Methyl salicylate production in tomato affects biotic interactions

The role of methyl salicylate (MeSA) production was studied in indirect and direct defence responses of tomato (Solanum lycopersicum) to the spider mite Tetranychus urticae and the root‐invading fungus Fusarium oxysporum f. sp. lycopersici, respectively. To this end, we silenced the tomato gene encoding salicylic acid methyl transferase (SAMT). Silencing of SAMT led to a major reduction in SAMT expression and MeSA emission upon herbivory by spider mites, without affecting the induced emission of other volatiles (terpenoids). The predatory mite Phytoseiulus persimilis, which preys on T. urticae, could not discriminate between infested and non‐infested SAMT‐silenced lines, as it could for wild‐type tomato plants. Moreover, when given the choice between infested SAMT‐silenced and infested wild‐type plants, they preferred the latter. These findings are supportive of a major role for MeSA in this indirect defence response of tomato. SAMT‐silenced tomato plants were less susceptible to a virulent strain of F. oxysporum f. sp. lycopersici, indicating that the direct defense responses in the roots are also affected in these plants. Our studies show that the conversion of SA to MeSA can affect both direct and indirect plant defence responses.     

Priming of inducible defenses protects Norway spruce against tree-killing bark beetles

Plants can form an immunological memory known as defense priming, whereby exposure to a priming stimulus enables quicker or stronger response to subsequent attack by pests and pathogens. Such priming of inducible defenses provides increased protection and reduces allocation costs of defense. Defense priming has been widely studied for short-lived model plants such as Arabidopsis, but little is known about this phenomenon in long-lived plants like spruce. We compared the effects of pretreatment with sublethal fungal inoculations or application of the phytohormone methyl jasmonate (MeJA) on the resistance of 48-year-old Norway spruce (Picea abies) trees to mass attack by a tree-killing bark beetle beginning 35 days later. Bark beetles heavily infested and killed untreated trees but largely avoided fungus-inoculated trees and MeJA-treated trees. Quantification of defensive terpenes at the time of bark beetle attack showed fungal inoculation induced 91-fold higher terpene concentrations compared with untreated trees, whereas application of MeJA did not significantly increase terpenes. These results indicate that resistance in fungus-inoculated trees is a result of direct induction of defenses, whereas resistance in MeJA-treated trees is due to defense priming. This work extends our knowledge of defense priming from model plants to an ecologically important tree species.     

Curcumin stimulates angiogenesis through VEGF and expression of HLA‐G in first‐trimester human placental trophoblasts

Curcumin has a protective role in placental diseases like preeclampsia and preterm birth. Very little is known about its functional effects on growth, angiogenesis, and epigenetic activities of human first trimester placenta. HTR8/SVneo trophoblasts cells were used as model for human first trimester placenta. Effects of curcumin (≥80%) in these cells were investigated using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), radioactive thymidine uptake, quantitative real‐time polymerase chain reaction (qRT‐PCR), promoter DNA methylation, qRT‐PCR array, tube formation, wound healing, and immunoblot assays. PC3 (prostate cancer), JEG‐3 (trophoblast), and HMEC‐1 (endothelial) cells were used as control in various experiments. Unlike in PC3 cells, curcumin stimulated growth, proliferation, and viability in HTR8/SVneo cells. Curcumin increased tube formation, and messenger RNA (mRNA) expression of angiogenic factors such as vascular endothelial growth factor A (VEGFA) and protein expression of proangiogenic factor VEGF receptor‐2 and fatty acid‐binding protein‐4 (FABP4) in these cells. Curcumin‐stimulated tube formation was associated with an increased expression of VEGFR2 and FABP4. The stimulatory effects of curcumin were inhibited by VEGFR2 (SU5416) and FABP4 (BMS309403) inhibitors. Curcumin also significantly increased both mRNA and protein expression of HLA‐G in HTR8/SVneo cells. Curcumin increased mRNA expression of DNMT3A and NOTCH signaling system whereas down‐regulated mRNA expression of HSD11β2. Curcumin enhanced hypomethylation of gene promoters against oxidative stress and DNA damage pathway mediators. Curcumin promotes cell growth, migration, and thus angiogenic potential of these cells. Increased expression of HLA‐G by curcumin, hitherto unknown, is a novel finding since HLA‐G not only favors the immune environment for invasive trophoblasts but also positively modulates angiogenesis.     

甲基硬脂酸酯和E-β-罗勒烯对中华蜜蜂育王质量的影响

为研究育王过程中添加蜜蜂幼虫信息素成分甲基硬脂酸酯和E-β-罗勒烯对中华蜜蜂育王质量的影响,本试验结合免移虫育王技术,在幼虫60～64 h时向王台内注入1μL配置好的信息素(浓度梯度分别为0%、0.1%、1.0%、10.0%),待蜂王出房后测定蜂王个体指标及蜂王卵巢Vg、hex70b和hex110基因的表达水平。与空白对照相比,添加10.0%浓度的E-β-罗勒烯组蜂王的初生重和单侧卵巢管数显著增加,同时Vg、hex70b和hex110基因表达水平也显著上升;添加1.0%的E-β-罗勒烯蜂王单侧卵巢管数量显著增加,Vg和hex110基因的表达量也显著上升;添加甲基硬脂酸酯对培育蜂王的初生重、胸部指标和卵巢相关基因表达均无显著影响,但1.0%和10.0%的甲基硬脂酸酯使蜂王卵巢管数量显著减少。结果表明在中蜂育王过程中添加E-β-罗勒烯可以在一定程度上提高蜂王的质量。     

广藿香FPPS基因原核表达及茉莉酸甲酯对FPPS表达量的影响

法尼基焦磷酸合酶(farnesyl diphosphate synthase,FPPS)是广藿香甲羟戊酸途径中萜类物质生物合成的关键酶,其催化异戊二烯焦磷酸(IPP)和二甲基烯丙基焦磷酸(DMAPP)合成萜类物质前体法尼基焦磷酸。为了进一步研究广藿香萜类合成途径的分子机制,该文通过逆转录聚合酶链式反应获得FPPS基因的cDNA序列,利用生物信息学软件预测FPPS编码蛋白的理化性质、结构和功能。结果表明:(1)该序列的开放阅读框全长1 050 bp,编码349个氨基酸,预测分子量为40 KD,等电点为5.43,存在一个结构域,参与异戊二烯化合物的合成,不存在信号肽,亚细胞定位于细胞质;系统发育分析结果显示,广藿香FPPS氨基酸序列和丹参(Salvia miltiorrhiza)、撒尔维亚(S. officinalis)的氨基酸序列亲缘关系最近。(2)使用无缝克隆技术构建pET-32b-FPPS原核表达载体,并导入菌株BL21(DE3)中,考察不同浓度异丙基-β-D-硫代半乳糖苷(IPTG)对诱导融合蛋白的表达量的影响。结果发现融合表达蛋白以包涵体形式存在沉淀中,4个浓度的IPTG诱导蛋白表达效果差异不明显。(3)采用荧光定量技术分析0.10、0.25 mmol·L-1MeJA对FPPS基因表达水平的影响,发现0.10 mmol·L~(-1)MeJA诱导后FPPS基因的表达量趋势是先升高后降低再升高再降低; 0.25 mmol·L~(-1)MeJA诱导后FPPS基因的表达量趋势是先降低后升高再降低。因此,推测植物体内MeJA浓度的变化能影响FPPS基因的表达,高浓度具抑制作用,低浓度具促进作用。本研究为广藿香萜类合成途径的研究奠定基础,以及为后续基因功能验证提供理论参考。     

铁皮石斛DoSMT2基因的功能分析

为了揭示铁皮石斛(Dendrobium officinale)甾醇C-24甲基转移酶2基因(DoSMT2)在甾醇代谢过程的功能,该研究通过根癌农杆菌介导法将来源于铁皮石斛的DoSMT2基因转化烟草(Nicotiana tabacum),并采用qRT-PCR技术检测DoSMT2基因在转基因烟草叶片中的表达,采用气相色谱质谱法分析菜油甾醇和谷甾醇的含量。结果显示:(1)成功获得DoSMT2基因的开放阅读框(1 119 bp),并成功构建正义植物表达载体质粒pCXSN-DoSMT2,经农杆菌介导的烟草叶盘转化法转化烟草并鉴定,获得4株阳性转基因烟草植株。(2)Southern blot结果表明,4株转基因烟草植株都有1条杂交信号带,而非转基因烟草植株没有,说明外源DoSMT2基因都以单拷贝整合到4株转基因烟草基因组中。(3)qRT-PCR检测显示,非转基因烟草未检测到外源DoSMT2基因的表达,4株转基因烟草都能检测到DoSMT2基因的表达,且表达水平差异极显著,各株系表达量高低依次为P3P1P2(P4)。(4)气相色谱质谱分析显示,转DoSMT2基因烟草叶片的菜油甾醇含量均极显著低于非转基因烟草叶片,而谷甾醇含量均极显著高于非转基因烟草叶片。研究表明,DoSMT2具有催化24-亚甲基胆甾烯醇转化形成24-亚乙基胆甾烯醇活性。     

Oxidative modification of citrate synthase by peroxyl radicals and protection with novel antioxidants

In mammals, aging is linked to a decline in the activity of citrate synthase (CS; E.C. 2.3.3.1), the first enzyme of the citric acid cycle. We used 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), a water-soluble generator of peroxyl and alkoxyl radicals, to investigate the susceptibility of CS to oxidative damage. Treatment of isolated mitochondria with AAPH for 8–24?h led to CS inactivation; however, the activity of aconitase, a mitochondrial enzyme routinely used as an oxidative stress marker, was unaffected. In addition to enzyme inactivation, AAPH treatment of purified CS resulted in dityrosine formation, increased protein surface hydrophobicity, and loss of tryptophan fluorescence. Propyl gallate, 1,8-naphthalenediol, 2,3-naphthalenediol, ascorbic acid, glutathione, and oxaloacetate protected CS from AAPH-mediated inactivation, with IC₅₀ values of 9, 14, 34, 37, 150, and 160?μM, respectively. Surprisingly, the antioxidant epigallocatechin gallate offered no protection against AAPH, but instead caused CS inactivation. Our results suggest that the current practice of using the enzymatic activity of CS as an index of mitochondrial abundance and the use of aconitase activity as an oxidative stress marker may be inappropriate, especially in oxidative stress-related studies, during which alkyl peroxyl and alkoxyl radicals can be generated.     

A derivatization method for the simultaneous detection of glucosinolates and isothiocyanates in biological samples

Various analytical methods have been established to quantify isothiocyanates (ITCs) that derive from glucosinolate hydrolysis. However, to date there is no valid method applicable to pharmacokinetic studies that detects both glucosinolates and ITCs. A specific derivatization procedure was developed for the determination of ITCs based on the formation of a stable N-(tert-butoxycarbonyl)-l-cysteine methyl ester derivative, which can be measured by high-performance liquid chromatography with ultraviolet detection after extraction with ethylacetate. The novel method, which is also applicable to the indirect determination of glucosinolates after their hydrolysis by myrosinase, was established for the simultaneous determination of glucoraphanin and sulforaphane. By derivatization, the sensitivity of ITC detection was increased 2.5-fold. Analytical recoveries from urine and plasma were greater than 75% and from feces were approximately 50%. The method showed intra- and interday variations of less than 11 and 13%, respectively. Applicability of the method was demonstrated in mice that received various doses of glucoraphanin or that were fed a glucoraphanin-rich diet. Besides glucoraphanin and sulforaphane, glucoerucin and erucin were detected in urine and feces of mice. The novel method provides an essential tool for the analysis of bioactive glucosinolates and their hydrolysis products and, thus, will contribute to the elucidation of their bioavailability.     

Radiotracer Experiments on Biological Volatilization of Organic Iodine from Coastal Seawaters

Biological volatilization of iodine from seawaters was studied using a radiotracer technique. Seawater samples were incubated aerobically in serum bottles with radioactive iodide tracer (¹²⁵I), and volatile organic and inorganic iodine were collected with activated charcoal and silver wool trap, respectively. Iodine was volatilized mainly as organic iodine, and inorganic iodine volatilization was not observed. Influence of light intensity on the volatilization was determined, but no significant differences were observed under light (70,000 lux) and dark conditions. The effect of the chemical form of iodine on the volatilization was determined, and the results suggested that volatilization preferentially occurs from iodide (I^?) but not from iodate (IO₃ ^?). Volatilization did not occur when the samples were autoclaved or filtered through a 0.22-μm pore size membrane filter. Incubation of the samples with antibiotics caused decreased volatilization. Conversely, enhanced volatilization was observed when the samples were incubated with yeast extract. Fifty-nine marine bacterial strains were then randomly isolated from marine environments, and their iodine-volatilizing capacities were determined. Among these, 19 strains exhibited significant capacities for volatilizing iodine. 16S ribosomal RNA gene comparisons indicated that these bacteria are members of Proteobacteria (α and γ subdivisions) and Cytophaga-Flexibacter-Bacteroides group. One of the strains, strain C-19, volatilized 1 to 2% of total iodine during cultivation, and the gaseous organic iodine was identified as methyl iodide (CH₃I). These results suggest that organic iodine volatilization from seawaters occurs biologically, and that marine bacteria participate in the process. Considering that volatile organic iodine emitted from the oceans causes atmospheric ozone destruction, biological iodine volatilization from seawater is of great importance. Our results also contribute to prediction of movement and diffusion of long-lived radioactive iodine (¹²⁹I) in the environment.