconds, actual recovery of O₂-evolving activityoccurred more slowly in a few minutes. It was inferred thatphotoreactivation in Tris-inactivated thylakoids is not a simplephotooxidation of Mn2²⁺ but involves more complicated processeswhich are coupled to the Mehlertype electron transport fromPSII to oxygen via PSI. (Received July 11, 1994; Accepted August 23, 1996)     

Phytochrome E Controls Light-Induced Germination of Arabidopsis

Germination of Arabidopsis seeds is light dependent and under phytochrome control. Previously, phytochromes A and B and at least one additional, unspecified phytochrome were shown to be involved in this process. Here, we used a set of photoreceptor mutants to test whether phytochrome D and/or phytochrome E can control germination of Arabidopsis. The results show that only phytochromes B and E, but not phytochrome D, participate directly in red/far-red light (FR)-reversible germination. Unlike phytochromes B and D, phytochrome E did not inhibit phytochrome A-mediated germination. Surprisingly, phytochrome E was required for germination of Arabidopsis seeds in continuous FR. However, inhibition of hypocotyl elongation by FR, induction of cotyledon unfolding, and induction of agravitropic growth were not affected by loss of phytochrome E. Therefore, phytochrome E is not required per se for phytochrome A-mediated very low fluence responses and the high irradiance response. Immunoblotting revealed that the need of phytochrome E for germination in FR was not caused by altered phytochrome A levels. These results uncover a novel role of phytochrome E in plant development and demonstrate the considerable functional diversification of the closely related phytochromes B, D, and E.     

Singlet Oxygen Is the Major Reactive Oxygen Species Involved in Photooxidative Damage to Plants

Reactive oxygen species act as signaling molecules but can also directly provoke cellular damage by rapidly oxidizing cellular components, including lipids. We developed a high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry-based quantitative method that allowed us to discriminate between free radical (type I)- and singlet oxygen (¹O₂; type II)-mediated lipid peroxidation (LPO) signatures by using hydroxy fatty acids as specific reporters. Using this method, we observed that in nonphotosynthesizing Arabidopsis (Arabidopsis thaliana) tissues, nonenzymatic LPO was almost exclusively catalyzed by free radicals both under normal and oxidative stress conditions. However, in leaf tissues under optimal growth conditions, ¹O₂ was responsible for more than 80% of the nonenzymatic LPO. In Arabidopsis mutants favoring ¹O₂ production, photooxidative stress led to a dramatic increase of ¹O₂ (type II) LPO that preceded cell death. Furthermore, under all conditions and in mutants that favor the production of superoxide and hydrogen peroxide (two sources for type I LPO reactions), plant cell death was nevertheless always preceded by an increase in ¹O₂-dependent (type II) LPO. Thus, besides triggering a genetic cell death program, as demonstrated previously with the Arabidopsis fluorescent mutant, ¹O₂ plays a major destructive role during the execution of reactive oxygen species-induced cell death in leaf tissues.Plant leaves capture sun-derived light energy to drive CO₂ fixation during photosynthesis. During this process, leaves need to cope with photooxidative stress when the balance between energy absorption and consumption is disturbed. Excess excitation energy in the photosystems (PSI and PSII) leads to the inhibition of photosynthesis via the production of various reactive oxygen species (ROS) at different spatial levels of the cell (Apel and Hirt, 2004; Asada, 2006; Van Breusegem and Dat, 2006). Both exposure to high light intensities and decreased CO₂ availability direct linear electron transfer toward the reduction of molecular oxygen, generating superoxide radicals (O₂^−.) at PSI (the Mehler reaction). Superoxide dismutation generates hydrogen peroxide (H₂O₂), which is detoxified in the chloroplast by ascorbate peroxidases. As such, this so-called water-water cycle participates in the dissipation of excess energy (Asada, 2006). Decreased CO₂ availability affects the first step in CO₂ fixation by shifting the carboxylation of Rubisco by the Rubisco carboxylase-oxygenase enzyme toward oxygenation, a process called photorespiration. This leads, through the action of glycolate oxidase, to peroxisomal H₂O₂ production that is counteracted by catalases. Finally, when the intersystem electron carriers are overreduced, triplet excited P680 in the PSII reaction center as well as triplet chlorophylls in the light-harvesting antennae are produced, with the production of singlet oxygen (¹O₂) as a consequence (Krieger-Liszkay, 2005). In photosynthetic membranes, ¹O₂ is quenched by carotenoids and tocopherols. When antioxidant mechanisms are overwhelmed, increased cellular ROS levels trigger signal transduction events related to stress signaling and programmed cell death (Mittler et al., 2004; Van Breusegem and Dat, 2006). On the other hand, excessive ROS accumulation damages pigments, proteins, nucleic acids, and lipids (Halliwell and Gutteridge, 2007), thereby contributing to or executing cell death.Since under environmental stress conditions different ROS are produced simultaneously, a causal link between the accumulation of a specific ROS and its signaling or damaging effects has always been difficult to establish. In recent years, the production of various transgenic Arabidopsis (Arabidopsis thaliana) plants with compromised levels of specific antioxidant enzymes and the identification of the conditional fluorescent (flu) mutant provided important tools to assess the specific effects of O₂^−., H₂O₂, and ¹O₂ within a particular subcellular compartment (Dat et al., 2003; op den Camp et al., 2003; Pnueli et al., 2003; Rizhsky et al. 2003; Vandenabeele et al., 2004; Wagner et al., 2004; Queval et al., 2007). For example, with catalase-deficient [Cat(−)] plants, the signaling effects of increased photorespiratory H₂O₂ levels could be identified (Dat et al., 2003; Vandenabeele et al., 2004; Queval et al., 2007). Similarly, in the conditional flu mutant increased plastid ¹O₂ levels were shown to induce a genetic program leading to cell death (op den Camp et al., 2003; Wagner et al., 2004). Nevertheless, whereas careful monitoring of gene expression on the whole-genome level enables to pinpoint specific signaling capacities for diverse ROS (Mittler et al., 2004; Gadjev et al., 2006), it remained impossible to discriminate between the oxidative damaging effects on cellular components of different ROS.One consequence of ROS formation is lipid peroxidation (LPO; Halliwell and Gutteridge, 2007). Two nonenzymatic reaction types lead to specific patterns of oxidized membrane polyunsaturated fatty acids (PUFAs; Stratton and Liebler, 1997; Montillet et al., 2004; Mueller et al., 2006). Type I reactions are initiated by free radicals (FRs) having high redox potential, such as hydroxyl radicals (^.OH) or organic oxyl and peroxyl radicals, and type II reactions are the result of ¹O₂ action. Notably, O₂^−. and H₂O₂ are not sufficiently reactive to oxidize any PUFA. However, both ROS can be nonenzymatically converted to ^.OH through Fenton-type reactions in the presence of transition metal ions such as Fe²⁺ (Halliwell and Gutteridge, 2007). Both type I and type II reactions lead to the formation of respective oxygenated fatty acids. Here, we propose a novel and quantitative approach to distinguish between FR- and ¹O₂-mediated LPO in plants by quantifying type II oxidation-specific hydroxy fatty acids with HPLC-tandem mass spectrometry (MS/MS), allowing us to monitor the relative contribution of LPO caused by PSI-dependent O₂^−./H₂O₂, photorespiratory H₂O₂, and photosynthetic ¹O₂ during photooxidative stress and cell death. We demonstrate that nonenzymatic LPO in leaves is almost exclusively mediated by ¹O₂ and that photooxidative stress-dependent cell death involves ¹O₂ production in its final stage.     

Phosphatidylglycerol Composition Is Central to Chilling Damage in the Arabidopsis fab1 Mutant

The Arabidopsis (Arabidopsis thaliana) fatty acid biosynthesis1 (fab1) mutant has increased levels of the saturated fatty acid 16:0, resulting from decreased activity of 3-ketoacyl-ACP synthase II. In fab1 leaves, phosphatidylglycerol, the major chloroplast phospholipid, contains >40% high-melting-point molecular species (HMP-PG; molecules that contain only 16:0, 16:1-trans, and 18:0 fatty acids)—a trait associated with chilling-sensitive plants—compared with <10% in wild-type Arabidopsis. Although they do not exhibit short-term chilling sensitivity when exposed to low temperatures (2°C to 6°C) for long periods, fab1 plants do suffer collapse of photosynthesis, degradation of chloroplasts, and eventually death. To test the relevance of HMP-PG to the fab1 phenotype, we used transgenic 16:0 desaturases targeted to the endoplasmic reticulum and the chloroplast to lower 16:0 in leaf lipids of fab1 plants. We produced two lines that had very similar lipid compositions except that one, ER-FAT5, contained high HMP-PG, similar to the fab1 parent, while the second, TP-DES9*, contained <10% HMP-PG, similar to the wild type. TP-DES9* plants, but not ER-FAT5 plants, showed strong recovery and growth following 75 d at 2°C, demonstrating the role of HMP-PG in low-temperature damage and death in fab1, and in chilling-sensitive plants more broadly.

In higher plants, the chloroplast membranes that host the light harvesting and electron transport processes of photosynthesis have a characteristically high number of double bonds in the glycerolipid acyl chains. Only ∼10% of the fatty acids that compose the hydrophobic core of the thylakoid bilayer lack double bonds altogether, whereas >80% are polyunsaturated, having two or three double bonds (Ohlrogge et al., 2015). The photosynthetic light reactions produce reactive oxygen species as by-products, and these can degrade polyunsaturated fatty acids, so it is assumed that highly unsaturated membranes are required to support photosynthesis (McConn and Browse, 1998).The glycerolipids in chloroplast membranes are synthesized by two separate pathways. (Browse et al., 1986; Ohlrogge and Browse, 1995). Synthesis de novo of fatty acids takes place in the stroma of chloroplasts, producing 16:0 esterified to acyl carrier protein (ACP). A large proportion of this 16:0-ACP is elongated by 3-keto-acyl-ACP synthase II (KASII) to 18:0-ACP, which is in turn desaturated by stearoyl ACP desaturase to produce 18:1-ACP (Lindqvist et al., 1996; Carlsson et al., 2002). The fatty acids from 16:0-ACP and 18:1-ACP may be used within the chloroplast in the prokaryotic pathway (Kunst et al., 1988; Kim and Huang, 2004) to produce phosphatidic acid (PA). Some of this PA intermediate is used for synthesis of phosphatidylglycerol (PG; Ohlrogge and Browse, 1995; Wada and Murata, 2007), which is the only chloroplast glycerolipid that is produced solely by the prokaryotic pathway. In some plants, including Arabidopsis (Arabidopsis thaliana), PA is also converted to diacylglycerol (DAG), which is the precursor for the synthesis of the other chloroplast glycerolipids, monogalactosyldiacylglycerol (MGD), digalactosyldiacylglycerol (DGD), and sulfoquinovosyldiacylglycerol (SQD; Browse et al., 1986; Ohlrogge and Browse, 1995; Ohlrogge et al., 2015).The second route for chloroplast glycerolipid synthesis, the eukaryotic pathway, begins with export of 16:0 and 18:1 from the chloroplast as CoA thioesters. (Li et al., 2015). In the endoplasmic reticulum (ER), these fatty acids are rapidly incorporated into phosphatidylcholine (PC) by acyl exchange (Bates et al., 2007), and are also used (via PA and DAG intermediates) for the synthesis of all the phospholipids of the extrachloroplast membranes of the cell (Ohlrogge et al., 2015). In addition however, the DAG moiety of PC can be returned to the chloroplast and contribute to the production of MGD, DGD, and SQD required for thylakoid synthesis (Benning, 2009; Roston et al., 2012). The ER-to-chloroplast flux of lipid is reversible to some extent (Browse et al., 1989, 1993).With the exception of the first Δ9 double bond in 18:1-ACP, all the double bonds in the acyl chains are introduced after the initial synthesis of glycerolipid molecules. In Arabidopsis, this involves the action of seven fatty acid desaturases that are integral membrane proteins in the chloroplast and ER (Ohlrogge and Browse, 1995; Wallis and Browse, 2010). Characterization of Arabidopsis fatty acid desaturation (fad) mutants deficient in one or more of these desaturases has shown that the high level of thylakoid unsaturation is essential to photosynthetic function (Murakami et al., 2000; Routaboul et al., 2000). For example, fad2 fad6 double-mutant plants are unable to synthesize polyunsaturated fatty acids and cannot grow autotrophically; however, when grown on Suc as a carbon source, the double mutants are robust plants showing strong leaf and root development (McConn and Browse, 1998). These results indicate that the vast majority of receptor-mediated and transport-related membrane functions required to sustain the organism and induce proper development are adequately supported in the absence of polyunsaturated lipids; photosynthesis is the one process that requires high levels of polyunsaturation. Mutants with smaller changes in unsaturation are often similar to the wild type under typical growth-chamber conditions and reveal their phenotypes only under more extreme conditions (Wallis and Browse, 2002, 2010). Several mutants grow more slowly and become chlorotic at temperatures in the range 2°C to 10°C (Hugly and Somerville, 1992; Routaboul et al., 2000), indicating a role for fatty acid composition in maintaining photosynthesis at these low temperatures.Like other species native to temperate regions, Arabidopsis is chilling resistant and able to grow at temperatures close to 0°C. By contrast, many tropical and subtropical plant species are chilling sensitive and suffer sharp reductions of photosynthesis and extensive tissue damage after even short exposure to low temperatures. Many of the world’s most important crops, including rice (Oryza sativa), maize (Zea mays), and soybean (Glycine max) are chilling sensitive, so a better understanding of the biochemical and genetic factors contributing to this sensitivity has the potential to enhance sustainable food production (Nishida and Murata, 1996; Iba, 2002; Thakur et al., 2010). One hypothesis proposes that chilling sensitivity is a result of the fatty acid composition of chloroplast PG. It is based on the observation that many chilling-sensitive plants contain >30% of PG molecules with only saturated or trans unsaturated fatty acids—16:0, 18:0, and 16:1-Δ3trans (16:1t)—at both the sn-1 and sn-2 positions of the glycerol backbone, referred to as high-melting-point molecular species (HMP-PG; Murata, 1983; Barkan et al., 2006). This name alludes to the fact that HMP-PG species can induce a phase change from liquid crystalline (typical of biological membranes) to gel phase at temperatures well above 0°C and thereby disrupt membrane and cellular function (Murata and Yamaya, 1984). Chilling-resistant plants have <10% HMP species in chloroplast PG (Murata et al., 1982; Murata, 1983; Roughan, 1985).One perspective on the role of HMP-PG in plant temperature responses has come from our investigations of the fatty acid biosynthesis1 (fab1) mutant of Arabidopsis. In this mutant, a hypomorphic mutation in the gene encoding KASII reduces elongation of 16:0-ACP to 18:0-ACP (Carlsson et al., 2002), producing plants that have increased levels of 16:0 in all membrane glycerolipids (Wu et al., 1994). In particular, fab1 plants contain HMP-PG at levels (∼40% to 50% of total PG) similar to those of many chilling-sensitive plant species (Wu and Browse, 1995). Nevertheless, the fab1 mutant does not show typical symptoms of chilling sensitivity and is unaffected, in comparison to wild-type controls, by a range of chilling treatments that kill chilling-sensitive plants; instead, fab1 plants only show a collapse of photosynthesis after >10 d of exposure to 2°C, with the plants dying after several weeks at low temperature (Wu and Browse, 1995; Wu et al., 1997).We have previously screened for genetic suppressors of the fab1 low-temperature phenotype. Most, though not all, of the suppressor mutations substantially reduce the proportion of saturated fatty acids in PG, consistent with the notion that HMP-PG causes eventual death of fab1 plants in the cold (Barkan et al., 2006; Kim et al.,2010; Gao et al., 2015). However, all the suppressors have additional changes, relative to fab1, in the fatty acid compositions of membrane lipids that prevent a clear linkage between reductions in HMP-PG and improved low-temperature survival.Here, we have taken a new approach to investigating the role of HMP-PG in damage and death of fab1 plants at chilling temperatures by using a 16:0-CoA desaturase from Caenorhabditis elegans, FAT-5 (Watts and Browse, 2000), and a glycerolipid desaturase, DES9*15, derived from a cyanobacterial enzyme by directed evolution (Bai et al., 2016). When expressed in the fab1 mutant background, both the FAT-5 enzyme targeted to the ER and the DES9*15 enzyme targeted to the chloroplast reduced leaf 16:0 to near-wild type levels. The fatty acid compositions of individual leaf lipids in plants of both transgenic lines were very similar, with the sole exception of PG. Plants expressing the FAT-5 desaturase retained high levels of HMP-PG, similar to fab1, while plants expressing the DES9*15 enzyme had HMP-PG lowered to levels close to those of the wild type. Like the fab1 mutant, fab1 plants expressing a 16:0 desaturase in the ER lost photosynthetic function over 28 d of exposure to 2°C and showed little capacity for recovery and growth after longer periods at low temperature. By contrast, plants expressing a 16:0 desaturase targeted to the chloroplast retained substantial photosynthetic function, even after 75 d at 2°C, and were subsequently able to resume growth, flower, and set seed upon return to 22°C. These results provide the most direct evidence yet that high levels of HMP-PG cause gradual loss of photosynthesis and eventual death of plants at chilling temperatures.     

Acclimation of Arabidopsis thaliana to the light environment: the role of photoreceptors

The regulation by light of the composition of the photosynthetic apparatus was investigated in photomorphogenic mutants of Arabidopsis thaliana (L.) Heynh. cv. Landsberg erecta. Leaf chlorophyll, photosynthesis, photosystem II function, and ribulose-1,5-bisphosphate carboxylase-oxygenase and photosystem II contents were determined for plants grown under high- or low-irradiance growth regimes. Although certain mutant lines had altered chloroplast composition compared to the wild type, all photoreceptor mutants tested were capable of light-dependent changes in chloroplast composition and photosynthetic function, indicating that photoreceptors do not play a central role in the regulation of acclimation at the level of the chloroplast. However, the clear acclimation defect in a det1 signal transduction mutant indicates that photoreceptor-controlled responses either share regulatory components with acclimation, or are important in the expression of components which in turn regulate acclimation. We suggest that the COP/DET/FUS regulatory cluster is a focus for multiple signal transduction pathways, including some of the metabolic signals which form the basis for the acclimatory response. Received: 22 April 1999 / Accepted: 6 June 1999     

Acclimation of Arabidopsis thaliana to the light environment: regulation of chloroplast composition

The regulation by light of the composition of the photosynthetic apparatus was investigated in Arabidopsis thaliana (L.) Heynh. cv. Landsberg erecta. When grown in high- and low-irradiance white light, wild-type plants and photomorphogenic mutants showed large differences in their maximum photosynthetic rate and chlorophyll a/b ratios; such changes were abolished by growth in red light. Photosystem I (PSI) and PSII levels were measured in wild-type plants grown under a range of light environments; the results indicate that regulation of photosystem stoichiometry involves the specific detection of blue light. Supplementing red growth lights with low levels of blue light led to large increases in PSII content, while further increases in blue irradiance had the opposite effect; this latter response was abolished by the hy4 mutation, which affects certain events controlled by a blue-light receptor. Mutants defective in the phytochrome photoreceptors retained regulation of photosystem stoichiometry. We discuss the results in terms of two separate responses controlled by blue-light receptors: a blue-high-fluence response which controls photosystem stoichiometry; and a blue-low-fluence response necessary for activation of such control. Variation in the irradiance of the red growth light revealed that the blue-high-fluence response is attenuated by red light; this may be evidence that photosystem stoichiometry is controlled not only by photoreceptors, but also by photosynthetic metabolism.Abbreviations BHF blue-high-fluence - BLF blue-low-fluence - Chl chlorophyll - FR far-red light - LHCII light-harvesting complex of PSII - P_max maximum photosynthetic rate - R red light - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase This work was supported by Natural Environment Research Council Grant No. GR3/7571A. We would like to thank H. Smith (Botany Department, University of Leicester) and E. Murchie (INRA, Versailles) for helpful discussions.     

Acclimation of Arabidopsis thaliana to the light environment: changes in photosynthetic function

Arabidopsis thaliana (L.) Heynh. cv. Landsberg erecta was grown under light regimes of differing spectral qualities, which results in differences in the stoichiometries of the two photosynthetic reaction centres. The acclimative value of these changes was investigated by assessing photosynthetic function in these plants when exposed to two spectrally distinct actinic lights. Plants grown in an environment enriched in far-red light were better able to make efficient use of non-saturating levels of actinic light enriched in long-wavelength red light. Simultaneous measurements of chlorophyll fluorescence and absorption changes at 820 nm indicated that differences between plants grown under alternative light regimes can be ascribed to imbalances in excitation of photosystems I and II (PSI, PSII). Measurements of chlorophyll fluorescence emission and excitation spectra at 77 K provided strong evidence that there was little or no difference in the composition or function of PSI or PSII between the two sets of plants, implying that changes in photosynthetic stoichiometry are primarily responsible for the observed differences in photosynthetic function.Abbreviations Chl chlorophyll - FR far-red light - HF highirradiance FR-enriched light (400 mol·m^–2·s^–1, RFR = 0.72) - HW high-irradiance white light (400 mol·m^–2 1·1 s^–1RFR = 1.40) - LHCI, LHCII light-harvesting complex of PSI, PSII - qO quenching of dark-level chlorophyll fluorescence - qN non-photochemical quenching of variable chlorophyll fluorescence - qP photochemical quenching of variable chlorophyll fluorescence - R red light - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase We thank Dr. Sasha Ruban for assistance with the 77 K fluorescence measurements and for helpful discussions. This work was supported by Natural Environment Research Council Grant GR3/7571A.     

The Ene Reaction of Singlet Oxygen with Olefins

The reactions of singlet oxygen (¹O₂) with cis and trans butenes-1,1,1-d₃, at—80°C in Freon-11, show a product isotope effect (k_H/k_D) of 1.38 and 1.25 respectively. Isomerization of the starting materials or formation of dioxetanes were not observed during the course of the photooxygenation. Together with the isotope effects on the reactions of tetramethylethylene-d₆ isomers with singlet oxygen, these results require the reversible formation of a perepoxide or charge transfer intermediate.     

The Ene Reaction of Singlet Oxygen with Olefins

The reactions of singlet oxygen (¹O₂) with cis and trans butenes-1,1,1-d₃, at—80°C in Freon-11, show a product isotope effect (k_H/k_D) of 1.38 and 1.25 respectively. Isomerization of the starting materials or formation of dioxetanes were not observed during the course of the photooxygenation. Together with the isotope effects on the reactions of tetramethylethylene-d₆ isomers with singlet oxygen, these results require the reversible formation of a perepoxide or charge transfer intermediate.     

Photosynthetic Properties of Photosystem Ⅱ in Arabidopsis thaliana Ipa1 Mutant

In a previous study, we characterized a high chlorophyll fluorescence Ipal mutant of Arabidopsis thallana, in which approximately 20% photosystem （PS） Ⅱ protein is accumulated. In the present study, analysis of fluorescence decay kinetics and thermoluminescence profiles demonstrated that the electron transfer reaction on either the donor or acceptor side of PSII remained largely unaffected in the Ipa1 mutant. In the mutant, maximal photochemical efficiency （Fv/Fm, where Fm is the maximum fluorescence yield and Fv is variable fluorescence） decreased with increasing light intensity and remained almost unchanged in wildtype plants under different light conditions. The Fv/Fm values also increased when mutant plants were transferred from standard growth light to low light conditions. Analysis of PSll protein accumulation further confirmed that the amount of PSll reaction center protein is correlated with changes in Fv/Fm in Ipal plants. Thus, the assembled PSll in the mutant was functional and also showed increased photosensitivity compared with wild-type plants.     

Photosynthetic Properties of Photosystem II in Arabidopsis thaliana lpa1 Mutant

In a previous study, we characterized a high chlorophyll fluorescence lpa1 mutant of Arabidopsis thaliana, in which approximately 20% photosystem (PS) II protein is accumulated. In the present study, analysis of fluorescence decay kinetics and thermoluminescence profiles demonstrated that the electron transfer reaction on either the donor or acceptor side of PSII remained largely unaffected in the lpa1 mutant. In the mutant, maximal photochemical efficiency (Fv/Fm, where Fm is the maximum fluorescence yield and Fv is variable fluorescence) decreased with increasing light intensity and remained almost unchanged in wild-type plants under different light conditions. The Fv/Fm values also increased when mutant plants were transferred from standard growth light to low light conditions. Analysis of PSII protein accumulation further confirmed that the amount of PSII reaction center protein is correlated with changes in Fv/Fm in lpa1 plants. Thus, the assembled PSII in the mutant was functional and also showed increased photosensitivity compared with wild-type plants.(Author for correspondence. Tel: +86 (0)10 6283 6256; Fax: +86 (0)10 8259 9384; E-mail: zhanglixin@ibcas.ac.cn)     

Mitochondrial Ubiquitin Ligase MITOL Ubiquitinates Mutant SOD1 and Attenuates Mutant SOD1-induced Reactive Oxygen Species Generation

We have previously identified a novel mitochondrial ubiquitin ligase, MITOL, which is localized in the mitochondrial outer membrane and is involved in the control of mitochondrial dynamics. In this study, we examined whether MITOL eliminates misfolded proteins localized to mitochondria. Mutant superoxide dismutase1 (mSOD1), one of misfolded proteins, has been shown to localize in mitochondria and induce mitochondrial dysfunction, possibly involving in the onset and progression of amyotrophic lateral sclerosis. We found that in the mitochondria, MITOL interacted with and ubiquitinated mSOD1 but not wild-type SOD1. In vitro ubiquitination assay revealed that MITOL directly ubiquitinates mSOD1. Cycloheximide-chase assay in the Neuro2a cells indicated that MITOL overexpression promoted mSOD1 degradation and suppressed both the mitochondrial accumulation of mSOD1 and mSOD1-induced reactive oxygen species (ROS) generation. Conversely, the overexpression of MITOL CS mutant and MITOL knockdown by specific siRNAs resulted in increased accumulation of mSOD1 in mitochondria, which enhanced mSOD1-induced ROS generation and cell death. Thus, our findings indicate that MITOL plays a protective role against mitochondrial dysfunction caused by the mitochondrial accumulation of mSOD1 via the ubiquitin–proteasome pathway.     

Arabinose Kinase-Deficient Mutant of Arabidopsis thaliana

A mutant of Arabidopsis thaliana that is sensitive to exogenous l-arabinose has been isolated. Comparisons of growth of the wild type, mutant, and F1 and F2 progeny of crosses showed the arabinose-sensitive phenotype is semidominant and segregates as a single Mendelian locus. Crosses of the mutant to marker strains showed the mutation is linked to the eceriferum-2 locus on chromosome 4. In vivo incorporation of exogenous labeled l-arabinose into ethanol-insoluble polysaccharides was greatly reduced in the mutant with a concomitant accumulation of free labeled arabinose. Enzyme assays of crude plant extracts demonstrated a defect in arabinose kinase activity in the mutant.