Acylated anthocyanins from red radish (Raphanus sativus L.)

Twelve acylated anthocyanins were isolated from the red radish (Raphanus sativus L.) and their structures were determined by spectroscopic analyses. Six of these were identified as pelargonidin 3-O-[6-O-(E)-feruloyl-2-O-beta-D-glucopyranosyl]-(1-->2)-beta-D-glucopyranoside]-5-O-(beta-D-glucopyranoside), pelargonidin 3-O-[6-O-(E)-caffeoyl-2-O-(6-(E)-feruloyl-beta-D-glucopyranosyl)-(1-->2)-beta-D-glucopyranoside]-5-O-(beta-D-glucopyranoside), pelargonidin 3-O-[6-O-(E)-p-coumaroyl-2-O-(6-(E)-caffeoyl-beta-D-glucopyranosyl)-(1-->2)-beta-D-glucopyranoside]-5-O-(beta-D-glucopyranoside), pelargonidin 3-O-[6-O-(E)-feruloyl-2-O-(6-(E)-caffeoyl-beta-D-glucopyranosyl)-(1-->2)-beta-D-glucopyranoside]-5-O-(beta-D-glucopyranoside), pelargonidin 3-O-[6-O-(E)-p-coumaroyl-2-O-(6-(E)-feruloyl-beta-D-glucopyranosyl)-(1-->2)-beta-D-glucopyranoside]-5-O-(beta-D-glucopyranoside), and pelargonidin 3-O-[6-O-(E)-feruloyl-2-O-(2-(E)-feruloyl-beta-D-glucopyranosyl)-(1-->2)-beta-D-glucopyranoside]-5-O-(beta-D-glucopyranoside).     

Rapid identification of pickle yeasts by fluorescent PCR and microtemperature-gradient gel electrophoresis

Monitoring the yeast populations within pickle soaking fluid is imperative for ensuring optimum taste, but these analyses have proven time-consuming and expensive, limiting their industrial application. Here, yeasts were identified in the soaking fluid from Japanese radish pickles using fluorescent PCR amplification of the variable D1/D2 region of the 26S rDNA, followed by analysis with microtemperature-gradient gel electrophoresis (micro-TGGE). This smaller version of the normal TGGE apparatus is capable of analyzing samples 10- to 20-fold faster without sacrificing data quality. Each primer set was labeled with a different fluorescent dye, allowing easy isolation of the various PCR products and identification of the bands corresponding to the various yeasts. The results indicate that fluorescent PCR and micro-TGGE may be a useful new method for rapid, easy monitoring of yeast flora in various food industries. This new method can be used on a daily basis to provide overviews of yeast flora during pickle production, allowing producers to quickly grasp pickle readiness at a single glance.     

Protective role of exogenous spermidine against paraquat toxicity in radish chloroplasts

When radish chloroplasts were pretreated with 1 mM spermidine (Spd) and then exposed to 30 M paraquat (PQ), they improved their tolerance to subsequent PQ-induced oxidative damages. That included the decreases in the contents of chlorophyll, protein, and ascorbate, as well as the increases in malondialdehyde (MDA) and H₂O₂ levels. Analysis of antioxidant enzymes showed that Spd pretreatment effectively prevented the PQ-induced decreases in the total activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX). In contrast, the normally enhanced activities of dehydroascorbate reductase (DHAR) and glutathione reductase (GR) in PQ-treated chloroplasts were reversed by Spd pretreatment In a native gel assay, the Cu/ZnSOD isozyme, which disappeared under the PQ alone treatment, was significantly recovered when tissues were pretreated with Spd. The dominant APX4 isozyme activity, which was preferentially decreased in response to PQ alone treatment, was also strongly reactivated by earlier Spd exposure. Therefore, we suggest that Spd could play a substantial role in protecting the radish chloroplasts from PQ stress. Furthermore, the enhancement of the Cu/ZnSOD and APX4 isozymes by Spd pretreatment seems to be responsible for prevention of the PQ-induced decreases in the total activities of SOD and APX, thereby providing a tolerance to PQ toxicity.     

Toxic peptides in Frazer's fraction interact with the actin cytoskeleton and affect the targeting and function of intestinal proteins

Celiac disease (CD) is a multisystemic autoimmune inflammation of the intestinal tract induced by wheat gluten and related cereals in HLA-DQ2/8 positive individuals. An essential role in the pathogenesis of CD is played by a fraction of the peptic-tryptic digest of gluten, Frazer's Fraction (FF). Here, we investigate the effects of FF on the integrity of intestinal cells with particular emphasis on brush border membrane (BBM) components, their subsequent trafficking and endocytosis.Caco-2 cells were incubated with FF at different concentrations. Thereafter, several protein and lipid components of treated and untreated cells were analysed at the molecular, functional and cellular levels. The control employed tryptic-peptic digests of ovalbumin.Our results show that FF directly interacts with actin in an alternating manner eliciting substantial alterations in its integrity and extent in the BBM. These alterations lead to an impaired trafficking of SI to the apical membrane and reduction in its enzymatic function. ApN and DPPIV follow a transcytotic pathway and are only partly affected by FF. By contrast, the trafficking of LPH remains unaffected concomitant with its actin-independent trafficking pattern. Finally, the endocytic pathway is substantially blocked in FF-treated cells leading to an accumulation of cholesterol, and sphingolipids in the BBM.We conclude that FF deteriorates the actin cytoskeleton in Caco-2 leading to reduced protein sorting and hampered endocytic events with subsequent alterations in the protein and lipid composition of the BBM. The reduced levels of the disaccharidase SI in the BBM suggest a potential pathomechanism of carbohydrate malabsorption in CD.     

Differential expression of two SOD (Superoxide dismytase) genes from small radish (Rhaphanus sativus L. var.sativus)

We have isolated two Superoxide dismutase cDNA clones (RsCu/ZnSod andRsFeSod) from small radish (Raphanus sativus L.) by cDNA library screening.RsCu/ZnSod is 563 bp long, with an open reading frame of 153 amino acids, and corresponds to a protein of predicted molecular mass 15.1 kDa and a pl of 5.44. The 823-bp RsFeSod has an ORF of 213 amino acids, corresponding to a protein of predicted molecular mass 25.4 kDa and a pl of 8.77. Their nucleotide and deduced amino acid sequences show the highest homology with those ofArabidopsis. Genomic Southern blot analysis, using each cDNA clone as probe, has revealed that the SOD genes are present as at least two copies in the small radish genome. Nondenaturing polyacrylamide gels for SOD activity has demonstrated the presence of several isozymes, depending on the organ type and developmental stage. TheseRsSod genes also have differential expression patterns in response to treatments with white light, xenobiotics, UV, osmoticums, plant hormones, and salicylic acid. Therefore, we suggest that they are involved in an antioxidative defense mechanism against stress induced by environmental change.     

萝卜基因组DNA的RAMP-PCR体系优化

以萝卜为材料,对基因组DNA的RAMP分析体系中的Mg²⁺、dNTPs和引物浓度进行优化。分别设计3个浓度梯度：Mg²⁺为 0.75、1.5、3.0 mmol·L^-1;dNTPs为 0.05、0.15、0.3 mmol·L^-1;引物为 0.065、0.2、0.4 μmol·L^-1,并对合适退火温度进行研究。筛选出的RAMP优化体系为(20 μL):dNTPs 0.15 mmol·L^-1,Mg²⁺ 1.5 mmol·L^-1,引物0.2～0.4 μmol·L^-1,DNA 10 ng,Taq E 0.8U;PCR扩增程序为94℃ 3 min,94℃ 1 min,45℃ 1 min,72℃ 1.5 min,42个循环, 72℃ 8 min。运用此体系,进行引物组合筛选,并对7个萝卜品种的遗传多样性与品种鉴定进行RAMP标记分析。     

The role of the persistent fruit wall in seed water regulation in Raphanus raphanistrum (Brassicaceae)

Background and Aims

Dry fruits remain around the seeds at dispersal in a number of species, especially the Brassicaceae. Explanations for this vary, but usually involve mechanisms of innate dormancy. We speculate that, instead, a persistent fruit may give additional protection through control of dehydration, to species growing in arid or Mediterranean environments where water is sporadic.

Methods

X-rays and weight measurements were used to determine the extent to which Raphanus raphanistrum seeds within mature fruits imbibe water, and germination tests determined the roles of the fruit and seed coat in seed dormancy. Rates of water uptake and desiccation, and seedling emergence were compared with and without the fruit. Finally, germinability of seeds extracted from fruits was determined after various periods of moist conditions followed by a range of dry conditions.

Key Results

Most seeds rapidly take up water within the fruit, but they do not fully imbibe when compared with naked seeds. The seed coat is more important than the dry fruit wall in maintaining seed dormancy. The presence of a dry fruit slows emergence from the soil by up to 6–8 weeks. The fruit slows the rate of desiccation of the seed to a limited extent. The presence of the fruit for a few days during imbibition somehow primes more seeds to germinate than if the fruit is absent; longer moist periods within the pod appear to induce dormancy.

Conclusions

The fruit certainly modifies the seed environment as external conditions change between wet and dry, but not to a great extent. The major role seems to be: (a) the physical restriction of imbibition and germination; and (b) the release and then re-imposition of dormancy within the seed. The ecological significance of the results requires more research under field conditions.     

萝卜地方优良品种高制种潜力雄性不育系转育方法研究

不育系的制种潜力是决定制种产量的重要因素。在前期选育过程中对不育系制种潜力关注不够,往往导致所选育的品种制种产量低甚至无法制种,造成品种无法推广。我国拥有丰富的优良地方品种萝卜资源,但是存在着种质退化、栽培面积缩减、栽培区域小而分散的问题。培育杂交种是提升优良地方品种品质、维持栽培面积和扩大栽培地域的有效途径。本研究将Ogura胞质转育到26份优良地方品种萝卜资源,发现其中16份种质资源在Ogura胞质中的不育率达到或接近100%;建立了一种基于网纱隔离和壁蜂授粉的鉴定方法,用于分析这16份优良地方品种萝卜资源的保持系及其Ogura不育系的制种产量与潜力,发现练丝萝卜和五月红两份资源的不育系和保持系都具有较高的角果产量,有望转育成高制种潜力的不育系亲本,红灯笼、大青皮和心里美3份资源中都分离出具有较高种子产量的胞质雄性不育单株,但是其保持系种子产量很低,需要提高保持系的种子产量;小红袍水萝卜和扶沟萝卜的不育系和保持系的种子产量都处于中等水平,具有优化和应用的潜力;红水萝卜、青皮脆等5份资源不适合Ogura胞质不育系制种;狗头罐、绛县白、象牙白3份资源是自交不亲和材料。本研究发现,Ogura胞质的不同品种以及同一品种不同单株间制种产量差异巨大,说明从转育初代开始逐代监测和筛选制种潜力是非常必要的。本研究介绍一种简易高效鉴定及筛选萝卜胞质不育系制种潜力的方法,并用此方法初步筛选出一批高制种潜力的萝卜地方品种Ogura胞质雄性不育材料。