Isolation and characterization of a novel thermostable non-specific lipid transfer protein-like antimicrobial protein from motherwort (Leonurus japonicus Houtt) seeds

In screening for potent antimicrobial proteins from plant seeds, a novel heat-stable antimicrobial protein, designated LJAMP2, was purified from seeds of the motherwort (Leonurus japonicus Houtt), a medicine herb, with a procedure involving cation exchange chromatography on a CM FF column, and reverse phase HPLCs on C8 column and C18 column. LJAMP2 exhibited a molecular mass of 6.2 kDa determined. Automated Edman degradation determined the partial N-terminal sequence of LJAMP2 to be NH2-AIGCNTVASKMAPCLPYVTGKGPLGGCCGGVKGLIDAARTTPDRQAVCNCLKTLAKSYSG, which displays homology with plant non-specific lipid transfer proteins (nsLTPs). In vitro bioassays showed that LJAMP2 inhibits the growth of a variety of microbes, including filamentous fungi, bacteria and yeast. The growth of three phytopathogenic fungi, Alternaria brassicae, Botrytis maydis, and Rhizoctonia cerealis, are inhibited at 7.5 μM of LJAMP2, whereas Bacillus subtilis is about 15 μM. The IC₅₀ of LJAMP2 for Aspergillus niger, B. maydis, Fusarium oxysporum, Penicillium digitatum and Saccharomyces cerevisiae are 5.5, 6.1, 9.3, 40.0, and 76.0 μM, respectively.     

Suitable water temperature for seed storage of Zostera japonica for subtropical seagrass bed restoration

Restoration of Zostera japonica is needed. Laboratory culture experiments to know the germination characteristics might be helpful for implementation of actual restoration. As a part of germination experiments, we explored suitable water temperature for long-term storage of Z. japonica seeds. This work was based on earlier reports of Zostera marina, which presumably has similar physiological properties to Z. japonica. This study consisted of two experiments: (1) preservation experiments to investigate the fate of stored Z. japonica seeds and (2) germination experiments to investigate the germination potential of the stored seeds. The results of the preservation experiments suggested that seed condition, that is, germinated, degraded, unstable, stable, etc., showed variations between the seeds stored at 4 and 23 °C. The majority of the seeds stored at 4 °C were germinated, while those at 23 °C seemed to be degraded, presumably by bacteria and mold. The germination experiments suggested high germination potential of seeds stored at 4 °C even after 302 days had elapsed. In conclusion, including previously reported results on Z. marina, low temperature was suitable for the preservation of seeds to maintain germination potential.     

Somatic embryo and root regeneration from quince leaves cultured in ventilated vessels or under different oxygen and carbon dioxide levels

Summary The influence of the gaseous composition of the atmosphere inside culturing vessels on somatic embryogenesis and on adventitious root formation was investigated in the quince clone (Cydonia ablonga Mill.) BA29. Leaves taken from in vitro-grown shoots were cultured in glass Petri dishes and exposed to ventilation with atmospheric air (flow rate 25 ml min⁻¹) for 0, 5, 10, 20, and 40 d. Twenty days of ventilation reduced the frequency of embryogenic leaves and a further decrease was observed after 40d of treatment. Conversely, adventitious root formation in the ventilated dishes was never different from the untreated cultures. In a second test, leaves were incubated in atmospheres containing different levels of oxygen (0, 5.0, 10.0, and 21.0%) or carbon dioxide (0, 0.04, 0.15, 1.5, and 3.0%). Anoxia conditions almost completely inhibited somatic embryo and adventitious root formation, but without compromising callus formation and explant viability. In contrast, embryo and root regeneration occurred even in totally CO₂-free atmosphere. Oxygen seemed to influence somatic embryogenesis according to a quadratic response; a similar relationship was also observed for root regeneration. Instead, no clear trend could be inferred between embryo or root regeneration and CO₂ levels. Furthermore, in dishes flushed with gas mixtures containing oxygen or carbon dioxide somatic embryo formation was almost always lower than in confined dishes. A different result was observed for root regeneration, since the number of roots was never lower than in the control and increased appreciably with 3.0% CO₂. These results demonstrate that atmosphere composition of the culture head-space can influence somatic embryogenesis in quince. The finding that both vessel ventilation and atmosphere replacement with different gas mixtures reduced somatic embryo formation suggests that gaseous compounds, different from O₂ an CO₂, present in the gaseous environment may promote embryogenesis in this species.     

Purification, structure and immunobiological activity of an arabinan-rich pectic polysaccharide from the cell walls of Prunus dulcis seeds

The structure and bioactivity of a polysaccharide extracted and purified from a 4M KOH + H3BO3 solution from Prunus dulcis seed cell wall material was studied. Anion-exchange chromatography of the crude extract yielded two sugar-rich fractions: one neutral (A), the other acidic (E). These fractions contain a very similar monosaccharide composition: 5:2:1 for arabinose, uronic acids and xylose, respectively, rhamnose and galactose being present in smaller amounts. As estimated by size-exclusion chromatography, the acidic fraction had an apparent molecular mass of 762 kDa. Methylation analysis (from the crude and fractions A and E), suggests that the polysaccharide is an arabinan-rich pectin. In all cases, the polysaccharides bear the same type of structural Ara moieties with highly branched arabinan-rich pectic polysaccharides. The average relative proportions of the arabinosyl linkages is 3:2:1:1 for T-Araf:(1-->5)-Araf:(1-->3,5)-Araf:(1-->2,3,5)-Araf. The crude polysaccharide extract and fractions A and E induced a murine lymphocyte stimulatory effect, as evaluated by the in vitro and in vivo expression of lymphocyte activation markers and spleen mononuclear cells culture proliferation. The lymphocyte stimulatory effect was stronger on B- than on T-cells. No evidence of cytotoxic effects induced by the polysaccharide fractions was found.     

受白粉菌诱导大麦抗感等基因系蛋白质变化的双向电泳分析

分别对接种与否的大麦抗—感白粉病等基因系—叶期幼苗取材进行蛋白质双向电泳分析。结果表明,病原的侵入使抗—感两系在30Kd以下的低分子量区域的蛋白质发生了明显变化。接种48小时之后,抗病系在pH5.5、6.0、6.8及8.8附近出现了对照中所没有的蛋白质,而在pH6.0和8.8附近的蛋白质则较对照有减小的趋势;感病系在pH6.0附近蛋白质明显增多,在pH8.8处不仅在量上有大幅度提高,而且种类也有增加。结果还表明,抗—感系间在未接种的情况下双向电泳图谱也有差异,接种之后由于感病系在pH8.8处蛋白质的特异性合成,使抗—感两系间的差异缩小。     

Repellent efficacy of essential oils and plant extracts against Tribolium castaneum and Plodia interpunctella

This study was conducted to investigate the repellent efficacy of essential oils (Origanum vulgare, Pimpinella anisum, and Tanacetum cinerariifolium) and four plant extracts (Agastache rugosa, Capsicum annuum, Citrus reticulata, and Ginkgo biloba) against Tribolium castaneum (adults and larvae) and Plodia interpunctella (larvae). Gas chromatography/mass spectrometry analysis revealed the presence of carvacrol, anethole, and jasmolin I as the predominant constituent in O. vulgare, P. anisum, and T. cinerariifolium, respectively. Furthermore, ethyl hexopyranoside, 9,12‐octadecadienoic acid, cyclopentanol, and 2‐cresol were identified in A. rugosa, C. annuum, C. reticulata, and G. biloba, respectively. The repellent efficacy of each essential oil, plant extract, and the combination of oils was evaluated using a specially designed cylinder trap for 120 h. Among the three oils, O. vulgare and T. cinerariifolium had greatest repellent efficacy against P. interpunctella larvae. T. cinerariifolium exhibited effective repellence against the adults and larvae of T. castaneum. Therefore, O. vulgare (O) and T. cinerariifolium (T) were selected for further investigation of combined effects. Two essential oils were mixed in three different ratios of OT1 (1:3), OT2 (1:1), and OT3 (3:1). The repellent efficacies of OT1 and OT2 against the adults of T. castaneum were significantly greater than that of OT3. OT1 was effective against the larvae of T. castaneum, whereas OT2 was effective against the larvae of P. interpunctella. OT1 enhanced the repellent efficacy by approximately five times against larvae of T. castaneum, compared with that of T. cinerariifolium. Overall, OT1 was selected as the best repellent substance against all the tested insects.     

A glucose-tolerant β-glucosidase from Prunus domestica seeds: Purification and characterization

A glucose-tolerant β-glucosidase was purified to homogeneity from prune (Prunus domestica) seeds by successive ammonium sulfate precipitation, hydrophobic interaction chromatography and anion-exchange chromatography. The molecular mass of the enzyme was estimated to be 61 kDa by SDS-PAGE and 54 kDa by gel permeation chromatography. The enzyme has a pI of 5.0 by isoelectric focusing and an optimum activity at pH 5.5 and 55 °C. It is stable at temperatures up to 45 °C and in a broad pH range. Its activity was completely inhibited by 5 mM of Ag⁺ and Hg²⁺. The enzyme hydrolyzed both p-nitrophenyl β-d-glucopyranoside with a K_m of 3.09 mM and a V_max of 122.1 μmol/min mg and p-nitrophenyl β-d-fucopyranoside with a K_m of 1.65 mM and a V_max of 217.6 μmol/min mg, while cellobiose was not a substrate. Glucono-δ-lactone and glucose competitively inhibited the enzyme with K_i values of 0.033 and 468 mM, respectively.     

Inhibition of mugineic acid-ferric complex uptake in barley by copper, zinc and cobalt

The interfering effects of copper, zinc, and cobalt on the uptake of mugineic acid-ferric complex were studied in barley ( Hordeum vulgare , cv. Minorimugi) grown in nutrient solution. Short-term uptake experiments of 3 h were performed utilizing both ionic and mugineic acid-complex forms of each metal at two different concentrations. Copper was most effective in decreasing iron uptake when added in an ionic form at either concentration. The inhibition order at higher concentrations followed Cu(II) > Zn(II) ≥ Co(II), Co(III), which is consistent with the stability constants of these metal complexes with mugineic acid. The displacement of iron from its mugineic acid complex by these metals is suggested as a probable explanation for the decreased iron uptake. The inhibitory effect of metal complexes with mugineic acid on iron uptake was only found in cases with higher concentrations of Cu(II) and Zn(II) complexes. Deformation of the specific iron transport system in the plasma membrane due to their adsorption may be responsible for this effect.     

Contemporary seasonal and altitudinal variations of leaf structural features in oregano (Origanum vulgare L.)

The effects of elevation (200, 950 and 1760 m) and season (April-October) on leaf morphological, anatomical, ultrastructural, morphometrical and photosynthetic parameters were studied in Origanum vulgare plants. Observations aimed at the determination of the alterations in leaf structure and function associated with differential growth and adaptation of plants. Raising elevation results in a progressive decrease of plant height. During the growing period, summer plants are taller than spring and autumn plants at all elevations examined. In high-altitude populations (O. vulgare ssp. vulgare), the blade size becomes reduced in June leaves as compared with October leaves, while it does not change remarkably in low-altitude populations (O. vulgare ssp. hirtum). Leaf thickness remains more or less stable during the growing period. Expanded leaves in June and October at 200 m elevation contain dark phenolics only in their epidermis, whereas leaves of August are densely filled with phenolics in all of their tissues. In June at 1760 m elevation, leaves are devoid of phenolics, which, however, occur in the epidermis of the leaves in August and October. At higher altitudes, larger mesophyll chloroplasts with more starch grains are present in June leaves, whereas in August and October leaves chloroplasts are smaller with fewer starch grains. Leaf stomata and non-glandular hairs increase in number from the lowland to the upland habitats, whereas glandular hairs decrease in number. During the growing season, the density of stomata and of glandular and non-glandular hairs progressively increases. In the low- and mid-altitude oregano populations, leaf chlorophyll a content and PSII activity significantly increase in October, whereas they simultaneously decrease in the high-altitude population, suggesting a phenomenon of chilling-induced photoinhibition. The highest photochemical efficiency of PSII appears in the mid-altitude population (having characteristics intermediate between those of O. vulgare ssp. hirtum and ssp. vulgare) where environmental conditions are more favourable. This conclusion is also confirmed by the observation that the 950 m O. vulgare population has larger and thicker leaves with highly developed palisade and spongy parenchymas.     

青阳参种子的萌发

青阳参（Cynanchum otophyllum）种子在11月成熟时有休眠习性。收获后将其种子种植在自然温室内,到第二年的春天种子才会萌发,且大多数种子在3月28至4月4日间萌发,这期间的日平均最高和最低温度分别为19．0℃和9．9℃。层积能有效地打破青阳参种子的休眠,休眠种子通过大约1周的层积便能萌发。种子在有光的条件下层积1周后转移到25／15℃的黑暗条件下萌发率可达到75．4％。青阳参种子不论在有光的条件下还是在黑暗环境中层积2～3周后转入30／20和25／15℃进行变温处理,其萌发率最低能达到66．4％,而转入20／10℃变温处理其萌发率最多只能达到20．1％,但若层积6周,即便在20／10℃变温处理的情况下其萌发率也可以达到65．3％以上。