Salicylic acid and methyl jasmonate improve chilling tolerance in cold-stored lemon fruit (Citrus limon)

Chilling injury (CI) is associated with the degradation of membrane integrity which can be aligned to phenolic oxidation activated by polyphenol oxidase (PPO) and peroxidase (POD), enzymes responsible for tissue browning. Phenylalanine ammonia-lyase (PAL) is a further enzyme prominent in the phenolic metabolism that is involved in acclimation against chilling stress. It was hypothesized that treatment with methyl jasmonate (MJ) and salicylic acid (SA) may enhance chilling tolerance in lemon fruit by increasing the synthesis of total phenolics and PAL by activating the key enzyme regulating the shikimic acid pathway whilst inhibiting the activity of POD and PPO. Lemon fruit were treated with 10 μM MJ, 2 mM SA or 10 μM MJ plus 2 mM SA, waxed, stored at −0.5, 2 or 4.5 °C for up to 28 days plus 7 days at 23 °C. Membrane integrity was studied by investigating membrane permeability and the degree of membrane lipid peroxidation in lemon flavedo following cold storage. The 10 μM MJ plus 2 mM SA treatment was most effective in enhancing chilling tolerance of lemon fruit, significantly reducing chilling-induced membrane permeability and membrane lipid peroxidation of lemon flavedo tissue. This treatment also increased total phenolics and PAL activity in such tissue while inhibiting POD activity, the latter possibly contributing to the delay of CI manifestation. PPO activity was found to be a poor biochemical marker of CI. Treatment with 10 μM MJ plus 2 mM SA resulted in an alteration of the phenolic metabolism, enhancing chilling tolerance, possibly through increased production of total phenolics and the activation of PAL and inhibition of POD.     

苯丙氨酸脱氨酶cDNA在大肠杆菌中的克隆与表达及酶法合成L-苯丙氨酸

利用基因重组技术 ,在大肠杆菌中克隆并表达苯丙氨酸脱氨酶 (PAL) (EC4 .3 .1 .5) ,并应用此酶转化肉桂酸生成L 苯丙氨酸。方法是将欧芹苯丙氨酸脱氨酶cDNA亚克隆到组成型表达载体pMG3 6e启动子P3 2下游 ,以菌落PCR法鉴定插入片段的大小和方向都正确的克隆 ,进而以HPLC检测肉桂酸浓度的方法鉴别重组质粒有催化肉桂酸生成L 苯丙氨酸的酶活力。结果获得能表达PAL酶活性的阳性克隆 ,在pH1 0 ,含 1 .0 %肉桂酸、8.0mol/L氨的转化液中 ,3 0℃反应 2 0h ,肉桂酸重量转化率可达 60 %。该基因工程菌有希望用于工业化生产L 苯丙氨酸。     

Phenylalanine Ammonia-Lyase Genes Involved in Anthocyanin Synthesis and the Regulation of its Expression in Suspension Cultured Carrot Cells

D and is also induced rapidly and transiently by transfer of cells to fresh medium and lowering the cell density. From the carrot genomic library, four clones of PAL genes, gDcPAL1,2,3 and 4, were obtained. Analyses of nucleotide sequences revealed that only the gDcPAL3 gene is responsible for the induction of anthocyanin synthesis by 2,4-D. Several cis-elements, boxes M, P, A, L, and G, exist in the proximal promoter region of gDcPAL3. Transient expression experiments in carrot protoplasts using deletion mutants of the proximal promoter region of gDcPAL3 gene showed that boxes M and L, both of which contain core sequences of the Myb binding sites, might play an important role in gDcPAL3 promoter activity. Four myb cDNAs, Dcmyb8,10,12 and 14 were obtained from a carrot subtracted cDNA library and their structure and expression patterns were analyzed. In addition to the analysis of the proximal region of gDcPAL3 promoter, the possibility of the regulation of gene expression by genomic DNA structure and chromatin modification in metabolic differentiation is discussed. Received 10 June 2000/ Accepted in revised form 1 July 2000     

土壤干旱胁迫对紫叶矮樱叶片呈色的影响

以盆栽3 a紫叶矮樱叶片为试材,采用称重控水法,设对照(土壤含水量18.11%)、轻度干旱(土壤含水量14.72%)、中度干旱(土壤含水量11.32%)和重度干旱(土壤含水量7.92%)4个处理组,研究不同土壤干旱条件下紫叶矮樱叶片呈色色素含量、可溶性糖含量、PAL酶活性的变化规律及其对叶片呈色的影响.结果表明:轻度干旱,随胁迫时间的延长叶片中花青苷、类黄酮、叶绿素、可溶性糖含量、PAL酶活性和a*增加, L*和b*降低;中度和重度干旱,随胁迫时间的延长花青苷、类黄酮、叶绿素、类胡萝卜素、可溶性糖含量、PAL酶活性和a*先增加再降低,L*和b*先减小再增大.短时间的干旱能够提高紫叶矮樱的叶片色泽,中度干旱15 d或重度干旱12 d,是紫叶矮樱叶色发生明显转变的关键时期;花青苷含量的变化是影响紫叶矮樱叶色变化的主要原因.     

Transformation of Rhodosporidium toruloides

Rhodosporidium toruloides protoplasts could be transformed, in the presence of polyethylene glycol (PEG), at frequencies of approx. 1 X 10(3) transformants/micrograms of DNA. The plasmid used, pHG2, which contains the phenylalanine ammonia-lyase (PAL)-coding gene (PAL) of R. toruloides, could replicate as an unstable plasmid in the yeast, or could integrate at the PAL locus to give stable transformants. Plasmids that function in R. toruloides were constructed using either the PAL gene or LEU2 gene of Saccharomyces cerevisiae as dominant selectable markers. R. toruloides transformed with pHG8, which contains both genes, coinherited the two markers. It is also shown that the 2mu replicon of S. cerevisiae does not function in R. toruloides; neither is the PAL gene expressed in S. cerevisiae.     

Phenolic metabolism in root slices of selected carrot cultivars

Carrot root slices, stored for 4 days at 20 °C reacted with a strong accumulation of total phenols, especially chlorogenic acid. A significant accumulation of isocoumarin content within the peel was observed in stored slices. Synthesis of phenols was accompanied by an increase in phenylalanine ammonia lyase activity, wound induced respiration and ethylene production. The great variability among the studied four cultivars was found concerning isocoumarin synthesis, PAL activity, respiration rate and ethylene evolution, but less distinct in the case of chlorogenic acid accumulation. The carrot slices obtained from freshly harvested roots were more sensitive to mechanical damage and short-term storage than those prepared from roots previously stored.     

Phenylalanine Ammonia-Lyase Activity in Soybean Roots Extract Measured by Reverse-Phase High Performance Liquid Chromatography

Abstract: The high performance liquid chromatography (HPLC) technique was applied to measure phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity in soybean ( Glycine max L. Merril cv. BR16) roots. t -Cinnamate, the catalytic product of the PAL reaction was quantified at 275nm by isocratic elution with methanol: water through an ODS(M) column. Comparative experiments were carried out with 1.0 mM ferulic acid, an inducer of PAL activity. The results suggest that liquid chromatography is a rapid and sensitive method to analyze PAL activity in non-purified extract.     

Physico-chemical principles of cAMP-dependent protein phosphorylation. Catalysis of phosphoryl group transfer to nucleophilic agents

The specificity of pig brain protein kinase towards high- and low-Mr 'analogs' of protein substrate was studied. Solvolysis of the phosphoenzyme intermediate by various nucleophilic agents is shown. The transition state structure of the phosphoryl transfer reaction is discussed.