Sequential Induction of mRNAs for Phenylalanine Ammonia-lyase in Slices of Potato Tuber

Induction of the mRNA that encodes for phenylalanine ammonia-lyase(PAL, EC 4.3.1.5 [EC] ), which catalyzes the first reaction in thebiosynthesis of a wide variety of phenylpropanoid natural productsfrom phenylalanine, was investigated in. wounded tuber tissuesof the potato (Solanum tuberosum L. cv. Irish Cobbler). Northernblot analysis showed that hybridizable RNA was not present inunwounded tissue, but the amount of hybridizable PAL-specificmRNA increased rapidly in the polysomal RNA fraction with asharp, high peak at the early stage (0 h to 6 h) and two broadlower peaks at the later stage (6 h to 48 h) of the wound response.Addition of actinomycin D to the tissue prevented the appearanceof hybridizable mRNA in the total RNA fraction, confirming thatthe increase resulted from synthesis of PAL mRNA de novo. Levelsof translatable PAL mRNA activity in vitro increased in thepolysomal RNA fraction in parallel with the changes in levelsof hybridizable mRNA, with a subsequent increase in levels ofPAL subunit polypeptides and enzymatic activity in wounded tissues.PAL subunits synthesized both in vivo and in vitro had the samemolecular masses, of about 79 kDa, on SDS-polyacrylamide gelelectrophoresis, but isoelectric focusing revealed the presenceof isoforms of the native tetrameric enzyme with different pIvalues and changes in the relative levels of the isoforms afterwounding. Furthermore, two-dimensional gel electrophoresis ofPAL subunits synthesized in vitro showed that at least eightmRNAs that encoded subunit isoforms with different pI valueswere expressed sequentially after wounding. (Received May 24, 1990; Accepted October 24, 1990)     

Sequential Induction of Phenylalanine Ammonia-lyase and a Lyase-inactivating System in Potato Tuber Disks

The light induced synthesis of phenylalanine ammonia-lyase in disks cut from potato tubers is very sensitive to cycloheximide. Synthesis is inhibited 50% in disks cultured on 5 μm cycloheximide instead of water and almost completely in disks aged in the presence of 10 μm inhibitor. Inhibition is irreversible. Fresh disks exposed only 1 hour to 10 μm cycloheximide do not synthesize enzyme during the subsequent 24 hours.     

Induction of Phenylalanine Ammonia-lyase in Xanthium Leaf Disks. Photosynthetic Requirement and Effect of Daylength

A cycloheximide-sensitive increase in the activity of phenylalanine ammonia-lyase (EC 4.3.1.5) occurs in Xanthium leaf disks exposed to light. Radioactive ammonia-lyase has been isolated by means of sucrose density gradient centrifugation and starch gel electrophoresis from disks fed l-isoleucine-U-(14)C or l-arginine-U-(14)C. The incorporation of radioactive amino acids into phenylalanine ammonia-lyase together with the inhibitory effects of cycloheximide indicate that the observed increase in enzyme activity involves the induction of lyase synthesis.The light-dependent synthesis of the ammonia-lyase is completely inhibited by 50 mum 3-(4-chlorophenyl)-1,1-dimethylurea (CMU) indicating that photosynthesis is involved. Only a trace quantity of some photosynthetic product must be needed because half light saturation occurs at very low intensity (ca. 30 ft-c). Exogenous carbohydrate is also required for continuing enzyme synthesis over a 72 hr period. But carbohydrate does not replace the photosynthetic requirement in darkness.Enzyme formed in light disappears rapidly from disks placed in the dark. The decay of ammonia-lyase activity follows first order kinetics. The half-life of the lyase ranged from 6 to 15 hr in leaf material used. Cyoloheximide inhibits the decay of lyase activity. Thus the maintenance of turnover in Xanthium leaf disks requires de novo synthesis of protein. That turnover, i.e., degradation as well as synthesis of lyase protein occurs is suggested by the apparent loss of radioactive ammonia-lyase from leaf disks placed in darkness. Light-induced synthesis coupled with rapid turnover can produce a diurnal fluctuation of ammonia-lyase activity in Xanthium leaf disks. Alternating periods of enzyme synthesis and degradation were observed in disks exposed to 24 hr cycles of light and dark. The average level of enzyme activity maintained in the tissue was directly related to the length of the light period. Induction of lyase synthesis was also observed in excised leaves and to a lesser extent in leaves of whole plants.     

一种适合从柑橘果皮提取总RNA的方法

柑橘果皮由于富含果胶、酚类物质等干扰RNA分离的物质,较难提取到纯度高的RNA。本试验建立了一种适于从柑橘果皮提取RNA的方法,从脐橙和蕉柑两种柑橘的果皮提取总RNA,经凝胶电泳、紫外分光光度法检测所提RNA的品质。研究结果表明,该法所提RNA条带清晰、无降解。OD260/OD280接近2.0,具有较高的纯度。RT-PCR试验结果进一步表明,该法提取的RNA纯度高,完全能够用于后续的分子生物学研究。     

百合鳞茎苯丙氨酸解氨酶提取条件的优化

研究兰州百合(Lilium davidii var. unicolor)鳞茎内PAL的最佳提取条件结果表明：pH 8.8的硼砂缓冲液为最适缓冲液;百合鳞茎的PAL不耐酸碱,更不耐酸;最适反应温度为40℃;随着水浴时间延长和水浴温度提高,PAL活性逐渐降低,但40℃水浴30 min后,酶活性仍保留47%;最适底物浓度为0.032 mol·L^-1;磨样时加入PVP 0.5 g,能够明显提高PAL活性。     

Mineral Content of the Pulp and Peel of Various Citrus Fruit Cultivars

Biological Trace Element Research - The aim of the study was to compare the mineral content between the peel and the pulp of citrus fruits and to determine which citrus fruit, among orange (Citrus...     

从基因工程乳酸菌中高效提取苯丙氨酸解氨酶的方法研究

为从高表达苯丙氨酸解氨酶(PAL)的基因工程乳酸乳球菌NZ3900/pNZ8149-palxt中有效提取所表达的PAL酶,针对乳酸乳球菌的特点,结合各种物理、化学、生物学等方法,探索出一条简便、高效的技术路线.对提取物进行了酶活性测定、分析及细菌活性鉴定.结果表明,该混合法可以完全导致细菌死亡,所提取的酶蛋白结构完整,其酶活性达到了等量活菌的92.5%,且该方法中所用到的各种试剂对体外培养细胞的生长无明显影响,为PAL酶在科研及生物医药领域的应用提供了可靠来源.     

MPTA对柑橘果皮类胡萝卜素形成的影响

以宽皮柑橘“本地早”为试材 ,研究了 2 (4 甲基苯氧 )三乙基胺 (MPTA)处理对柑橘果实外观颜色的影响 ,并探讨其与相关类胡萝卜素消长的关系及与糖含量变化的可能联系。结果表明 :MPTA处理促进柑橘果皮组织中类胡萝卜素 (主要为番茄红素 )的生物合成 ,使果皮颜色由淡橙黄色转为红色 ;随着番茄红素的积累 ,其上游前体物质葡萄糖含量下降 ,而其下游产物β 隐黄质、玉米黄素的合成不受影响     

柑橘果皮颜色的形成与类胡萝卜素组分变化的关系

分析了果皮分别为红、橙和黄颜色的柑橘品种“满头红”、“尾张”和“胡柚”果实着色过程中果皮类胡萝卜素组分及含量的变化。结果表明,随着果实的发育和成熟,果皮叶绿素含量下降,α-胡萝卜素、β—胡萝卜素含量逐渐下降直至消失,而β—隐黄质、β-柠乌素、玉米黄质等叶黄素成分含量逐渐上升,果实逐渐褪绿并呈现其特征色泽。“胡柏”果皮中类胡萝卜素总量及其橙、红色类胡萝卜素成分β-隐黄质和β-柠乌素积累少是其颜色淡、呈黄色的主要原因;三种果实的颜色差异并非由于果皮类胡萝卜素总量的差异,而主要是由于果皮不同类胡萝卜素成分组成比例不同。“满头红”以积累红色的β-柠乌素为主,而“尾张”则以积累橙色的β-隐黄质为主。     

Induction of Phenylalanine Ammonia-lyase in Strawberry Leaf Disks: Action Spectra and Effects of Wounding, Sucrose, and Light

The increase in phenylalanine ammonia-lyase (PAL) activity in strawberry (Fragaria vesca var. WSU-1232) leaf disks required wounding, sucrose, and light and was cycloheximide-sensitive. In injured leaves and in leaf disks, the highest PAL activity was detected nearest the wounded tissues. Without wounding, no increase in activity was observed when leaves were cultured in sucrose and light.     

Effect of Ethylene on the Phenylalanine Ammonia-lyase Activity of Carrot Tissues

Ethylene stimulated the activity of l -phenylalanine ammonia-lyase (PAL) in carrot (Daucus carota L.) tissues. It is also known to induce the formation of isochlorogenic acid. However, the induction patterns were different for isochlorogenic acid and PAL activity. Ethylene action seems to be indirect, as it did not affect the PAL activity of cell-free extracts.     

Properties of Phenylalanyl-tRNA Synthetase and Phenylalanine Ammonia-lyase of Pine Suspension Cultures

Phenylalanyl-tRNA synthetase and phenylalanine ammonia-lyase activities were demonstrated in partially purified extracts of pine (Pinus elliottii) suspension cultures. The optimum pH for the phenylalanyl-tRNA synthetase reaction was 7.5 and the optimum ATP and Mg²⁺ concentrations were 1.0 and 15 mM respectively. Pine, calf liver and yeast tRNA were inadequate substitutes for pea tRNA in the synthetase reaction mixtures. The optimum pH for the phenylalanine ammonia-lyase reaction was 9.0. The K_m for phenylalanine was approximately 6.6 × 10^?5M. The activity of both enzymes in the partially purified extracts was unstable on storage.     

Relationship of Phenylalanine Ammonia-lyase Activity to o-Hydroxycinnamic Acid Content in Melilotus alba

Phenylalanine ammonia-lyase activity was investigated in preparations representing various parts of sweetclover (Melilotus alba Desr.) plants of CuCu and cucu genotypes. In contrast to other plant parts, very young leaves and stems of CuCu plants displayed high phenylalanine ammonia-lyase activity. Initial leaf samples from CuCu plants were approximately 3 times as high in enzyme activity as leaves from cucu plants, but stems were only slightly higher in activity. Defoliation of the plants resulted in decreased enzyme activity, increased o-hydroxycinnamic acid content, and essentially no difference in enzyme activity between the genotypes. It appears that phenylalanine ammonia-lyase activity in leaves is not primarily controlled by the Cu/cu alleles and that the reaction catalyzed by this enzyme is not the limiting step in o-hydroxycinnamic acid synthesis.     

温州蜜柑果实发育期叶片和果皮吸收32P的研究

应用同位素示踪技术,研究了宫川温州蜜柑果实发育期叶片、果皮对³²P的吸收速率和转化形式。结果表明:在湖南气候条件下,叶片、果皮能以不同速率利用其表面³²P并进一步转化为蛋白质、核酸、醚溶性磷和磷脂。果皮吸收³²P的性能弱于叶片,没有出现第二次吸收高峰,而是随油胞数量增加、蜡质层加厚、细胞老化而逐渐减弱,且吸收的³²P绝大部分仍滞留于果皮中。     

柑橘钙结合蛋白基因的克隆及其在果皮褐变中的表达分析

从柑橘果皮褐变相关基因的差减cDNA文库中,筛选了一个与钙结合蛋白基因家族同源的EST片段,通过RACE技术克隆了其全长cDNA序列(CsCAB,GenBank登录号EF010854).CsCAB基因全长984 bp,含有一个621 bp的开放阅读框,编码207个氨基酸,预测蛋白质分子量为22.95 kD,理论等电点为4.5;序列分析结果显示CsCAB与钙结合蛋白具有很高的同源性,且具有钙结合蛋白的保守结构域EF-hand.Northern blot结果显示CsCAB在褐变果皮中上调表达,说明该基因与柑橘果实的果皮褐变有密切关系.     

砀山酥梨果实苯丙氨酸解氨酶基因cDNA克隆及序列分析

为了研究梨石细胞形成与木质素合成的关系,以砀山酥梨果实为材料,通过基于同源性的RT—PCR方法,克隆木质素合成途径中的关键酶苯丙氨酸解氨酶（PAL）基因。同时利用基因数据库资料对克隆的PAL序列进行比较分析。结果表明,从砀山酥梨果实中克隆的PAL基因eDNA片段长度为585bp,推断其编码195个氨基酸序列。该序列包含与其它植物PAL相同的活性催化位点,经序列分析和同源性比对,该氨基酸序列与其它植物PAL氨基酸序列的同源性在90％以上。系统进化树分析表明,砀山酥梨PAL氨基酸序列与蔷薇科的甜樱桃PAL氨基酸序列聚类关系最近。     

Increase in Free and Bound Abscisic Acid during Natural and Ethylene-induced Senescence of Citrus Fruit Peel

Free and bound abscisic acid and neutral growth inhibitors were detected in citrus fruit peel using two bioassays and gasliquid chromatography. None of the inhibitor components seems to be directly associated with chloroplasts or chromoplasts in citrus fruit peel.     

The Effect of L-Phenylalanine on Phenylalanine Ammonia-lyase and Cell Division in Artichoke Callus Culture

5 x 10^–5 M L-phenylalanine overcame the inhibitory effectof white light on cell division in artichoke callus culturesand increased extractable phenylalanine ammonia-lyase (PAL)activity compared to cultures grown in the presence of 5 x 10^–4M phenylalanine The lower concentration of the amino acid alsoenhanced rates of uptake and incorporation of ¹⁴C labelled phenylalaninethroughout G₁ and S. Differences between the two concentrationswere greatest during S with a 4-fold increase in uptake anda 3-fold increase in incorporation It is suggested thereforethat the capacity of 5 x10^–5 M phenylalanine to offsetthe light effect is due to an indirect stimulatory effect onamino acid and protein metabolism Increased levels of extractablePAL activity would then be reflected by this general stimulationof protein synthesis. Helianthus tuberosus L, Jerusalem artichoke, callus culture, cell division, phenylalanine ammonia-lyase