烟草中多酚氧化酶(PPO)的特征

烟草中的多酚氧化酶介导的褐变会影响烟叶和烟丝的色泽和内在质量,因此对其特性的研究,以及活性的控制成为多年来的研究热点。本文从其生物发生模型、分子结构、生物化学和光谱学特征与植物抗病和机械损伤的关系,多酚氧化酶的抑制、多酚氧化酶的应用等方面着手,对近几年来烟草中PPO研究的最新成果进行总结和回顾,对一些有争议的问题进行了探讨,并对未来PPO研究的方向和领域进行了展望。     

Isolation of a full-length cDNA encoding polyphenol oxidase from sugarcane, a C4 grass

Polyphenol oxidase (PPO) activity in sugarcane (a C4 grass) was highest in the growing point and declined down the stalk. Sugarcane PPO with an apparent molecular mass of 45 kDa was purified to homogeneity from immature stem tissue. Western analysis of sugarcane extracts with a polyclonal antibody raised to this protein suggested it resulted from cleavage of a 60 kDa protein during purification. The antibody was used to screen a sugarcane stem cDNA library. A full-length PPO clone (sugppol) was characterised and shown to encode a 67 kDa precursor protein comprising a plastid transit sequence of 8 kDa and a mature PPO protein of 59 kDa. High levels of expression ofsugppol were detected in the growing point of the stalk and in the immature tissue immediately below it, but no message was detected in RNA from mature stem or leaf. Comparison with other PPO sequences indicated thatsugppol was significantly different to PPO genes in C3 dicotyledonous plants.     

The overexpression of an alternative oxidase gene triggers ozone sensitivity in tobacco plants

The alternative oxidase (AOX) of plant mitochondria transfers electrons from the ubiquinione pool to oxygen without energy conservation and prevents the formation of reactive oxygen species (ROS) when the ubiquinone pool is over-reduced. Thus, AOX may be involved in plant acclimation to a number of oxidative stresses. To test this hypothesis, we exposed wild-type (WT) Xanthi tobacco plants as well as Xanthi plants transformed with the Bright Yellow tobacco AOX1a cDNA with enhanced (SN21 and SN29), and decreased (SN10) AOX capacity to an acute ozone (O3) fumigation. As a result of 5 h of O3 exposition (250 nL L(-1)), SN21 and SN29 plants surprisingly showed localized leaf damage, whereas SN10, similarly to WT plants, was undamaged. In keeping with this observation, WT and SN21 plants differed in their response to O3)for the expression profiles of catalase 1 (CAT1), catalase 2 (CAT2), glutathione peroxidase (GPX) and ascorbate peroxidase (APX) genes, and for the activity of these antioxidant enzymes, which were induced in WT. Concomitantly, although ozone induced H2O2 accumulation in WT and in all transgenic lines, only in transgenics with high AOX capacity the H2O2 level in the post-fumigation period was high. The alternative pathway of WT plants was strongly stimulated by O3, whereas in SN21 plants, the respiratory capacity was always high across the treatment. The present results show that, far from exerting a protective role, the overexpression of AOX triggers an increased O3 sensitivity in tobacco plants. We hypothesize that the AOX overexpression results in a decrease of mitochondrial ROS level that in turn alters the defensive mitochondrial to nucleus signalling pathway that activates ROS scavenging systems.     

Molecular cloning and characterization of the polyphenol oxidase gene from sweetpotato

Polyphenol oxidase is the enzyme responsible for enzymatic browning in sweetpotato that decreases the commercial value of sweetpotato products. Here we reported the cloning and characterization of a new cDNA encoding PPO from sweetpotato, designated as IbPPO (GeneBank accession number: AY822711). The full-length cDNA of IbPPO is 1984 bp with a 1767 bp open reading frame (ORF) encoding a 588 amino acid polypeptide with a calculated molecular weight of 65.7 kDa and theoretical pI of 6.28. The coding sequence of IbPPO was also directly amplified from the genomic DNA of sweetpotato that demonstrated that IbPPO was an intron-free gene. The computational comparative analysis revealed that IbPPO showed homology to other PPOs of plant origin and contained a 50 amino acid plastidial transit peptide at its N-terminal and the two conserved CuA and CuB copper-binding motifs in the catalytic region of IbPPO. A highly conserved serine-rich motif was firstly found in the transit peptides of plant PPO enzymes. Then the homology based structural modeling of IbPPO showed that IbPPO had the typical structure of PPO: the catalytic copper center was accommodated in a central four-helix bundle located in a hydrophobic pocket close to the surface. Finally, the results of the semiquantitative RT-PCR analysis of IbPPO in different tissues demonstrated that IbPPO could express in all the organs of sweetpotato including mature leaves, young leaves, the stems of mature leaves (petioles), the storage roots, and the veins but at different levels. The highest-level expression of IbPPO was found in the veins, followed by storage roots, young leaves and mature leaves; and the lowest-level expression of IbPPO was found in petioles. The present researches will facilitate the development of antibrown sweetpotato by genetic engineering. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 6, pp. 1006–1012. The article was submitted by the authors in English.     

马铃薯多酚氧化酶基因克隆及反义表达载体构建

从甘农薯1号马铃薯试管苗叶片中提取总DNA,根据Genebank报道的马铃薯多酚氧化酶基因(PPT32)序列,设计合成了带有BamHⅠ、SacⅠ特定酶切位点的2对特异引物,通过PCR获得l840bP和476bP的扩增产物。测序结果表明,l840bp的扩增产物与Oenebank中发表的序列一致,476bp的扩增产物正是欲扩增的马铃薯多酚氧化酶基因的同源片段。将2个扩增产物反向连接到表达载体PC3中,通过双酶切鉴定后,按直接导入法实现反义基因表达载体质粒向农杆菌EHA105的导人;并经PCR鉴定证明,此质粒已整合到农杆菌Ti上。     

岷江流域王百合和通江百合多酚氧化酶同功酶的研究

应用聚丙烯酰胺凝胶垂直平板电泳技术,对岷江流域王百合3个采样点的14个样品和通江百合4个采样点的4个样品的多酚氧化酶(PPO)同功酶进行了研究。结果表明,两种百合的多酚氧化酶在种间和种内均有一定差异。酶谱的聚类分析在遗传相似系数约0.5处,将两种百合18个样品分成5个类群,其中王百合的13个样品聚成了4类,样品12则与通江百合的4个样品聚成了1类;5个类群在外部形态上也呈现出相似性。     

Transcriptional regulation of a pineapple polyphenol oxidase gene and its relationship to blackheart

Two genes encoding polyphenol oxidase (PPO) were isolated from pineapple (Ananas comosus[L.] Merr. cv. Smooth Cayenne). Sequence analyses showed that both contained a single intron and encoded typical chloroplast-localized PPO proteins, the sequences of which corresponded to two pineapple PPO cDNAs, PINPPO1 and PINPPO2, recently described by Stewart et al. (2001). Southern blot analyses suggested that pineapple contained only two PPO genes. Analysis of expression of PINPPO1 promoter GUS fusion constructs showed this promoter had a low basal activity and was cold- and wound-inducible, consistent with known mRNA expression profiles. Striking homologies to gibberellin response complexes (GARC) were observed in sequences of both the PINPPO1 and PINPPO2 promoters. Transient assays in mature pineapple fruit and stable expression in transgenic tobacco showed that PINPPO1 promoter-GUS fusions were indeed gibberellin (GA) responsive. A role for the element within the putative GARCs in mediating GA-responsiveness of the PINPPO1 promoter was confirmed by mutational analysis. PINPPO2 was also shown to be GA-responsive by RT-PCR analysis. Mutant PINPPO1 promoter-GUS fusion constructs, which were no longer GA-inducible, showed a delayed response to cold induction in pineapple fruit in transient assays, suggesting a role for GA in blackheart development. This was supported by observations that exogenous GA(3) treatment induced blackheart in the absence of chilling. Sequences showing homology to GARCs are also present in some PPO promoters in tomato, suggesting that GA regulates PPO expression in diverse species.     

Regulation of cytokinin oxidase activity in tobacco callus expressing the T-DNA ipt gene

There are indications that the cytokinin content in transgenic tissues expressing the cytokinin biosynthetic ipt gene is under metabolic control, which prevents the accumulation of cytokinins to lethal levels. The objective of this study was to investigate the relationships between the content of endogenous cytokinins and the activity of cytokinin oxidase (which is believed to be a copper-containing amine oxidase, EC 1.4.3.6.) in ipt transgenic tobacco callus. In addition, the effect of exogenously applied N-benzyladenine (BA) on this relationship was examined. Endogenous cytokinin concentrations were measured in callus of Nicotiana tabacum L. cv. Petit Havana SRI transformed with the ipt of Agrobacterium tumefaciens under the control of a light-inducible promoter and in non-transformed tissue using LC-tandem mass spectrometry. The activity of cytokinin oxidase was estimated by measuring the conversion of [2,8-³H]N⁶-(Δ²-isopentenyl)adenine to [³H]adenine by enzyme preparations in vitro. The 14-day-old ipt-transformed callus contained a 25-fold higher amount of cytokinins as compared to the non-transformed tissue. Mainly zeatin- and dihydrozeatin-types of cytokinins (free bases, ribosides, nucleotides and O-glucosides) accumulated in the ipt transgenic tissue. The cytokinin pool of both ipt-transformed and non-transformed tissues consisted predominantly of cytokinins that are either resistant to cytokinin oxidase attack (nucleotides and O-glucosides of cytokinins and cytokinins bearing N⁶-saturated side chain) or have a low affinity for the enzyme (zeatin and its riboside). The former represented 71.6 and 74.8% and the latter 27.7 and 24.4% of the pool of endogenous cytokinins in ipt-transformed and non-transformed tissues, respectively. Enzyme preparations from ipt-transformed tissue exhibited 1.5-fold higher cytokinin oxidase activity compared with that observed in control tissues. Application of exogenous BA affected the total levels of cytokinins of the two tissue lines in different ways. The cytokinin content increased by 1.7- and 1.5-fold in ipt-transformed tissues 6 and 12 h after BA application, respectively, while it declined in the non-transformed control by 1.6- to 2.0-fold between 3 and 12 h after BA application. The increase in cytokinin content in the ipt callus is due to an increase of zeatin- and dihydrozeatin-type cytokinins (nucleotides, ribosides and free bases) leading to an enhanced accumulation of O-glucosides after 12 h. Following BA treatment, the cytokinin oxidase activity increased up to 1.8-fold in ipt-transformed and 1.6-fold in non-transformed tissues. The levels of isopentenyl-type cytokinins were near the detection limit; however, the enhancement of cytokinin oxidase activity after BA treatment in both tissue lines was correlated with the content of preferred substrate of the enzyme, N⁶-(Δ²-isopentenyl)adenosine.     

Expression of the Talaromyces flavus glucose oxidase gene in cotton and tobacco reduces fungal infection,but is also phytotoxic

Glucose oxidase secreted by the fungus Talaromyces flavus generates, in the presence of glucose, hydrogen peroxide that is toxic to phytopathogenic fungi responsible for economically important diseases in many crops. A glucose oxidase gene from T. flavus, was modified with a carrot extensin signal peptide and fused to either a constitutive or root-specific plant promoter. T1 tobacco plants expressing the enzyme constitutively were protected against infection by the seedling pathogen Rhizoctonia solani. Constitutive expression in tobacco was associated with reduced root growth, and slow germination on culture medium, and with reduced seed set in glasshouse conditions. Several independent transformed cotton plants with a root-specific construct expressed high glucose oxidase activity in the roots, excluding the root tip. Selected T3 homozygous lines showed some protection against the root pathogen, Verticillium dahliae, but not against Fusarium oxysporum. High levels of glucose oxidase expression in cotton roots were associated with reduced height, seed set and seedling germination and reduced lateral root formation. If this gene is to be of value for crop protection against pathogens it will require precise control of its expression to remove the deleterious phenotypes. This revised version was published online in June 2006 with corrections to the Cover Date.     

苹果多酚氧化酶的纯化与表征

运用丙酮浸漬干燥、磷酸盐缓冲液提取、低温离心、硫酸铵沉淀、DEAE-Sephadex(A-50)、Sephadex(G-75) 和DEAE-celluse(DE-52)层析等方法从苹果中分离获得一种新的含铜酶蛋白,该酶被命名为多酚氧化酶Ⅱ(polyphenol oxidase Ⅱ, PPOⅡ),纯化倍数是215,纯化收率是23%.PAGE、SDS-PAGE和MALDI-TOF 等技术用于测定所获的酶的纯度和分子量.在PAGE和SDS-PAGE 均显示一条带,表明PPOⅡ只由一个亚基组成,且已达到单一组分(MALDI-TOF的结果更证实了这一点).SDS-PAGE 和 MALDI-TOF 的结果都表明PPO的分子量为 38204 Da.pH值对酶活性和稳定性研究的结果显示,从pH值4.0～7.0随着pH值的增加,酶活性也不断增加;从pH值 7.0～11.0, 酶活性不断降低.PPOⅡ的最适pH值为6.6最适温度为30℃.     

施钾与蚜害处理后马铃薯叶片中多酚氧化酶活性的变化

蚜虫危害是影响马铃薯Solanum tuberosum产量和品质的重要因素之一, 而多酚氧化酶（polyphenol oxidase, PPO）与植物的抗性密切相关。为了阐明施钾条件下马铃薯与桃蚜Myzus persicae的关系, 本实验通过比色法、 iTRAQ技术和蛋白免疫印迹法研究了对照（不施钾, 不接虫）、 接虫、 施钾以及施钾＋接虫4种处理后马铃薯叶片中多酚氧化酶活性的变化。结果表明： 施钾显著降低桃蚜种群数量。随着桃蚜发育期延长, 桃蚜的种群数量显著低于对照, 且6 g/株施钾量对桃蚜种群数量的抑制效果最强。以6 g/株作为施钾量, 对不同处理后马铃薯叶片中多酚氧化酶活性研究显示, 施钾、 接虫+施钾处理均使马铃薯叶片中PPO活性显著提高, 分别比对照增加了44%和67%。通过液相色谱 质谱/质谱联用仪（LC-MS/MS） 分析, 接虫、 施钾、 接虫+施钾处理均不同程度上调了PPO蛋白表达量。Western杂交结果显示： 施钾、 接虫+施钾处理显著增加了PPO的相对表达量, 且接虫+施钾处理使该相对表达量达到最高。结果说明, 施钾、 接虫+施钾处理通过诱导马铃薯叶片中的PPO活性, 从一个侧面提高了马铃薯抗蚜虫能力。     

TohpreproHypSys- A Gene Expression and Defense Protein Activity in the Tobacco Wounding Response

Tobacco hydroxyproline-rich glycopeptide systemin precursor A (TobpreproHypSys-A), from which TobHypSys I and II are released, plays a crucial role in defense responses. Here, we investigated the expression otTobpreproHypSys- A and the activity of defense proteins in tobacco organs during wounding. Expression was induced more rapidly in upper, non-wounded leaves than in lower, wounded leaves. At 24 h after mechanical wounding, expression was tow in the roots, but increased in the stems and flowers, although to a lesser extent than in the leaves. At 3 or 10 d after insect-wounding, expression did not differ among organs, suggesting thatTobpreproHypSys- A could be induced globally and continuously throughout such stress. During that period, the activity of two defense proteins — PPO and PI — was consistent with the expression ofTobpreproHyp- Sys- A in various organs. This indicates that those proteins also could be regulated by TobHypSys, both globally and continuously.     

黑芝麻多酚氧化酶的重组表达及其酶学特性

为明确黑芝麻多酚氧化酶的酶学性质,利用大肠杆菌Escherichia coli原核表达了黑芝麻多酚氧化酶(Black sesame polyphenol oxidase,BsPPO).将合成的基因构建至pMAL-c5x载体,并在大肠杆菌中进行表达,对重组蛋白进行分离纯化及融合标签切除,获得的BsPPO蛋白用于酶学性质探...     

The purification and spectral properties of polyphenol oxidase I fromNicotiana tabacum

Polyphenol oxidase plays a key role in plant defense systems. We report the first-time purification of polyphenol oxidase (PPO 1.14.18.1) from fresh leaves of tobacco (Nicotiana tabacum) using acetone powder, ammonium sulfate precipitation, and column chromatography with DEAE-Sephadex A-50, CM-Sephadex C-50, and Sephadex G-75. PPO I was purified approximately 71-fold (3200 U/mg). The MALDI-TOF-MS spectrum showed that the enzyme was purified to a pure protein with a molecular weight of 35700 Da. The optimum pH of PPO I was 7, the optimum temperature was 40°C, and the Km value was 6.8 mM using catechol as the substrate at pH 6.5 and with 0.05 M H₃PO₄−NaOH buffer. The maximum emission peak of PPO I was 339 nm with 16 nm of blue-shifted compared with 355 nm of free tryptophan. The UV/VIS spectra and the absence of an EPR signal are indicative of type-3 coppers, but not type-1 or type-2 coppers. PPO I and mushroom PPO have the same active center for a pair of coupled antiferromagnetic copper ions.