Reptilian transferrins: evolution of disulphide bridges and conservation of iron-binding center

Transferrins, found in invertebrates and vertebrates, form a physiologically important family of proteins playing a major role in iron acquisition and transport, defense against microbial pathogens, growth and differentiation. These proteins are bilobal in structure and each lobe is composed of two domains divided by a cleft harboring an iron atom. Vertebrate transferrins comprise of serotransferrins, lactoferrins and ovotransferrins. In mammals serotransferrins transport iron in physiological fluids and deliver it to cells, while lactoferrins scavenge iron, limiting its availability to invading microbes. In oviparous vertebrates there is only one transferrin gene, expressed either in the liver to be delivered to physiological fluids as serotransferrin, or in the oviduct with a final localization in egg white as ovotransferrin. Being products of one gene sero- and ovotransferrin are identical at the amino-acid sequence level but with different, cell specific glycosylation patterns. Our knowledge of the mechanisms of transferrin iron binding and release is based on sequence and structural data obtained for human serotransferrin and hen and duck ovotransferrins. No sequence information about other ovotransferrins was available until our recent publication of turkey, ostrich, and red-eared turtle (TtrF) ovotransferrin mRNA sequences [Ciuraszkiewicz, J., Olczak, M., Watorek, W., 2006. Isolation, cloning and sequencing of transferrins from red-eared turtle, African ostrich and turkey. Comp. Biochem. Physiol. 143 B, 301-310]. In the present paper, ten new reptilian mRNA transferrin sequences obtained from the Nile crocodile (NtrF), bearded dragon (BtrF), Cuban brown anole (AtrF), veiled and Mediterranean chameleons (VtrF and KtrF), sand lizard (StrF), leopard gecko (LtrF), Burmese python (PtrF), African house snake (HtrF), and grass snake (GtrF) are presented and analyzed. Nile crocodile and red-eared turtle transferrins have a disulphide bridge pattern identical to known bird homologues. A partially different disulphide bridge pattern was found in the Squamata (snakes and lizards). The possibility of a unique interdomain disulphide bridge was predicted for LtrF. Differences were found in iron-binding centers from those of previously known transferrins. Substitutions were found in the iron-chelating residues of StrF and TtrF and in the synergistic anion-binding residues of NtrF. In snakes, the transferrin (PtrF, HtrF and GtrF) N-lobe "dilysine trigger" occurring in all other known transferrins was not found, which indicates a different mechanism of iron release.     

24份甜樱桃材料自交不亲和S基因型鉴定

甜樱桃品种绝大部分自交不亲和,限制了甜樱桃的正确评价和合理利用,因此自交不亲和基因型的鉴定对于生产具有重要意义。以24个甜樱桃主栽品种为材料,用5对蔷薇科李属引物组合对24个甜樱桃品种进行了S等位基因的PCR扩增,克隆S基因的扩增片段,用核酸序列在Gen Bank上搜索,确定了5种S基因的核酸序列和大小。结果表明:Pru C2+Pru C4R引物组合扩增效果最好;在琼脂糖凝胶上位置相同的扩增带其核酸序列相同,是同一种S基因;5种S基因扩增片段的大小分别是S1为800 bp,S3为762 bp,S4为962bp,S5为300 bp,S6为456 bp,S9为650 bp;24个甜樱桃S基因型是红手球、早红宝石为S1S3,拉宾斯S1S4',红宝石S1S6,布鲁克斯S1S9,那翁S3S4,秦林、泰安大紫、先锋、早大果、丽珠、美早、5-106、左滕锦、桑提娜为S3S6,黑珍珠、红灯、萨米脱、秦樱为S3S9,胜利为S5S9,明珠、红蜜、雷尼、滨库为S6S9。     

A comparative histological study between normal and fasciated shoots of <Emphasis Type="Italic">Prunus avium</Emphasis> generated <Emphasis Type="Italic">in vitro</Emphasis>

A combination of Murashige and Skoogs medium and N⁶–benzyladenine (BA) at various concentrations (0, 0.1, 0.25, 0.5, 0.75, 1.0, 1.25 and 1.5 mg l^–1) was supplied to shoot tips from root cuttings of a 50-year-old wild-cherry tree (Prunus avium). The concentration of BA in the growing medium was a determining factor with respect to the number of proliferated shoots per explant in vitro.Normal and fasciated shoots were generated when BA was present at 0.5, 0.75, 1.0 and 1.25 mg l^–1 in the medium and the mean numbers of normal shoots per explant were 3.63, 5.37, 8.93 and 7.30 respectively, and those of the fasciated shoots per explant were 0.03, 0.1, 0.47 and 0.4 respectively. Anatomical analysis by confocal microscopy of sections of paraffin-embedded specimens revealed that the cell structure and organization of the cortex and vascular cylinder in the fasciated shoots was similar to that in normal shoots. However, the cross-sectional area of stem of the fasciations was apparently greater than that of the normal shoots. In particular, the volume of vascular tissues, of pith and of some individual parenchyma cells in the cortex and pith was apparently greater in fasciated shoots than in normal shoots. Increases in cytokinesis and morphogenetic activity, such as the development of callus-like regions and the formation of adventitious shoots, were observed in the cortex and pith throughout the fasciations. The fasciated shoots had numerous buds and initiating new shoots at their apices while normal shoots had a single dominant axial bud.     

Transformation of Montmorency sour cherry (Prunus cerasus L.) and Gisela 6 (P. cerasus × P. canescens) cherry rootstock mediated by Agrobacterium tumefaciens

Sour cherry (Prunus cerasus L.) scion cv. Montmorency and rootstock cv. Gisela 6 (P. cerasus x P. canescens) were transformed using Agrobacterium tumefaciens strain EHA105:pBISN1 carrying the neomycin phosphotransferase gene (nptII) and an intron interrupted ss-glucuronidase (GUS) reporter gene (gusA). Whole leaf explants were co-cultivated with A. tumefaciens, and selection and regeneration of transformed cells and shoots of both cultivars was carried out for 12 weeks on selection medium containing 50 mg l(-1) kanamycin (Km) and 250 mg l(-1) timentin. These media were [Quoirin and Lepoivre (Acta Hortic 78:437-442, 1977)] supplemented with 0.5 mg l(-1) benzylaminopurine (BA) + 0.05 mg l(-1) indole-3-butyric acid (IBA), and woody plant medium [Lloyd and McCown (Proc Int Plant Prop Soc 30:421-427, 1980)] containing 2.0 mg l(-1) BA + 1.0 mg l(-1) IBA for cv. Montmorency and cv. Gisela 6, respectively. Seven out of 226 (3.1%) explants of cv. Montmorency and five out of 152 (3.9%) explants of cv. Gisela 6 produced 30/39 GUS- and PCR-positive shoots from the cut midribs via an intermediate callus. Southern analysis of the GUS- and PCR-positive transformants confirmed stable integration of the transgenes with 1-3 copy numbers in the genomes of seven lines of cv. Montmorency and five of cv. Gisela 6. The selected transformants have a normal phenotype in vitro.     

Crystal macropattern development in Prunus serotina (Rosaceae, Prunoideae) leaves

BACKGROUND AND AIMS: Prunus, subgenus Padus, exhibits two completely different calcium oxalate crystal macropatterns in mature leaves. Foliar macropattern development has been described previously in P. virginiana, representing one version. Prunus serotina, in the group exhibiting the second macropattern, is described here. The goal was to describe developmental details for comparison with P. virginiana, and to extend the sparse current knowledge of crystal macropatterns. METHODS: Leaves at various developmental stages were removed from local trees and from herbarium specimens. Early leaf stages and freehand leaf and stem sections were mounted directly in aqueous glycerine; larger leaves were processed whole or in representative pieces in household bleach, dehydrated in alcohol/xylol, and mounted in Permount. Crystals were detected microscopically between crossed polarizers. KEY RESULTS: Bud scales have a dense druse population. Druses appear first at the stipule tip and proliferate basipetally but soon stop forming; growing stipules therefore have a declining density of druses. Druses appear at the tip of leaves <1 mm long, then proliferate basipetally in the midrib. Lamina druses appear in the distal marginal teeth of leaves 3 cm long; from here they proliferate basipetally and towards midrib along major veins. In about two-thirds-grown leaves (6-9 cm length) druses are all adaxial to veins of most orders; a shift occurs then to formation of prisms, which appear first abaxial to, then all around, veins. Mature leaves have virtually all prisms encrusting all major veins, more sparsely along smaller minor veins. Late season leaves form epitactic crystals on existing prismatics. CONCLUSIONS: The developing and mature macropattern of P. serotina is almost the reverse of the pattern described previously in P. virginiana, and shows that two closely related species can develop radically different modes of crystallization. The few detailed macropattern studies to date reveal striking variations that indicate a new level of organization that must be integrated with the anatomical, physiological and molecular approaches that have been dominant so far.     

Micropropagation of mature <Emphasis Type="Italic">Cornus mas</Emphasis> ‘Macrocarpa’

Sprouting axillary buds sampled from a mature 27-year-old shrub of Cornus mas ‘Macrocarpa’ were used as starting material for in vitro culture establishment. Multiple shoot cultures, grown on basal woody plant medium with the pH adjusted to 5.6–5.7 and supplemented with 6-benzylaminopurine in combination with 1-naphthaleneacetic acid, were capable of continuous axillary and adventitious shoot proliferation up to 1 year. Later on, growth ceased, shoot tip necrosis appeared and shoot cultures died. Transfer of living shoots onto modified woody plant medium with the pH adjusted to 6.8–7.0 led to vigorous growth of multiple shoot cultures without any loss of multiplication rates or decreased vitality for several years. The use of 6-benzylaminopurine in combination with 1-naphthaleneacetic acid proved superior to the application of thidiazuron which induced a frequent formation of short and fasciated shoots. 1-naphthaleneacetic acid promoted in vitro adventitious rooting frequency up to 73.3%, whereas indole-3-butyric acid was not effective. Ex vitro acclimatized plants did not show any visually detectable morphological variation.     

田间应用拮抗酵母菌对甜樱桃果实采后病害的防治效果

研究了应用拮抗酵母菌丝孢酵母(Trichosporon pullulans(Lindner.)Diddens et Lodder)、罗伦隐球酵母(Cryptococcus laurentii(Kuffer.)Skinner)和粘红酵母(Rhodotorula glutinis(Fresenius)Harrison)后拮抗菌在果实表面的繁殖能力以及对不同贮藏条件下甜樱桃(Pranus avivum L.cv.Hongdeng)果实采后病害的防治效果。酵母菌的使用浓度为1×10~8CFU/mL。结果表明,田间3种拮抗菌都能够在果实表面增值,但是只有C.laurentii和R.glutinis能够持续稳定地生长。C.laurentii的抑病效果最好,它对田间环境和采后低温低氧及高CO_2都具有很强的适应能力。     

Triacontanol-supported micropropagation of woody plants

 The effectiveness of triacontanol in the micropropagation of two woody, economically important fruit plant species was investigated. Triacontanol was added to the routine multiplication and rooting media of apple (Malus domestica cv. JTE-E4) and sour cherry (Cerasus fruticosa cv. Probocskai) rootstocks at concentrations of 2, 5, 10 and 20 μg/l. It was found to increase the number of shoots and the fresh weight of apple in the multiplication phase and to enhance root number and chlorophyll content in the rooting phase. The addition of indole-3-butyric acid (IBA) to the media further improved the effect of triacontanol. A less pronounced effect could be seen in the multiplication phase of sour cherry, although there was an enhancement of shoot proliferation. In the rooting phase, however, the application of triacontanol caused a significant increase in the number of roots per plant, and this effect was further improved when triacontanol was combined with 0.5 mg indole-3-butyric acid/l. Received: 29 March 2000 / Revision received: 1 September 2000 / Accepted: 4 September 2000