Effect of phosphate on aluminium-inhibited growth and signal transduction pathways in Coffea arabica suspension cells

In acid soils, aluminium (Al) toxicity and phosphate (Pi) deficiency are the most significant constraints on plant growth. Al inhibits cell growth and disrupts signal transduction processes, thus interfering with metabolism of phospholipase C (PLC), an enzyme involved in second messenger production in the cell. Using a Coffea arabica suspension cell model, we demonstrate that cell growth inhibition by Al toxicity is mitigated at a high Pi concentration. Aluminium-induced cell growth inhibition may be due to culture medium Pi deficiency, since Pi forms complexes with Al, reducing Pi availability to cells. Phosphate does not mitigate inhibition of PLC activity by Al toxicity. Other enzymes of the phosphoinositide signal transduction pathway were also evaluated. Aluminium disrupts production of second messengers such as inositol 1,4,5-trisphosphate (IP₃) and phosphatidic acid (PA) by blocking PLC activity; however, phospholipase D (PLD) and diacylglycerol kinase (DGK) activities are stimulated by Al, a response probably aimed at counteracting Al effects on PA formation. Phosphate deprivation also induces PLC and DGK activity. These results suggest that Al-induced cell growth inhibition is not linked to PLC activity inhibition.     

Aluminum induces changes in oxidative burst scavenging enzymes in Coffea arabica L. suspension cells with differential Al tolerance

The accumulation of reactive oxygen species (ROS) and concomitant oxidative stress have been considered deleterious consequences of aluminum toxicity. However, several lines of evidence suggest that ROS can function as important signaling molecules in the plant defense system for protection from abiotic stress and the acquisition of tolerance. The role of ROS-scavenging enzymes was assayed in two different coffee cell suspension lines. We treated L2 (Al-sensitive) and LAMt (Al-tolerant) Coffea arabica suspension cells with 100 μM AlCl₃ and observed significant differences in catalase activity between the two cell lines. However, we did not observe any differences in superoxide dismutase or glutathione reductase activity in either cell line following Al treatment. ROS production was diminished in the LAMt cell line. Taken together, these results indicate that aluminum treatment may impair the oxidative stress response in L2 cells but not in LAMt cells. We suggest a possible role for Al-induced oxidative bursts in the signaling pathways that lead to Al resistance and protection from Al toxicity.     

Differential gene expression in embryogenic and non-embryogenic clusters from cell suspension cultures ofCoffea arabica

Somatic embryogenesis (SE) is a very useful system for studying the differentiation process in plants and involves gene regulation at several levels. During SE induction in Coffea arabica cv. Catura Rojo two types of cell clusters, embryogenic (EC) and non-embryogenic (NEC), were observed. The goal of this work was to compare the most relevant characteristics between EC and NEC for a better understanding of the mechanism driving SE. Morphohistological observations indicated a correlation between the morphological features of clusters and their embryogenic competence. On the other hand, no variation at the DNA level, studied by AFLP, were found to explain the disparity in embryogenic competence of clusters, but gene expression, observed by RNA differential display, and SDS-PAGE showed differences that can explain that disparity. Our results lead us to propose that differential gene expression can modulate the embryogenic capacity of coffee cells and that the number of genes turned off in somatic cells to allow for the change from a somatic to an embryogenic state, is higher than those genes that are turned on.     

Trigonelline biosynthesis and the pyridine nucleotide cycle in Coffea arabica fruits: Metabolic fate of [carboxyl-C]nicotinic acid riboside

Trigonelline is a major component in coffee seeds and may contribute to the bitter taste of the resultant beverage. To determine the trigonelline biosynthetic pathway in coffee fruits, we investigated the metabolic fate of [carboxyl-¹⁴C]nicotinic acid riboside and in situ activity of related enzymes. Exogenously supplied [carboxyl-¹⁴C]nicotinic acid riboside was rapidly converted to nicotinic acid mononucleotide and was utilized for NAD synthesis. Nicotinic acid riboside was also used for trigonelline synthesis, but this process took longer than NAD synthesis. These results indicate that an efficient nicotinic acid riboside salvage system functions in coffee fruits, and that trigonelline is synthesized mainly from nicotinic acid produced by the degradation of NAD.     

Aluminium induces changes in organic acids metabolism in Coffea arabica suspension cells with differential Al-tolerance

The primary Al-tolerance mechanism in plants involves exudation and/or accumulation of specific organic acid species, which form non-phytotoxic complexes with Al³⁺ under physiological conditions. An evaluation was done of the role of organic acids in the tolerance mechanism of a cell suspension line of coffee Coffea arabica that exhibits Al-tolerance (LAMt) but for which the metabolic tolerance mechanism remains unknown. Significant differences existed in malate dehydrogenase and citrate synthase activities (key enzymes in organic acids metabolism) between protein extracts (day 7 of culture cycle) of the L2 (Al-sensitive) and LAMt (Al-tolerant) cells when cell suspensions were treated with 100 μM AlCl₃. HPLC analysis showed that the suspension cells of both lines exudate malate when incubated in a minimal solution but that exudation was not enhanced by treatment with AlCl₃ (100 μM). This is the first study demonstrating that plant Al-tolerance may be associated with down-regulation of malate dehydrogenase and citrate synthase activities.     

Caffeine metabolism in Coffea arabica and other species of coffee

High performance liquid chromatography has been used to measure the quantities of caffeine, theobromine, and theophylline in aqueous extracts of endosperm from immature and mature fruits of Coffea arabica and six other species of Coffea. Caffeine was the alkaloid present in largest amounts and, with one exception, in concentrations that were broadly similar in immature and mature fruit. The highest concentrations of caffeine were found in C. canephora at 35.1 and 24.5 mg g⁻¹, respectively, in immature and mature endosperm. The lowest concentrations were in C. bengalensis, where caffeine was not detected in extracts from mature fruit. [8-³H]Caffeine was metabolised relatively slowly by immature endosperm of C. arabica and C. canephora. In contrast, C. dewevrei, C. eugenioides, C. stenophylla, C. salvatrix and C. bengalensis all appeared to metabolise [8-³H]caffeine much more rapidly, as the percentage recovery of the applied label was much lower and there was more extensive incorporation of radioactivity into theobromine, theophylline, 3-methylxanthine and two unidentified polar metabolites. The endogenous caffeine concentrations and the metabolism data indicate that there may be marked differences in the rate of turnover of caffeine in the various species of Coffea. Potential sources of material for the production of naturally decaffeinated coffee are discussed.     

Aluminium-induced phospholipid signal transduction pathway in Coffea arabica suspension cells and its amelioration by silicic acid

Coffee (Coffea arabica L.) is of economic importance worldwide. Its growth in organic-rich acidic soils is influenced by aluminium such that coffee yield may be impaired. Herein we have used the Al-sensitive C. arabica suspension cell line L2 to analyse the effect of two different Al species on the phosphoinositide signal transduction pathway. Our results have shown that the association of Al with coffee cells was affected by the pH and the form of Al in media. More Al was associated with cells at pH 4.3 than 5.8, whereas when Al was present as hydroxyaluminosilicates (HAS) the association was halved at pH 4.3 and unchanged at pH 5.8. Two signal transduction elements were also evaluated; phospholipase C (PLC) activity and phosphatidic acid (PA) formation. PLC was inhibited ( approximately 50%) when cells were incubated for 2 h in the presence of either AlCl(3) or Al in the form of HAS. PA formation was tested as a short-term response to Al. By way of contrast to what was found for PLC, incubation of cells for 15 min in the presence of AlCl(3) decreased the formation of PA whereas the same concentration of Al as HAS produced no effect upon its formation. These results suggest that Al is capable to exert its effects upon signal transduction as Al((aq))(3+) acting upon a mechanism linked to the phosphoinositide signal transduction pathway.     

中粒咖啡WRKY基因家族的生物信息学分析

WRKY转录因子是植物信号网络中不可缺少的部分,作为植物中最大的转录因子家族之一,在植物的多种应激反应中发挥着重要作用。该文利用生物信息学方法对中粒咖啡WRKY蛋白家族的理化特性及其分子进化进行了详细分析。结果表明:(1)CcWRKY蛋白氨基酸数量在103～994个之间,均无信号肽,推测其为非分泌性蛋白; 其二级结构以无规则卷曲为最主要的结构元件,三级结构主要分为6类,其中以CcWRKY15、CcWRKY25、CcWRKY37和CcWRKY42为主要成员的D类结构最稳定。(2)保守结构域及进化树分析结果显示,中粒咖啡WRKY基因家族含有49个成员,其中的10个成员归为WRKY第Ⅰ家族,34个成员归为WRKY第Ⅱ家族,5个成员归为WRKY第Ⅲ家族。(3)中粒咖啡 WRKY47基因与其他物种的系统进化分析结果显示,WRKY47与烟草亲缘关系最近,与非洲油棕(Elaeis guineensis)亲缘关系最远,说明WRKY47蛋白在生物进化过程中比较保守。该研究结果可为中粒咖啡WRKY基因家族分子功能的深层次研究提供一定的借鉴作用,对进一步探究中粒咖啡WRKY基因的功能、进化以及分子育种具有重要意义。     

The light/dark cycle operation with an hour-scale period enhances caffeine production by Coffea arabica cells

The intermittent light irradiation with an hour-scale period is used for producing caffeine by Coffea arabica cells. Three factors concerning the light/dark cycle operation such as light intensity, the length of the cycle (period), and the ratio of the illumination time to the dark time (light/dark ratio) were investigated to optimize the caffeine production efficiency regarding light consumption. The light/dark ratio of 1/1 enhanced caffeine production, reaching the same level as continuous light; thus, the intermittent light irradiation improved the production efficiency twofold. The production was not influenced by the period, but was determined by light intensity regardless of intermittent or continuous light irradiation.     

Biotransformation of steviol by cultured cells of eucalyptus perriniana and Coffea arabica

A new biotransformation product, steviol 19-β-gentiobiosyl ester, together with steviol 19-β-glucopyranosyl ester and steviol-13-O-β-glucopyranoside 19-β-glucopyranosyl ester (rubusoside), was isolated from Eucalyptus perriniana jar fermentor culture following the administration of steviol. Only rubusoside was isolated as a biotransforination product of steviol from Coffea arabica cell suspension culture.     

Plant regeneration of protoplasts isolated from suspension cultures of Solanum lycopersicoides

A protocol was developed for the isolation, culture and plant regeneration of protoplasts isolated from suspension cultures of Solanum lycopersicoides Dun. (LA 1990). Protoplasts were isolated by an overnight enzyme digestion, further purified by washing in W5 salts solution, and plated in two modified MS protoplast culture media with and without type VII agarose. The addition of agarose to the two culture media did not enhance plating efficiencies and shoot regeneration percentages and in some cases was even inhibitory. Unlike the experience with some other solanaceous species, the deletion of ammonium from the protoplast culture medium was not found to be beneficial. Protoplasts sustained continuous division in the modified MS media and up to 70% of the protoplast-derived calli readily regenerated shoots on MS salts and vitamins medium containing zeatin and GA.     

Phosphate solubilization potential and stress tolerance of Eupenicillium parvum from tea soil

Eupenicillium parvum was recorded for first time during isolation of phosphate-solubilizing microorganisms from the tea rhizosphere. The fungus developed a phosphate solubilization zone on modified Pikovskaya agar, supplemented with tricalcium phosphate. Quantitative estimation of phosphate solubilization in Pikovskaya broth showed high solubilization of tricalcium phosphate and aluminium phosphate. The fungus also solubilized North Carolina rock phosphate and Mussoorie rock phosphate, and exhibited high levels of tolerance against desiccation, acidity, salinity, aluminium, and iron. Solubilization of inorganic phosphates by the fungus was also observed under high stress levels of aluminium, iron, and desiccation, though the significant decline in phosphate solubilization was marked in the presence of aluminium than iron. The fungal isolate showed 100 % identity with E. parvum strain NRRL 2095 ITS 1, 5.8S rRNA gene and ITS 2, complete sequence; and 28S rRNA gene, partial sequence.     

Plant regeneration from cotyledonary protoplasts of cauliflower (Brassica oleracea L. var.botrytis L.)

Summary Protoplasts isolated enzymatically from precultured cotyledonary leaves ofB. oleracea var.botrytis and cultured in KM8p medium (Kao andMichayluk 1975) underwent sustained divisions in about 0.1% population to eventually produce callus, whereas mesophyll protoplasts from either field grown orin vitro raised plants failed to divide. The callus readily differentiated on Murashige-Skoog medium as modified for shoot culture (Binding 1974) to give rise to shoot and roots.     

酿酒酵母SRP40基因对细胞耐受性影响的研究

【目的】本论文研究酿酒酵母srp40³⁹突变基因对酵母细胞异丁醇耐受性的影响。【方法】首先,以酿酒酵母野生型W303-1A和突变株EMS39染色体DNA为模板克隆野生型SRP40基因和srp40³⁹突变基因;然后,将野生型SRP40基因和srp40³⁹突变基因分别连接到质粒YCplac22上,构建质粒YCplac22-SRP40和YCplac22-srp40³⁹。将质粒YCplac22-SRP40、YCplac22-srp40³⁹以及YCplac22空质粒分别转化入野生型酿酒酵母W303-1A中,分别得到W303-1A-SRP40工程菌、W303-1A-srp40³⁹工程菌和W303-1A-control工程菌。将3株工程菌分别置于含1.0%异丁醇、1.3%异丁醇、8.0%乙醇和0.5%异戊醇的CM培养基中进行发酵,测定细胞密度(OD₆₀₀)和生长情况,并计算2–10 h的比生长速率（μ）。将3株工程菌于55°C热激4 min后做稀释...     

Constitutive and aluminium-induced patterns of phenolic compounds in two maize varieties differing in aluminium tolerance

Aluminium tolerance in maize is mainly due to more efficient Al exclusion. Nonetheless, even in tolerant varieties Al can gain access into the cells. Detoxification by binding to strong organic ligands should therefore play a role also in plants with high Al exclusion capacity. To test this hypothesis in this study the concentrations of soluble, free and bound, phenolics were analyzed in roots of two maize varieties differing in Al tolerance. Exposure for 24 h to 50 μM Al in nutrient solution strongly inhibited root elongation in the sensitive variety 16 × 36, but not in the Al-tolerant variety Cateto. Cateto accumulated about half the concentration of Al in roots than 16 × 36 (analysis performed after root desorption with citrate). Roots of Al-tolerant Cateto contained higher concentrations of caffeic acid, catechol and catechin than roots of the sensitive variety. Exposure to Al induced the accumulation of taxifolin in roots of both varieties. However, Al-tolerant Cateto accumulated about twice the concentration than Al-sensitive 16 × 36 of this pentahydroxyfavonol. The molar ratio for phenolics with catecholate groups to Al was about unity in roots of Cateto, while in those of 16 × 36 the ratio was ten times lower. Both the fact that these phenolics are strong ligands for Al and their high antioxidant and antiradical activity suggest that these compounds may provide protection against the Al fraction that is able to surpass the exclusion mechanisms operating in the tolerant maize variety.     

Plant regeneration from protoplasts isolated from embryogenic suspension cultured cells of Cinnamomum camphora L.

An efficient and reproducible protocol is described for the regeneration of Cinnamomum camphora protoplasts isolated from cultured embryogenic suspension cells. Maximum protoplast yield (13.1±2.1×10⁶/g FW) and viability (91.8±3.8%) were achieved using a mixture of 3% (w/v) cellulase Onozuka R10 and 3% (w/v) macerozyme Onozuka R10 in 12.7% (w/v) mannitol solution containing 0.12% (w/v) MES, 0.36% (w/v) CaCl₂·2H₂O, and 0.011% (w/v) NaH₂PO₄·2H₂O. First divisions occurred 7–10 days following culture initiation. The highest division frequency (24.6±2.9%) and plating efficiency (6.88±0.8%) were obtained in liquid medium (MS) supplemented with 30 g l^–1 sucrose, 0.7M glucose, 0.1 mg l^–1 NAA, 1.0 mg l^–1 BA, and 1.0 mg l^–1 GA₃. After somatic embryo induction and then shoot induction, the protoplast-derived embryos produced plantlets at an efficiency of 17.5%. Somatic embryos developed into well-rooted plants on MS medium supplemented with 1.0 mg l^–1 3-indole butyric acid (IBA). Regenerated plants that transferred to soil have normal morphology.     

秋华柳和枫杨幼苗对镉的积累和耐受性

以秋华柳和枫杨当年实生幼苗为研究对象,采用向土壤添加外源镉(CdCl₂ · 2.5H₂O)的形式设置了0(对照组)、10 、20 、50、100 mg/kg 5个处理,研究了镉胁迫下秋华柳和枫杨幼苗的生长、生物量变化和根茎叶镉含量,并评价了两树种的耐性指数(Ti)、转移系数(Tf)和生物富集系数(BCF)。结果表明:(1)在镉含量为10 mg/kg时,秋华柳和枫杨幼苗基于生长和生物量参数的耐性指数(Ti)分别为91.72和91.62,与对照组相比无显著变化,其余各组(20、50、100 mg/kg)则显著低于对照植株(P<0.05);(2) 土壤镉浓度小于20mg/kg时,秋华柳植株茎、叶镉积累量分别高达61.73 mg/kg、163.04 mg/kg,根镉积累量为91.05 mg/kg;枫杨植株茎、叶镉积累量最高分别为7.9 mg/kg、5.25 mg/kg,仅为秋华柳茎、叶的12.8%和3.2%,根镉积累量高达190.68 mg/kg;(3) 除对照外,秋华柳幼苗各部分镉含量为叶＞根＞茎,转移系数(Tf)介于0.789-1.513之间,枫杨幼苗各部分镉含量为根＞茎＞叶,转移系数(Tf)介于0.037-0.044之间,远远小于秋华柳Tf;(4)秋华柳和枫杨幼苗在土壤镉浓度为10 mg/kg时具有很高的生长适应性和耐性,秋华柳根吸收的镉向地上部分转移能力、地上部分积累镉的能力都远远大于枫杨,生物富集系数(BCF)进一步证实了这一特性。研究证明,秋华柳植株具有很高的镉耐性、镉转移能力及地上部分积累镉的能力,适合于镉污染严重区域的植物修复。     

草菇过氧化氢酶基因(VvCAT1)异源表达及耐温度胁迫功能分析

[背景] 过氧化氢酶（catalase,CAT）参与真菌的生长发育,逆境胁迫时保护真菌免受氧化损伤。[目的] 实现草菇过氧化氢酶基因（VvCAT1）的异源表达,分析VvCAT1耐温度胁迫的功能。[方法] 克隆VvCAT1,构建过表达载体pBAR GPE1/VvCAT1,转化到大肠杆菌（Escherichia coli）菌株Stbl3中,异源表达草菇过氧化氢酶。测定温度胁迫后重组菌（pBAR GPE1/VvCAT1/Stbl3）与对照菌（pBAR GPE1/Stbl3）的过氧化氢酶活性和生长情况,验证VvCAT1的功能。[结果] 重组菌的CAT酶活性显著提高,生长情况显著优于对照菌。[结论] VvCAT1的导入及表达显著提高了大肠杆菌Stbl3的耐温度胁迫功能。     

Elicitation studies in cell suspension cultures of Cannabis sativa L.

Cannabis sativa L. plants produce a diverse array of secondary metabolites. Cannabis cell cultures were treated with biotic and abiotic elicitors to evaluate their effect on secondary metabolism. Metabolic profiles analysed by ¹H NMR spectroscopy and principal component analysis (PCA) showed variations in some of the metabolite pools. However, no cannabinoids were found in either control or elicited cannabis cell cultures. Tetrahydrocannabinolic acid (THCA) synthase gene expression was monitored during a time course. Results suggest that other components in the signaling pathway can be controlling the cannabinoid pathway.