Utilization of the genetic resources of wild species to create a nontransgenic high flavonoid tomato

Flavonoids represent a large and important group of plant natural products that are ubiquitous in the plant kingdom. Epidemiological studies have shown the health benefits of a diet high in flavonoids. However, the dietary intake of flavonoids in most western populations is limited, creating a need to find alternative food sources for these polyphenolic secondary metabolites. The domestication of many of our cultivated food crops has resulted in alterations in the biosynthetic pathways of many essential micronutrients and vitamins through inadvertent counterselection against nutritional traits in favor of agronomic ones. Flavonoids are nearly absent from fruits of cultivated tomato (Lycopersicon esculentum Mill.), a major vegetable in human diets. Previous attempts to restore the flavonoid pathway in tomato fruits have been limited to transgenic strategies, suggesting that the problem was intractable through traditional methods. Here, we describe for the first time a nontransgenic metabolic engineering approach to developing a high flavonoid tomato using a wild tomato species (Lycopersicon pennelliiv. puberulum) and demonstrate the opportunities for restoring functional pathways using the genetic resources of wild species, resulting in production of healthier foods.     

Validation of a tomato-specific gene, LAT52, used as an endogenous reference gene in qualitative and real-time quantitative PCR detection of transgenic tomatoes

Toward the development of reliable qualitative and quantitative Polymerase Chain Reaction (PCR) detection methods of transgenic tomatoes, one tomato (Lycopersicon esculentum) species specific gene, LAT52, was selected and validated as suitable for using as an endogenous reference gene in transgenic tomato PCR detection. Both qualitative and quantitative PCR methods were assayed with 16 different tomato varieties, and identical amplified products or fluorescent signals were obtained with all of them. No amplified products and fluorescent signals were observed when DNA samples from 20 different plants such as soybean, maize, rapeseed, rice, and Arabidopsis thaliana were used as templates. These results demonstrated that the amplified LAT52 DNA sequence was specific for tomato. Furthermore, results of Southern blot showed that the LAT52 gene was a single-copy gene in the different tested tomato cultivars. In qualitative and quantitative PCR analysis, the detection sensitivities were 0.05 and 0.005 ng of tomato genomic DNA, respectively. In addition, two real-time assays employing this gene as an endogenous reference gene were established, one for the quantification of processed food samples derived from nontransgenic tomatoes that contained degraded target DNA and the other for the quantification of the junction region of CaMV35s promoter and the anti-sense ethylene-forming enzyme (EFE) gene in transgenic tomato Huafan No. 1 samples. All of these results indicated that the LAT52 gene could be successfully used as a tomato endogenous reference gene in practical qualitative and quantitative detection of transgenic tomatoes, even for some processed foods derived from transgenic and nontransgenic tomatoes.     

乙烯受体基因LeETR1和LeETR4的克隆及在番茄果实中的表达

摘要：为研究野生型番茄（Lycopersicon esculentum Mill. cv . Lichun）和转反义ACS 番茄果实中乙烯受体基因LeETR1和LeETR4的表达与乙烯的关系，实验用RT-PCR方法扩增了乙烯受体基因LeETR1和LeETR4片段，用两片段作探针进行Northern 杂交。结果表明：两受体基因的表达在番茄果实成熟进程中变化不明显，LeETR4在同一时期的果实外果皮中表达水平低于其在辐射壁和中柱的表达。转反义ACS番茄果实中LeETR1和LeETR4的表达水平显著低于野生型番茄果实，外源乙烯处理转反义ACS番茄果实，促进两个受体基因的表达。可见果实中LeETR1和LeETR4的表达受乙烯调控的影响。     

Effect of increased cobalt treatments on sewage sludge amended soil: Nitrogen species in soil and tranference to tomato plants

The effects of increased cobalt additions (0, 50, 100 and 200 v mg v kg m 1 soil) in sewage sludge-amended soil on organic matter, N Kjeldahl, ammonium and nitrate were studied in this experiment. Three different rates of sewage sludge were applied (0, 30 and 60 v tn v ha m 1 ) to soil as main plots, using tomato (Lycopersicum esculentum Mill var. Ramy) such testing cultivation. Plant biomass and nitrogen content in tomato leaf were also monitored. The organic matter in the soil was clearly affected by the fertilization. N Kjeldahl, ammonium and nitrate were favoured by organic treatments. Co seemed to reduce the transformation of ammonium to nitrate on amended soils, with accumulation of ammonium forms, especially at the higher application rates of sewage sludge. This incidence of Co on nitrogen species in soil decreased with the time of experiment, probably due to the reduction of availability of the pollutant. Aerial biomass production and nitrogen content in leaf were increased for the organic fertilization compared to the control. Only very high levels of Co in soil reduced significantly the aerial biomass production of tomato plants in amended soils. Co seemed to induce a decrease of the nitrogen in leaf in the amended soils, but not for the non-fertilized soils.     

The effect on Cd contents and ethylene biosynthesis in tomato plants of adding cadmium sulphate to soil

Water, Air, & Soil Pollution - An experiment was carried out with increasing concentrations of Cd in soil on tomato (Lycopersicon esculentum Mill.) plants. The Cd contents of soil and in tomato...     

番茄果实成熟衰老相关蛋白酶鉴定

为进一步研究蛋白酶与成熟衰老的关系,试验以‘918’番茄(Lycopersicum esculentum Mill.cv.918)果实为材料,采用改进的含明胶的十二烷基硫酸钠聚丙烯酰胺凝胶电泳方法对番茄果实中的蛋白酶进行检测,结果发现番茄果实中有3个蛋白酶,命名为SP-1、SP-2和SP-3,其活性伴随着果实后熟衰老发生变化。乙烯处理可提高番茄果实蛋白酶SP-2的活性,使SP-1和SP-3活性被提前检出,而1-甲基环丙烯处理对蛋白酶起相反作用。番茄果实蛋白酶受果实后熟衰老和乙烯的双重调控,是衰老相关蛋白酶。该文为进一步研究蛋白酶与果蔬衰老的关系提供参考。     

复混肥中缩二脲含量对作物生长的影响

采用水培和盆栽法,研究了复混肥中不同含量的缩二脲对作物生长的影响.水培施肥水平为每升水溶液中复混肥(10-8-7)含量分别为0.5 g和1.0 g,即每升水溶液中含氮量达到56.05 mg和112.1 mg;土壤施肥水平为每千克土壤中复混肥(10-8-7)含量分别为0.5 g和1.0 g,即每千克土壤中含氮量达到56.05 mg和121.1 mg.水培试验结果表明:每升水溶液中,在适宜各供试作物生长的施肥条件下,复混肥中缩二脲对冬小麦、玉米、番茄、油菜、水稻的毒害作用的临界值分别为35、40、20、15、15 mg.盆栽试验结果表明:在适宜各供试作物生长的施肥条件下,缩二脲对冬小麦、玉米、番茄、油菜、水稻的毒害作用的临界值分别为所施复混肥(10-8-7)含量的1.6%、2.0%、2.0%、2.0%、2.0%.当高于这个临界值时,缩二脲对此5种作物会造成毒害作用.考虑到复混肥的实际制造过程中大量使用含氯化肥,氯离子含量波动大,建议国家在制定复混肥产品标准时缩二脲含量应限制在1%以下为宜.     

Quantification of chlorophyll content and classification of nontransgenic and transgenic tomato leaves using visible/near-infrared diffuse reflectance spectroscopy

Visible/near-infrared (vis/NIR) spectroscopy combined with multivariate analysis was used to quantify chlorophyll content in tomato leaves and classify tomato leaves with different genes. In this study, transgenic tomato leaves with antisense LeETR1 (n = 106) and their parent nontransgenic ones (n = 102) were measured in vis/NIR diffuse reflectance mode. Quantification of chlorophyll content was achieved by partial least-squares regression with a cross-validation prediction error equal to 2.87. Partial least-squares discriminant analysis was performed to classify leaves. The results show that differences between transgenic and nontransgenic tomato leaves do exist, and excellent classification can be obtained after optimizing spectral pretreatment. The classification accuracy can reach to 100% using the derivative of spectral data in the full and partial wavenumber range. These results demonstrate that vis/NIR spectroscopy together with chemometrics techniques could be used to quantify chlorophyll content and differentiate tomato leaves with different genes, which offers the benefit of avoiding time-consuming, costly, and laborious chemical and sensory analysis.     

Arsenic species: effects on and accumulation by tomato plants.

The uptake of arsenic (As) species by Lycopersicum esculentum, growing under soilless culture conditions, was studied. A 4 x 3 x 2 factorial experiment was conducted with four As species (arsenite, arsenate, methylarsonate, and dimethylarsinate), three As concentrations (1, 2, and 5 mg L(-)(1)) and two tomato cultivars (Marmande and Muchamiel). The phytoavailability and phytotoxicity were primarily determined by the As species. The concentrations of As in plant increased significantly with increasing As concentration in solution. Both MA and DMA showed a higher upward translocation than arsenite and arsenate, and treatments with MA and DMA clearly reduced plant growth and fruit yield. The As concentration in tomatoes treated with arsenite or arsenate were within the range considered normal in food crops; however, the As concentration in tomatoes treated with MA and DMA were close to or even above the maximum limit. When tomato plants are exposed to high concentrations of As in nutrient solutions, they may uptake As to concentrations unacceptable for human food.     

Determination of phenolic compounds and ascorbic acid in different fractions of tomato by capillary electrophoresis with electrochemical detection

Tomato (Lycopersicon esculentum Mill.), one of the most important crops worldwide, contains different classes of substances with antioxidant properties such as carotenoids, vitamin C, and phenolics. A method based on capillary electrophoresis with electrochemical detection has been developed to analyze ascorbic acid and phenolics in the peel, pulp, and seeds of tomatoes. Operating in a wall-jet configuration, a 300 microm diameter carbon disk electrode was used as the working electrode, which exhibits a good response at +0.90 V (vs saturated calomel electrode) for the analytes. Under optimum conditions, the analytes were baseline separated within 20 min in a 50 mmol/L borate buffer (pH 8.7). Notably, excellent linearity was obtained over 3 orders of magnitude with detection limits (S/N=3) ranging from 1x10(-8) to 2x10(-7) g/mL for all analytes. This proposed method has been successfully applied to monitor the content of ascorbic acid and phenolics in real samples, and the assay results were satisfactory.     

The flavonoids of tomatoes

Tomatoes ( Lycopersicon esculentum Mill.) have been recognized as an important source of dietary flavonoids because of a high consumption worldwide. The qualitative and quantitative flavonoid compositions of assorted tomato cultivars including individual quantitative contributions of the five most significant flavonoids have been determined in this work. The dihydrochalcone phloretin 3',5'-di-C-beta-glucopyranoside and the flavonol quercetin 3-O-(2'-O-beta-apiofuranosyl-6'-O-alpha-rhamnopyranosyl-beta-glucopyranoside) were identified for the first time in Solanaceae spp. and found to be among the main flavonoids in all cultivars. Phloretin 3',5'-di-C-glc is the first C-glycoside identified in tomatoes and also the first dihydrochalcone from this species. In addition, chalconaringenin, kaempferol 3-rutinoside, and quercetin 3-rutinoside (rutin), though previously reported to occur in tomato, were fully characterized by extensive use of 2D NMR techniques and high-resolution LCMS. The total flavonoid content of different tomato types varied from 4 to 26 mg 100 (-1) g FW with chalconaringenin as the predominant compound comprising 35 to 71% of the total flavonoid content. The individual quantities of quercetin 3-O-(2'- O-beta-apiofuranosyl-6'- O-alpha-rhamnopyranosyl-beta-glucopyranoside) and phloretin 3',5'-di-C-beta-glucopyranoside was similar to that of rutin in several cultivars.     

Identification and quantification of stilbenes in fruits of transgenic tomato plants (Lycopersicon esculentum Mill.) by reversed phase HPLC with photodiode array and mass spectrometry detection

Reversed-phase high-performance liquid chromatography (RP-HPLC) with photodiode array (PDA) and mass spectrometry (MS) detection was employed to study the accumulation of stilbenes and other naturally occurring polyphenol intermediates of flavonoid pathway in tomato fruits of plants genetically modified to synthesize resveratrol. The transgenic tomato fruits were obtained by overexpression of a grapevine gene encoding the enzyme stilbene synthase in tomato plants (Lycopersicon esculentum Mill.). Stilbenes and flavonoids, either glycosylated or free, were simultaneosly identified by electrospray interface (ESI)-MS in negative ionization mode and were quantified by PDA detection at the wavelength corresponding to their maximum absorbance. The two detectors were coupled online with an HPLC system utilizing a narrow-bore C18 reversed-phase column, which was eluted by a multistep gradient of increasing concentration of acetonitrile in water containing 0.5% (v/v) formic acid. The results of these analysis revealed that the genetic modification of the tomato plants originated different levels of accumulation of four stilbenes (i.e., trans- and cis-piceid and trans- and cis-resveratrol) in their fruit depending on the stages of ripening. Either at immature or at mature stages of ripening the stilbenes were preferentially accumulated in the fruit peel as the glycosylated form. The highest amount of trans-piceid and trans-resveratrol were found in the peel of fruits harvested at mature stage of ripening. The variations in the levels of rutin, naringenin, and chlorogenic acid found in the samples extracted from the fruits of transgenic tomato plants, in comparison to that determined in the control lines, seemed to be related to the genetic transformation, whose effect on the flavonoid biosynthetic pathway needs to be elucidated by additional studies.     

Structure-Activity Relationships (SAR) studies of benzoxazinones, their degradation products and analogues. phytotoxicity on standard target species (STS)

Benzoxazinones 2,4-dihydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (DIMBOA) and 2,4-dihydroxy-(2H)-1,4-benzoxazin-3(4H)-one (DIBOA) have been considered key compounds for understanding allelopathic phenomena in Gramineae crop plants such as corn (Zea mays L.), wheat (Triticum aestivum L.), and rye (Secale cereale L.). The degradation processes in the environment observed for these compounds, in which soil microbes are directly involved, could affect potential allelopathic activity of these plants. We present in this work a complete structure-activity relationships study based on the phytotoxic effects observed for DIMBOA, DIBOA, and their main degradation products, in addition to several synthetic analogues of them. Their effects were evaluated on standard target species (STS), which include Triticum aestivum L. (wheat) and Allium cepa L. (onion) as monocots and Lepidium sativum L. (cress), Lactuca sativa L. (lettuce), and Lycopersicon esculentum Will. (tomato) as dicots. This permitted us to elucidate their ecological role and to propose new herbicide models based on their structures. The best phytotoxicity results were shown by the degradation chemical 2-aminophenoxazin-3-one (APO) and several 2-deoxy derivatives of natural benzoxazinones, including 4-acetoxy-(2H)-1,4-benzoxazin-3(4H)-one (ABOA), 4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (D-DIBOA), and 4-hydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (D-DIMBOA). They showed high inhibitory activity over almost all species growth. The fact that APO is a degradation product from DIBOA with high phytotoxicity and stability makes it possible to assign an important ecological role regarding plant defense mechanisms. 2-Deoxy derivatives of natural benzoxazinones display a wide range of activities that allow proposing them as new leads for natural herbicide models with a 1,4-benzoxazine skeleton.     

废水污泥生物炭对圣女果生长、产量及重金属累积效应的对比评估

To investigate the potential effects of wastewater sludge and sludge biochar on growth, yield and metal bioaccumulation of cherry tomato ({\it Lycopersicon esculentum} L.), a pot experiment was carried out under greenhouse environment with three different treatments, control soil (CP), soil with wastewater sludge (SS) and soil with sludge biochar (SB), to reveal the comparative effect between the amendments of wastewater sludge and sludge biochar.~The soil used for pot experiment was Chromosol.~Wastewater sludge and sludge biochar produced through pyrolysis process at 550 $^\circ$C were applied at 10 t ha$^{-1}$. No significant difference was found in growth and production of cherry tomatoes between wastewater sludge and sludge biochar applications to the soil. The accumulation rates of metals in the fruits were lower in the treatment with sludge biochar than in the treatment with wastewater sludge. The study highlights the benefits of risk mitigation from toxic metal accumulation in fruits using wastewater sludge and sludge biochar as soil conditioners.     

Antioxidant activity of grains

Epidemiological studies have shown that consumption of whole grains and grain-based products is associated with reduced risk of chronic diseases. The health benefits of whole grains are attributed in part to their unique phytochemical composition. However, the phytochemical contents in grains have been commonly underestimated in the literature, because bound phytochemicals were not included. This study was designed to investigate the complete phytochemical profiles in free, soluble conjugated, and insoluble bound forms, as well as their antioxidant activities in uncooked whole grains. Corn had the highest total phenolic content (15.55 +/- 0.60 micromol of gallic acid equiv/g of grain) of the grains tested, followed by wheat (7.99 +/- 0.39 micromol of gallic acid equiv/g of grain), oats (6.53 +/- 0.19 micromol of gallic acid equiv/g of grain), and rice (5.56 +/- 0.17 micromol of gallic acid equiv/g of grain). The major portion of phenolics in grains existed in the bound form (85% in corn, 75% in oats and wheat, and 62% in rice), although free phenolics were frequently reported in the literature. Ferulic acid was the major phenolic compound in grains tested, with free, soluble-conjugated, and bound ferulic acids present in the ratio 0.1:1:100. Corn had the highest total antioxidant activity (181.42 +/- 0.86 micromol of vitamin C equiv/g of grain), followed by wheat (76.70 +/- 1.38 micromol of vitamin C equiv/g of grain), oats (74.67 +/- 1.49 micromol of vitamin C equiv/g of grain), and rice (55.77 +/- 1.62 micromol of vitamin C equiv/g of grain). Bound phytochemicals were the major contributors to the total antioxidant activity: 90% in wheat, 87% in corn, 71% in rice, and 58% in oats. Bound phytochemicals could survive stomach and intestinal digestion to reach the colon. This may partly explain the mechanism of grain consumption in the prevention of colon cancer, other digestive cancers, breast cancer, and prostate cancer, which is supported by epidemiological studies.     

Arsenic uptake,distribution, and accumulation in tomato plants: Effect of arsenite on plant growth and yield

The response of tomato (Lycopersicum esculentum Mill, cultivar Marmande) plants to different levels of arsenic (As) in nutrient solution was investigated—the processes of uptake, distribution and accumulation of As, and the effect of arsenite on yield and plant growth (plant height, diameter of stem, stem and root length, fresh and dry weight of root, stems, leaves, and fruit). The experiment was performed at three levels of As: 2, 5 and 10 mg/L [added as sodium arsenite (NaAsO₂)] in a nutrient solution, together with the corresponding control plants. Arsenic uptake depended on the As concentration in solution and As content in the roots increased as the time of treatment increased. The most important finding was the high toxicity of arsenite to roots. The concentration in stems, leaves, and fruit was correlated with the As level in the nutrient solution. Although the As level of 10 mg/L damaged the root membranes, resulting in a significant decrease in the upward transport of As. Arsenic exposure resulted in a drastic decrease in plant growth parameters (e.g., maximum decrease of 76.8% in leaf fresh weight) and in tomato fruit yield (maximum reduction of 79.6%). However, it is important to note that the As concentration in the fruits was not toxic or harmful for human consumption.     

温室有机土栽培番茄营养吸收特性研究

以腐熟玉米秸、麦秸、菇渣或锯末等农产废弃物及有机肥为有机化栽培的有机原料,添加土壤后配成有机土进行了番茄栽培试验,研究了植株对氮、磷、钾的吸收特性。结果表明,不同配方下番茄对氮磷钾的单株吸收量、养分利用率以及养分在植株不同部位的分配不同。全生育期N∶P2O5∶K2O=1∶0.194～0.375∶0.903～1.412。根据番茄产量计算出每生产100kg番茄果实的养分需要量为:N136.7～201.4g,P2O539.9～60.0g,K2O156.7～235.7g。综合不同有机土配比对番茄养分利用率、产量品质的影响,以大粪干+玉米秸+锯沫或菇渣(1∶2∶1)的配方较好。     

Liquid chromatographic determination and stability of the Fusarium mycotoxin moniliformin in cereal grains

Moniliformin is a mycotoxin produced by Fusarium subglutinans and other Fusarium species. A rapid, liquid chromatographic method for its determination in corn and wheat is described. Samples are extracted in acetonitrile-water (95 + 5); following defatting with n-hexane, an aliquot of the extract is evaporated and cleaned up on small C18 and neutral alumina columns successively. Reverse-phase liquid chromatography (LC) is conducted on a C18 column with 10 or 15% methanol or acetonitrile in aqueous ion-pair reagent as mobile phase, with detection by ultraviolet absorption at 229 and 254 nm. Average recoveries of moniliformin (potassium salt) added to ground corn and wheat at levels of 0.05-1.0 micrograms/g were 80% (n = 20) and 85% (n = 12), respectively, and the limit of detection was ca 0.01-0.18 micrograms/g, depending on LC conditions. Analysis of 24 samples of wheat, 4 samples of rye, and 12 samples of corn showed moniliformin in only 2 corn samples (0.06 and 0.2 micrograms/g). Moniliformin was also detected in a sample of artificially damaged (slashed) corn (0.2 micrograms/g) and selected kernels of corn that were field-inoculated with F. subglutinans and F. moniliforme (50 micrograms/g and 0.5 micrograms/g, respectively). In stability studies, moniliformin (potassium salt, 1 microgram/g) in ground corn and ground wheat heated at 50, 100, and 150 degrees C for 0.5-2 h decomposed moderately, e.g., 55% remained in corn after 0.5 h at 100 degrees C.     

Compositional equivalency of Cry1F corn event TC6275 and conventional corn (Zea mays L.)

Maize (Zea mays L.) plants have been transformed to express a Cry1F insecticidal crystal protein originally isolated from Bacillus thuringiensis Berliner. This protein controls lepidopteran pests of maize, including the European corn borer, Ostrinia nubilalis (Hübner). As part of the safety assessment for crops containing transgenes, a compositional analysis of the food and feed is conducted. This analysis is designed to detect unintended changes in the nutrient and antinutrient content of the raw commodities produced by the crop due to the insertion of the genes into the genomic DNA of the plant (pleotropic effects). Samples of transgenic and nontransgenic maize forage and grain were collected from six field sites located in the U.S. and Canada. Forage samples were analyzed for proximates and minerals, and grain was further analyzed for fatty acids, amino acids, vitamins, secondary metabolites, and antinutrients. Results demonstrated that maize expressing the Cry1F protein was equivalent to nontransgenic maize with respect to these important components. Comparison of the variability within the nontransgenic and transgenic hybrid, as compared to composition values reported in the literature, suggest that factors other than transgenes may contribute more substantially to the composition of crops.     

Contents of phenolic acids, alkyl- and alkenylresorcinols, and avenanthramides in commercial grain products

The contents of free and total phenolic acids and alk(en)ylresorcinols were analyzed in commercial products of eight grains: oat (Avena sativa), wheat (Triticum spp.), rye (Secale cerale), barley (Hordeum vulgare), buckwheat (Fagopyrum esculentum), millet (Panicum miliaceum), rice (Oryza sativa), and corn (Zea mays). Avenanthramides were determined in three oat products. Free phenolic acids, alk(en)ylresorcinols, and avenanthramides were extracted with methanolic acetic acid, 100% methanol, and 80% methanol, respectively, and quantified by HPLC. The contents of total phenolic acids were quantified by HPLC analysis after alkaline and acid hydrolyses. The highest contents of total phenolic acids were in brans of wheat (4527 mg/kg) and rye (4190 mg/kg) and in whole-grain flours of these grains (1342 and 1366 mg/kg, respectively). In other products, the contents varied from 111 mg/kg (white wheat bread) to 765 mg/kg (whole-grain rye bread). Common phenolic acids found in the grain products were ferulic acid (most abundant), ferulic acid dehydrodimers, sinapic acid, and p-coumaric acid. The grain products were found to contain either none or only low amounts of free phenolic acids. The content of avenanthramides in oat flakes (26-27 mg/kg) was about double that found in oat bran (13 mg/kg). The highest contents of alk(en)ylresorcinols were observed in brans of rye (4108 mg/kg) and wheat (3225 mg/kg). In addition, whole-grain rye products (rye bread, rye flour, and whole-wheat flour) contained considerable levels of alk(en)ylresorcinols (524, 927, and 759 mg/kg, respectively).