An optical biosensor for lysine based on the use of lysine decarboxylase and a cadaverine-sensitive membrane

We describe an optical biosensor for lysine based on the use of lysine decarboxylase and an optical transducer for detection of cadaverine which is formed as a result of enzymatic action. A plasticized PVC (polyvinyl chloride) membrane containing a lipophilic tartrate as the amine carrier acts as the optical cadaverine sensor. The transport of the cadaverine cation into the membrane is coupled to a transport of a proton (of the indicator dye) out of the membrane. This causes a spectral change of the indicator dye which can be related to the cadaverine concentration, provided the pH is kept constant. The enzymatic reaction is performed in an enzyme reactor which is part of a flow-through system. The dynamic range is from 0·1 to 100 mM for both cadaverine and lysine. While the cadaverine sensor is moderately selective (ethylamines, for example, interfere), the whole sensor system is highly specific for lysine, nicotine being the only major interferent.

Unlike other enzyme-based detection schemes where the production of CO₂ (in case of decarboxylates) or consumption of oxygen (in case of oxidases) is measured, this scheme is based on the measurement of the organic ammonium ion (cadaverin cation) formed in the enzymatic reaction. The major advantage of this approach is that in many real samples there is a rather low and fairly constant background of organic amines. This is in contrast to sensors based on the measurements of oxygen consumption (in the case of amino acid oxidases) or carbon dioxide production (using decarboxylases), where the background levels of the respective gases (which are ubiquitous) have to be kept constant in order to specifically measure only the concentration changes caused by the enzymatic reaction, or need to be measured in an independent assay.     

Cadaverine regulates biotin synthesis to modulate primary root growth in Arabidopsis

Cadaverine, a polyamine, has been linked to modification of root growth architecture and response to environmental stresses in plants. However, the molecular mechanisms that govern the regulation of root growth by cadaverine are largely unexplored. Here we conducted a forward genetic screen and isolated a mutation, cadaverine hypersensitive 3 (cdh3), which resulted in increased root-growth sensitivity to cadaverine, but not other polyamines. This mutation affects the BIO3-BIO1 biotin biosynthesis gene. Exogenous supply of biotin and a pathway intermediate downstream of BIO1, 7,8-diaminopelargonic acid, suppressed this cadaverine sensitivity phenotype. An in vitro enzyme assay showed cadaverine inhibits the BIO3-BIO1 activity. Furthermore, cadaverine-treated seedlings displayed reduced biotinylation of Biotin Carboxyl Carrier Protein 1 of the acetyl-coenzyme A carboxylase complex involved in de novo fatty acid biosynthesis, resulting in decreased accumulation of triacylglycerides. Taken together, these results revealed an unexpected role of cadaverine in the regulation of biotin biosynthesis, which leads to modulation of primary root growth of plants.     

产酸克雷伯氏菌赖氨酸脱羧酶的异源表达及粗酶性质

赖氨酸脱羧酶,可以催化赖氨酸脱羧生成戊二胺。戊二胺是重要的平台化合物,可以合成新型聚酰胺材料、脂肪族异氰酸酯等新材料。本研究对来自于产酸克雷伯氏菌的赖氨酸脱羧酶进行异源表达。以pUC18质粒为载体,将来源于产酸克雷伯氏菌的赖氨酸脱羧酶基因ldc克隆到大肠杆菌,得到菌株LN18。在添加0.5 mmol/L IPTG的LB培养基中,对LN18进行摇瓶培养,发酵液酶活可达到35 U/g发酵液,从发酵液制备的赖氨酸脱羧酶粗酶蛋白的酶活可以达到30 000 U/g粗蛋白。产酸克雷伯氏菌赖氨酸脱羧粗酶蛋白大小约80 kDa,粗酶的最适温度和pH值分别为55℃和5.5,与文献中报道的大肠杆菌的赖氨酸脱羧酶Cad A在pH 8.0几乎没有酶活不同,产酸克雷伯氏菌的赖氨酸脱羧酶在pH 8.0的酶活达到最优pH下酶活的30%以上。金属离子对酶活有一定的影响,Mg~(2+)对酶活有促进作用,Fe~(2+)、Zn~(2+)、Ca~(2+)有一定的抑制作用。     

A new balancing act: The many roles of melatonin and serotonin in plant growth and development

Melatonin and serotonin are indoleamines first identified as neurotransmitters in vertebrates; they have now been found to be ubiquitously present across all forms of life. Both melatonin and serotonin were discovered in plants several years after their discovery in mammals, but their presence has now been confirmed in almost all plant families. The mechanisms of action of melatonin and serotonin are still poorly defined. Melatonin and serotonin possess important roles in plant growth and development, including functions in chronoregulation and modulation of reproductive development, control of root and shoot organogenesis, maintenance of plant tissues, delay of senescence, and responses to biotic and abiotic stresses. This review focuses on the roles of melatonin and serotonin as a novel class of plant growth regulators. Their roles in reproductive and vegetative plant growth will be examined including an overview of current hypotheses and knowledge regarding their mechanisms of action in specific responses.     

Coupling the fermentation and membrane separation process for polyamides monomer cadaverine production from feedstock lysine

Nylon is a polyamide material with excellent performance used widely in the aviation and automobile industries, and other fields. Nylon monomers such as hexamethylene diamine and other monomers are in huge demand. Therefore, in order to expand the methods of nylon production, we tried to develop alternative bio‐manufacturing processes which would make a positive contribution to the nylon industry. In this study, the engineered E. coli‐overexpressing Lysine decarboxylases (LDCs) were used for the bioconversion of l‐lysine to cadaverine. An integrated fermentation and microfiltration (MF) process for high‐level cadaverine production by E. coli was established. Concentration was increased from 87 to 263.6 g/L cadaverine after six batch coupling with a productivity of 3.65 g/L‐h. The cadaverine concentration was also increased significantly from 0.43 g cadaverine/g l‐lysine to 0.88 g cadaverine/g l‐lysine by repeated batch fermentation. These experimental results indicate that coupling the fermentation and membrane separation process could benefit the continuous production of cadaverine at high levels.     

不同生长调节物质对水稻生长及镉积累的影响

比较脱落酸(ABA)、乙烯利(ETH)、水杨酸(SA)和茉莉酸甲酯(MeJ A)4种植物生长调节物质(PGR)对水稻生长及籽粒镉(Cd)积累的影响差异。试验采用重金属污染土种植水稻,于分蘖期、灌浆期各进行1次PGR叶面喷施处理,分析灌浆期叶片光合指标,丙二醛(MDA)含量以及收获期各部位生物量和Cd含量。结果表明:(1)低浓度ABA(5mg/L)可维持水稻正常产量;高浓度ABA(15mg/L)则导致产量下降。ETH对水稻地上部生长和单株产量有显著抑制作用,SA和MeJ A(0.56mg/L)均可保证地上部正常生长,维持正常产量。(2)外施4种PGR均抑制灌浆期叶片气孔开放,降低蒸腾速率和光合速率,抑制效果最明显的是高浓度MeJ A(0.56mg/L)。(3)在供试浓度范围内SA、低浓度ABA(5mg/L)以及高浓度MeJ A均可降低灌浆期叶片MDA含量,减少质膜过氧化水平。(4)4种PGR均可降低水稻籽粒Cd含量,其中低浓度ABA(5mg/L)抑制籽粒Cd积累的效应最佳。相关性分析结果表明,PGR抑制籽粒积累Cd的效应与地上部向籽粒转运Cd的调控机制有关,与蒸腾速率无显著相关关系。(5)综上所述,低浓度ABA(5mg/L)处理对水稻产量无影响,且籽粒Cd含量降低程度最大。适当浓度的PGR可降低水稻籽粒Cd含量,在中低度重金属污染农田生态修复实践中具有一定的应用前景,但必须精确控制PGR的处理时间和处理浓度,避免出现抑制生长和降低产量的负效应。     

Interplay of phytohormones and epigenetic regulation: A recipe for plant development and plasticity

Both phytohormone signaling and epigenetic mechanisms have long been known to play crucial roles in plant development and plasticity in response to ambient stimuli.Indeed,diverse signaling pathways mediated by phytohormones and epigenetic processes integrate multiple upstream signals to regulate various plant traits.Emerging evidence indicates that phytohormones and epigenetic processes interact at multiple levels.In this review,we summarize the current knowledge of the interplay between phytoho...     

Effect of plant growth regulators on in vitro fiber development from unfertilized and fertilized Egyptian cotton ovules

Unfertilized and fertilized ovules of Gossypium barbadense Giza 45 (extra long staple variety) were used to study the effect of plant growth substances (auxins, gibberellins and cytokinins) on in vitro fiber initiation and development. Kinetin, alone did not increase total fiber unit (TFU) of unfertilized ovules, while an increase in TFU value occurred when a constant level of IAA and GA₃ were used either separately or in combination in the liquid medium. GA₃ used alone, produced a higher TFU value than that produced by IAA, whilst, IAA with a constant level of GA₃ (5 M) produced the highest value of TFU. GA₃ with a constant level of IAA (5 M) produced a lower TFU value. Kinetin reduced the stimulatory effect of IAA and GA₃ on TFU value when used in combination with either substance. In fertilized ovules, the highest level of TFU was reached when IAA, with a constant level of GA₃, was added to the medium, whilst its lowest level was obtained when IAA was used alone. Estimation of in vitro fiber production, as well as the effect of growth substances used in different concentrations on in vitro fiber initiation and development from unfertilized and fertilized ovules of Egyptian cotton varieties Gossypium barbadense Giza 45 are discussed.     

Early lysine deficiency in young broiler chicks

The carry-over effect of a pre-starter diet (0 to 3 days of age) deficient in lysine on subsequent growth and body composition (3 to 10 days) was examined in two experiments on male broiler chicks raised in cages. In experiment 1, lysine deficiency was applied from 3 to 10 days after providing a balanced pre-starter control feed (D+, 1.40% lysine) or a lysine deficient feed (D−) during the first 3 days. Three levels of deficiency (A = 0.63%, B = 0.72%, C = 0.82%) were tested. Growth and feed intake were higher in D+ than in D− chicks ( P < 0.001). However, the feed conversion ratio from 3 to 10 days of age was higher in D+ chicks ( P < 0.001); pre-starter and starter feeds interacted ( P < 0.04) with the feed conversion of treatment D+/A = 2.07 being better than treatment D+/A = 2.61 ( P < 0.05). This suggests that chicks deficient from hatching exhibit a relatively lower sensitivity to lysine deficiency than chicks started on a control diet. In experiment 2, performance, slaughter parameters and body composition were analysed at 3 and 10 days of age, in chicks having received a lysine deficient feed (D0, 0.72% lysine), a control feed (D+, 1.40% lysine) or having been pair fed with control feed adjusted to D0 intake (PF) from 0 to 3 days of age, and then fed D0 ad libitum from 3 to 10 days of age. At 3 days, PF chicks had a higher body weight ( P < 0.05) than D0, and thus a better feed conversion. Body composition in relative values was little or not affected by dietary treatments, but the breast muscle weight at 3 days was higher in D+ and PF chicks compared with D0 ( P < 0.05) and this effect was even accentuated at 10 days of age. The present work confirms that early nutrition can have subsequent consequences on the adjustment of fast growing broiler chicks to their nutritional conditions. It also suggests that breast muscle development is a more reactive parameter than whole body composition in this kind of experiments.     

Arabidopsis microarrays identify conserved and differentially expressed genes involved in shoot growth and development from distantly related plant species

Expressed sequence tags (EST)-based microarrays are powerful tools for gene discovery and signal transduction studies in a small number of well-characterized species. To explore the usefulness of this technique for poorly characterized species, we have hybridized the 11,522-element Arabidopsis microarrays with labeled cDNAs from mature leaf and shoot apices from several different species. Expression of 23 to 47% of the genes on the array was detected, demonstrating that a large number of genes from distantly related species can be surveyed on Arabidopsis arrays. Differential expression of genes with known functions was indicative of the physiological state of the tissues tested. Genes involved in cell division, stress responses, and development were conserved and expressed preferentially in growing shoots.     

Identification of lysine residue involved in inactivation of brain glutamate dehydrogenase isoproteins by o-phthalaldehyde

Incubation of two types of glutamate dehydrogenase (GDH) isoproteins from bovine brain with o-phthalaldehyde resulted in a time-dependent loss of enzyme activity. The inactivation was partially prevented by preincubation of the GDH isoproteins with 2-oxoglutarate or NADH. Spectrophotometric studies indicated that the inactivation of GDH isoproteins with o-phthalaldehyde resulted in isoindole derivatives characterized by typical fluorescence emission spectra with a stoichiometry of one isoindole derivative per molecule of enzyme subunit. There were no differences between the two GDH isoproteins in sensitivities to inactivation by o-phthalaldehyde indicating that the microenvironmental structures of the GDH isoproteins are very similar to each other. Tryptic peptides of the isoproteins, modified with and without protection, identified a selective modification of one lysine as in the region containing the sequence L-Q-H-G-S-I-L-G-F-P-X-A-K for both GDH isoproteins. The symbol X indicates a position for which no phenylthiohydantoin-amino acid could be assigned. The missing residue, however, can be designated as an o-phthalaldehyde-labeled lysine since the sequences including the lysine residue in question have a complete identity with those of the other mammalian GDHs. Also, trypsin was unable to cleave the labeled peptide at this site. Both amino acid sequencing and compositional analysis identified Lys-306 as the site of o-phthalaldehyde binding within the brain GDH isoproteins.     

植物生长调节剂对银柴胡细胞生长特性的影响

利用细胞悬浮培养技术,研究不同激素对银柴胡细胞生长特性及过氧化氢酶和硝酸还原酶活性的影响。结果表明,第4种激素配比的培养液M₄(MS+6-BA 1.0 mg·L^-1+2,4-D 1.0 mg·L^-1+NAA 1.0 mg·L^-1)的细胞鲜重、干重,细胞数目均明显高于其他处理;M₄处理可有效增强银柴胡细胞硝酸还原酶的活性,提高氮素利用率;M₂(MS+6-BA 1.0mg·L^-1+NAA0.5 mg·L^-1)处理有利于增强银柴胡细胞过氧化氢酶活性。     

Evidence for plant growth promoting brassinosteroids in leaves of Thea sinensis

Steroids, which were active in inducing rice-lamina inclination, were separated from leaves of Thea sinensis and tentatively identified to be brassinolide and its 6-keto analogue castasterone, by mass fragmentographic analysis.     

植物生长发育中程序性细胞死亡

本文简要介绍了植物细胞凋亡的一些特点以及植物在营养生长和生殖生长过程中发生的细胞凋亡现象。指出细胞凋亡是植物生长发育过程中正常的生理现象。     

六种昆虫生长调节剂对葱蝇生长发育和繁殖力的影响

用灭蝇胺、氟铃脲等6种昆虫生长调节剂和对比药剂辛硫磷LC_40-50浓度处理葱蝇1龄和3龄幼虫,观察对其生长发育和繁殖的影响;并且研究了葱蝇成虫取食药剂对其繁殖力的控制效应。结果表明：6种药剂中除虫酰肼外,其余都对葱蝇幼虫发育历期、化蛹率、成虫产卵前期、成虫寿命、产卵量有很大影响,对其生长发育和繁殖表现出显著不利性。其中灭蝇胺和氟铃脲处理1龄幼虫后,存活幼虫的历期和成虫产卵前期各延长3天,化蛹率降低26.7%～30.0％,成虫寿命缩短22.3～24.3天,产卵量降低35.5％～49.9％;处理3龄幼虫主要导致产生畸形蛹,且正常羽化的成虫寿命降低18.0～20.67天,产卵量降低45.7%～57.8％。成虫连续取食用牛奶稀释的２ 000倍的药液,能导致产卵前期延长、产卵量减少、卵孵化率降低,幼虫死亡率极高,达95%以上。结果提示,利用成虫补充营养的特性,将灭蝇胺和氟铃脲等混入牛奶或糖水中诱集成虫取食,可有效降低成虫的繁殖力,有助于实现对葱蝇的无公害治理。     

Arabinogalactan-proteins: a class of extracellular matrix proteoglycans involved in plant growth and development

Arabinogalactanproteins (AGPs) are proteoglycans of the extracellular matrix o f most plants. Since the late 1980s, AGPs have attracted widespread attention from plant biologists following reports of their involvement in plant development. In particular, the use of monoclonal antibodies to carbohydrate epitopes of AGPs has demonstrated stage- and tissue-specificity and has led to suggestions that they are involved in tissue morphogenesis. The recent cloning of the genes for several AGP protein backbones allows us to consider new strategies to address their function. Here, we summarize our knowledge of AGPs and consider parallels with animal proteoglycans as a possible framework for future work.     

Increased production of cadaverine and anabasine in hairy root cultures of Nicotiana tabacum expressing a bacterial lysine decarboxylase gene

Several hairy root cultures of Nicotiana tabacum varieties, carrying two direct repeats of a bacterial lysine decarboxylase (ldc) gene controlled by the cauliflower mosaic virus (CaMV) 35S promoter expressed LDC activity up to 1 pkat/mg protein. Such activity was, for example, sufficient to increase cadaverine levels of the best line SR3/1-K1,2 from ca. 50 g (control cultures) to about 700 g/g dry mass. Some of the overproduced cadaverine of this line was used for the formation of anabasine, as shown by a 3-fold increase of this alkaloid. In transgenic lines with lower LDC activity the changes of cadaverine and anabasine levels were correspondingly lower and sometimes hardly distinguishable from controls. Feeding of lysine to root cultures, even to those with low LDC activity, greatly enhanced cadaverine and anabasine livels, while the amino acid had no or very little effect on controls and LDC-negative lines.