衰老叶片中叶绿素的降解

综述了近年来关于衰老叶片中叶绿素降解的研究情况,包括叶绿素代谢的中间产物、终产物、主要代谢途径、代谢酶及代谢途径在细胞内的定位及代谢调节方面的研究进展。     

Chlorophyll breakdown in oilseed rape

Chlorophyll catabolism accompanying leaf senescence is one of the most spectacular natural phenomena. Despite this fact, the metabolism of chlorophyll has been largely neglegted until recently. Oilseed rape has been used extensively as a model plant for the recent elucidating of structures of chlorophyll catabolites and for investigation of the enzymic reactions of the chlorophyll breakdown pathway. The key reaction which causes loss of green color is catalyzed in a two-step reaction by pheophorbide a oxygenase and red chlorophyll catabolite reductase. In this Minireview, we summarize the actual knowledge about catabolites and enzymes of chlorophyll catabolism in oilseed rape and discuss the significance of this pathway in respect to chlorophyll degradation during Brassica napus seed development. This revised version was published online in June 2006 with corrections to the Cover Date.     

Variation in the glucosinolate content of oilseed rape (Brassica napus L.) leaves

The glucosinolate content of oilseed rape {Brassica napus) leaves was monitored over the growth period 30–70 days after planting, and a comparison made between a single-low cultivar (low in erucic acid), Bienvenu, and a double-low cultivar (low in erucic acid and glucosinolate), Cobra. In older, fully-expanded leaves the glucosinolate concentration was very low (< 0.3 μmol/ml tissue water) and did not alter during the course of the experiment. In developing sixth leaves glucosinolate content increased rapidly and reached a maximum concentration (4–5 μmol/ml tissue water) 40 days after planting (6 days after leaf emergence). The concentration then declined, to about 1 μmol/ml after 60 days although the total glucosinolate content in leaves continued to increase until 50 days; much of the reduction in concentration was simply a result of leaf expansion. No major differences were seen between the two varieties in total glucosinolate content or in the individual compounds present. Cv. Cobra developed more quickly than cv. Bienvenu so direct comparison between leaves of the two cultivars was complex. When comparing the glucosinolate content of oilseed rape leaves, between cultivars or between treatments, it is vital to ensure that carefully matched leaves of comparable developmental age are selected.     

Local and systemic changes in arginine decarboxylase activity, putrescine levels and putrescine catabolism in wounded oilseed rape

* Here we report the effect of mechanical wounding on putrescine biosynthesis and catabolism in oilseed rape (Brassica napus ssp. oleifera). * The lamina of first leaves was wounded by crushing with forceps, and first and second leaves were harvested at various intervals over a 24 h period. Levels of free polyamines were measured and activities of enzymes of polyamine biosynthesis and catabolism were assayed in the harvested tissue. * Mechanical wounding of the first leaves led to significant, but transient, increases in arginine decarboxylase (ADC) activity and levels of free putrescine in the wounded first leaf and in unwounded second leaves. The increased putrescine appeared to be the result of a combination of increased ADC activity, coupled with reduced putrescine catabolism, as activity of the oxidative enzyme diamine oxidase was significantly reduced following wounding, both locally and systemically. * The role of the increased free putrescine in the wound response of oilseed rape is not known, although the possibility that it is used to form putrescine conjugates is worthy of further investigation.     

Localization of glyoxylate-cycle marker enzymes in peroxisomes of senescent leaves and green cotyledons

Crude particulate homogenates from leaves of barley (Hordeum vulgare L.), rice (Oryza sativa L.), leaf-beet (Beta vulgaris var.cicla L.) and pumpkin (Cucurbita pepo L.) cotyledons were separated on sucrose density gradients. The peroxisomal fractions appeared at a buoyant density of 1.25 g·cm^–3 and contained most of the activities of catalase (EC 1.11.1.6), and hydroxypyruvate reductase (EC 1.1.1.81) on the gradients. In peroxisomal fractions from detached leaves and green cotyledons incubated in permanent darkness we detected the presence of isocitrate lyase (EC 4.1.3.1) and malate synthase (EC 4.1.3.2), key enzymes of the glyoxylate cycle, and-oxidation activity (except in pumpkin). As proposed by H. Gut and P. Matile (1988, Planta176, 548–550) the glyoxylate cycle may be functional during leaf senescence, and the presence of two key enzymes indicates a transition from leaf peroxisome to glyoxysome; for pumpkin cotyledons in particular a double transition occurs (glyoxysome to leaf peroxisome during greening, and leaf peroxisome to glyoxysome during senescence).We are grateful to Professor P. Matile (Zürich, Switzerland) for his encouragement in pursuing this work.     

油菜烯醇酶基因的克隆与分析(英文)

烯醇酶(enolase)是糖酵解途径中的一个重要酶类,它能够催化磷酸甘油酸酯(2-PGA)生成磷酸烯醇丙酮酸酯(PEP)。我们通过RACE-PCR方法从油菜(Brassica napus L. )中克隆到了编码烯醇酶的全长基因。序列分析表明该基因全长cDNA为1624bp,拥有一个由444个氨基酸组成的开放读码框,所编码的蛋白质分子量为47.38kD,等电点为5.78。比较发现,油菜烯醇酶与已分离出的其他烯醇酶氨基酸序列有较高的同源性。Southern杂交结果显示烯醇酶以低拷贝形式在油菜基因组中存在。RT-PCR和Northern分析表明烯醇酶基因在100mmol/L盐浓度胁迫条件下表达量上升,而在低温诱导时表达量下降。该研究表明所克隆基因是植物烯醇酶基因家族的新成员。     

Antiviral activity of extracts from Morinda citrifolia leaves and chlorophyll catabolites,pheophorbide a and pyropheophorbide a,against hepatitis C virus

The development of complementary and/or alternative drugs for treatment of hepatitis C virus (HCV) infection is still needed. Antiviral compounds in medicinal plants are potentially good targets to study. Morinda citrifolia is a common plant distributed widely in Indo‐Pacific region; its fruits and leaves are food sources and are also used as a treatment in traditional medicine. In this study, using a HCV cell culture system, it was demonstrated that a methanol extract, its n‐hexane, and ethyl acetate fractions from M. citrifolia leaves possess anti‐HCV activities with 50%‐inhibitory concentrations (IC₅₀) of 20.6, 6.1, and 6.6 μg/mL, respectively. Bioactivity‐guided purification and structural analysis led to isolation and identification of pheophorbide a, the major catabolite of chlorophyll a, as an anti‐HCV compound present in the extracts (IC₅₀ = 0.3 μg/mL). It was also found that pyropheophorbide a possesses anti‐HCV activity (IC₅₀ = 0.2 μg/mL). The 50%‐cytotoxic concentrations (CC₅₀) of pheophorbide a and pyropheophorbide a were 10.0 and 7.2 μg/mL, respectively, their selectivity indexes being 33 and 36, respectively. On the other hand, chlorophyll a, sodium copper chlorophyllin, and pheophytin a barely, or only marginally, exhibited anti‐HCV activities. Time‐of‐addition analysis revealed that pheophorbide a and pyropheophorbide a act at both entry and the post‐entry steps. The present results suggest that pheophorbide a and its related compounds would be good candidates for seed compounds for developing antivirals against HCV.     

Polyethylene glycol and electric field treatment of plant protoplasts: characterization of some membrane properties

Petiolar protoplasts of a dihaploid line of winter oilseed rape Brassica napus L. ssp. oleifera were exposed to fusogenic polyethylene glycol (PEG) and electric field treatments. The surface properties and stability of membrane components of the treated protoplasts were investigated by contact angle measurements in aqueous two-phase systems and differential scanning calorimetry, respectively. The leakage of intracellular components was estimated with respect to amino acids, proteins and DNA. Both fusogenic treatments resulted in the same apparent changes in membrane surface hydrophobicity and the same destabilization of membrane components. However, the PEG-treated protoplasts were more leaky than both the control and the electric field-treated protoplasts. The results indicate that the molecular mechanisms of PEG- and electrical field-induced fusion are similar. However, the effects of the latter appear to be less harmful presumably because the parameters for electric field treatment are more easily controlled.     

Apparent induction of key enzymes of the glyoxylic acid cycle in senescent barley leaves

The activities of two key enzymes of the glyoxylic-acid cycle, isocitrate lyase and malate synthase, can barely be detected in mature, presenescent primary leaves of barley (Hordeum vulgare L.) but are apparently induced in senescent leaf tissue. Upon incubation of leaf segments in permanent darkness, the activities appear and increase dramatically up to the sixth day and thereafter decline. The glyoxylic-acid cycle may thus be functional during foliar senescence. The main period of galactolipid loss is characterized by RQ values as low as 0.63, indicating that long-chain fatty acids produced from thylakoidal acyl-lipids may be utilized for gluconeogenesis involving corresponding glyoxisomal metabolic pathways. Foliar senescence may be characterized by a peroxisomeglyoxysome transition analogous to the glyoxisome-peroxisome transition in greening cotyledons of fat-storing seeds.Abbreviations FW fresh weight - MGDG monogalactosyl diacylglycerol - RQ respiratory quotient     

Development and characteristics of myrosinase in Brassica napus during early seedling growth

Myrosinase (β-thioglucoside glucohydroase, E. C. 3.2.3.1) proteins with different physical, but similar kinetic characteristics exist in oilseed rape ( Brassica napus L. cv. Bienvenu) seedlings. Two protein fractions have been described which are immunologically, and therefore likely to be structurally, related. Myrosinase I, a dimeric 156 kDa glycosylated protein was purified to apparent homogeneity, and polyclonal antibodies were raised against this protein. Myrosinase II, in comparison, was significantly less glycosylated. The native protein had a molecular weight of approximately 188 kDa, with subunit M_r's of mainly 62 kDa and also 68 kDa. Total 'potential'enzyme activity (assayed in the presence of ascorbic acid activator) increased during early seedling growth. Immunoblot analysis of seedling proteins showed that this is directly related to an increase in the amount of myrosinase protein itself , predominantly myrosinase II proteins, which are not present in the dry seed. Myrosinase II protein is located exclusively in the cotyledons of 5-day-old seedlings, whilst myrosinase I is distributed throughout the seedling.     

The role of chlorophyllase in degreening canola (Brassica napus) seeds and its activation by sublethal freezing

Chlorophyllase mediates dephytylation of chlorophylls and pheophytins during seed degreening in canola ( Brassica napus cv. Westar). Degreening can be correlated with chlorophyllase activity in vitro, but it is difficult to demonstrate in vivo activity because of low levels of the dephytylated breakdown products during rapid degreening. If, however, degreening is inhibited by sublethal freezing, chlorophyllide and pheophorbide accumulation can be related to the action of chlorophyllase. Changes in the rate of in vitro dephytylation during degreening and the dramatic increase following freezing may indicate enzyme activation and de novo enzyme synthesis. Evidence from Western blots is presented in support of de novo synthesis. It is concluded that failure of the seed to degreen following sublethal freezing does not result from a reduction in chlorophyllase activity.     

Acetolactate synthase from Brassica napus: Immunological characterization and quaternary structure of the native enzyme

Acetolactate synthase (ALS, AHAS; EC 4. 1. 3. 18) from Brassica napus has been partially purified and characterized using polyclonal antibodies. Following denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis and western blot analysis, 65 and 66 kDa ALS subunit polypeptides were immunologically detected, along with a novel 36 kDa polypeptide which cross-reacted with the anti-ALS antibody. Partial peptide sequencing of the 36 kDa peptide revealed significant similarity to plant aldolase proteins. ALS activity from stromal extracts fractionated by gel filtration chromatography as a single species of estimated molecular mass of 124 kDa, while comparative sedimentation coefficient in glycerol gradients indicated a corresponding molecular mass of 132 kDa. The results suggest that the native enzyme is a dimer of 65 and/or 66 kDa subunits. Anion exchange chromatography resolved two classes of ALS activity of equal native molecular weight, but which exhibited different properties with respect to subunit structure, sensitivity to inhibition by chlorsulfuron and feedback inhibition by branched chain amino acids.     

不同硼效率甘蓝型油菜品种细胞壁中硼的分配

应用不同硼效率甘蓝型油菜品种 ,研究硼在细胞壁中的分配。硼主要结合在细胞壁中 ,缺硼显著提高硼在细胞壁中的分配比例。根系细胞壁硼含量显著低于叶片 ,但根系细胞壁硼占根系总硼量之比例显著高于叶片。同一品种根系及其细胞壁、老叶细胞壁硼含量受生育期影响较小 ,新叶及其细胞壁、老叶硼含量受生育期影响较大。在正常供硼条件下 ,硼高效品种根系细胞壁和叶片细胞壁硼含量均低于低效品种 ;正常和缺硼条件下 ,硼高效品种细胞壁硼占器官总硼量之比例均低于低效品种。说明硼低效品种需较多的硼构建细胞壁。     

Letting the gene out of the bottle: the population genetics of genetically modified crops

Genetically modified (GM) plants are rapidly becoming a common feature of modern agriculture. This transition to engineered crops has been driven by a variety of potential benefits, both economic and ecological. The increase in the use of GM crops has, however, been accompanied by growing concerns regarding their potential impact on the environment. Here, we focus on the escape of transgenes from cultivation via crop x wild hybridization. We begin by reviewing the literature on natural hybridization, with particular reference to gene flow between crop plants and their wild relatives. We further show that natural selection, and not the overall rate of gene flow, is the most important factor governing the spread of favorable alleles. Hence, much of this review focuses on the likely effects of transgenes once they escape. Finally, we consider strategies for transgene containment.     

Chlorophyll breakdown in senescent Arabidopsis leaves. Characterization of chlorophyll catabolites and of chlorophyll catabolic enzymes involved in the degreening reaction

During senescence, chlorophyll (chl) is metabolized to colorless nonfluorescent chl catabolites (NCCs). A central reaction of the breakdown pathway is the ring cleavage of pheophorbide (pheide) a to a primary fluorescent chl catabolite. Two enzymes catalyze this reaction, pheide a oxygenase (PAO) and red chl catabolite reductase. Five NCCs and three fluorescent chl catabolites (FCCs) accumulated during dark-induced chl breakdown in Arabidopsis (Arabidopsis thaliana). Three of these NCCs and one FCC (primary fluorescent chl catabolite-1) were identical to known catabolites from canola (Brassica napus). The presence in Arabidopsis of two modified FCCs supports the hypothesis that modifications, as present in NCCs, occur at the level of FCC. Chl degradation in Arabidopsis correlated with the accumulation of FCCs and NCCs, as well as with an increase in PAO activity. This increase was due to an up-regulation of Pao gene expression. In contrast, red chl catabolite reductase is not regulated during leaf development and senescence. A pao1 knockout mutant was identified and analyzed. The mutant showed an age- and light-dependent cell death phenotype on leaves and in flowers caused by the accumulation of photoreactive pheide a. In the dark, pao1 exhibited a stay-green phenotype. The key role of PAO in chl breakdown is discussed.