Purification and characterization of cyanogenic β-glucosidase from wild apricot (Prunus armeniaca L.)

β-Glucosidase catalyzes the sequential breakdown of cyanogenic glycosides in cyanogenic plants. The β-glucosidase from Prunus armeniaca L. was purified to 8-fold, and 20% yield was obtained, with a specific activity of 281 U/mg protein. The enzyme showed maximum activity in 0.15 M sodium citrate buffer, pH 6, at 35 °C with p-nitrophenylglucopyranoside as substrate. The β-glucosidase from wild apricot was used successfully for the saccharification of cellobiose into D-glucose. This enzyme has a V_max of 131.6 μmol min⁻¹ mg⁻¹ protein, K_m of 0.158 mM, K_cat of 144.8 s⁻¹, K_cat/K_m of 917.4 mM⁻¹ s⁻¹, and K_m/V_max of 0.0012 mM min mg μmole⁻¹, using cellobiose as substrate. The half-life, deactivation rate coefficient, and activation energy of this β-glucosidase were 12.76 h, 1.509 × 10⁻⁵ s⁻¹, and 37.55 kJ/mol, respectively. These results showed that P. armeniaca is a potential source of β-glucosidase, with high affinity and catalytic capability for the saccharification of cellulosic material.     

QTL analysis of resistance to sharka disease in the apricot (Prunus armeniaca L.) ‘Polonais’ × ‘Stark Early Orange’ F1 progeny

Different hypotheses on the genetic control of the resistance to the plum pox virus (PPV) have been reported in apricot, but there was a lack of agreement about the number of loci involved. In recent years, apricot genetic maps have been constructed from progenies derived from ‘Stark Early Orange’ or ‘Goldrich’, two main sources of resistance, three of these including the mapping of the PPV resistance loci. As the location of the locus was not precisely established, we mapped the PPV resistance loci using interval mapping (IM), composite interval mapping (CIM), and the Kruskal–Wallis non-parametric test in the F₁ progeny derived from a cross between the susceptible cv. ‘Polonais’ and ’Stark Early Orange’. Four genomic regions were identified as being involved in PPV resistance. One of these mapped to the upper region of linkage group 1 of ‘Stark Early Orange’, and accounted for 56% of the phenotypic variation. Its location was similar to the one previously identified in ‘Goldrich’ and Prunus davidiana. In addition, a gene strongly associated to these major quantitative trait loci (QTL) was found to be related to PPV infection. Two putative QTLs were detected on linkage groups 3 of ‘Polonais’ and 5 of both ‘Polonais’ and ‘Stark Early Orange’ with both parametric and non-parametric methods at logarithm of odds (LOD) scores slightly above the detection threshold. The last QTL was only detected in the early stage of the infection. PPV resistance is, thus, controlled by a major dominant factor located on linkage group 1. The hypothesis of recessive factors with lower effect is discussed.     

Micropropagation of sour cherry (Prunus cerasus L.) cv. Šumadinka

Sour cherry cv. umadinka (Prunus cerasus L.) is the leading Yugoslav cultivar for production orchards. A method of micropropagation has been developed for the purpose of growing umadinka on its own roots and for rapid multiplication.Aseptic cultures were initiated from shoot explants 1–2 mm long on Murashige & Skoog medium with (in mgl^-1) 6-benzylaminopurine (BAP): 1, indole-3-yl butyric acid (IBA): 1 and gibberellic acid (GA₃): 0–1.The best medium for proliferation was MS with (in mgl^-1): BAP 0.5, IBA 0.1, GA₃ 0.1, but media with (in mgl^-1): BAP 0.5, NAA 0.1, GA₃ 0.1 and BAP 1, NAA 0.1 and GA₃ 0.1 were also shown to be good. A higher degree of proliferation obtained with some media did not necessarily result in a better quality of plantlets produced.For rooting the best combination of culture medium was achieved with pretreatment 10 days in MS 1/2 with 1 mgl^-1 IBA, followed by transfer to a hormone-free medium after 5–10 days, resulting in 88% success.The rooted plants were planted in containers and acclimatized under mist, with over 90% of plants surviving transplantation.     

甜樱桃(Prunus avium L.)品种S基因型鉴定

根据蔷薇科S-RNase基因(S基因)高度保守区C2和RC4区设计一对特异引物PruC2和PruC4R,对甜樱桃品种的基因组DNA进行S基因特异PCR扩增。克隆S基因的扩增片段,核酸序列在GenBank上搜索,确定了4种S基因的核酸序列和大小。结果表明,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。4种S基因扩增片段的大小分别是：S1为677bp,S3为762bp,S4为945bp,S6为456bp。参试的自交不亲和品种的S基因型分别是：红灯、红艳、早红宝石和先锋相同,为S1S3;抉择、红丰和那翁相同,为S3S4;大紫为S1S6;长把红为S1S4;养老为S2S6;自交亲和品种外引7号和斯太拉为S3S4。     

Decay of cultivated apricot wood (Prunus armeniaca) by the ascomycete Hypocrea sulphurea,using solid state 13C NMR and off-line TMAH thermochemolysis with GC–MS

Chemical changes of polysaccharides and lignin in Prunus armeniaca decayed by the ascomycete fungus Hypocrea sulphurea were investigated. Solid-state ¹³C NMR spectra showed that polysaccharides were the main components of fresh and decayed wood. Decomposition of cellulose and hemicellulose by the fungus was minimal although a slight preference for crystalline as compared with amorphous cellulose was recognised. Comparison of the signal intensity of the resonance at 145 ppm in fresh and decomposed wood suggested that the fungus had decomposed tannin constituents. Thermochemolysis with tetramethylammonium hydroxide (TMAH) and product identification by gas chromatography–mass spectrometry revealed that the ratio of syringyl-type (S) units to guaiacyl-type (G) units decreased from 1.8 to 1.1 following fungal attack. Increases in both guaiacyl and syringyl acid-aldehyde ratios (Ad/Al)_G, (Ad/Al)_S together with an increase from 0.82 to 3.54 in the ratio of methyl 3,4-dimethoxybenzoate to the sum of 1-(3,4-dimethoxyphenyl)-1,2,3-trimethoxypropane (threo and erythro-isomers) Γ_G from the decayed wood confirmed oxidative Cα–Cβ cleavage for the mechanism of lignin decay by this ascomycete.     

The effects of external potassium and magnesium concentrations on the magnesium and potassium inflow rates and growth of micropropagated cherry rootstocks, “F.12/1” (Prunus avium L.) and “Colt” (Prunus avium L.) × Prunus pseudocerasus L.)

The effects (and interaction) of two solution concentrations of Mg (50, 500, μM) and two of K (250, 4250 μM) on the growth of micropropagated plants of “F. 12/1” and “Colt” were investigated using a flowing solution culture system. Magnesium inflow and growth of “Colt” and “F. 12/1” were inhibited to a similar extent by an increased concentration of K in the nutrient solution. However, the consequences of this inhibition were different. Reduced inflow of Mg in “F. 12/1” caused Mg deficiency symptoms at high and low concentrations of K, whereas this only occurred with a combination of high K concentration and low Mg concentration in “Colt”. The distribution of dry matter within the plant was significant in determining susceptibility to Mg deficiency. Since “F. 12/1” has a smaller root:shoot ratio than Colt it is unable to sustain the same concentration of Mg in leaves as “Colt” irrespective of external K concentration. The molar ratio of K:Mg in soil solutions should remain <8.5:1 in order to ensure maximum growth of “F. 12/1” and “Colt”. This revised version was published online in June 2006 with corrections to the Cover Date.     

鼠类对山杏(Prunus armeniaca)种子扩散及存活作用研究

虽然有关鼠类搬运森林种子的证据已很清楚,但这些被移走种子的存活情况却知之甚少.提出了一个新的标记和跟踪种子的方法--标签法,即将种子拴一带有编码的细长金属片,研究了北京东灵山地区山杏(Prunus armeniaca)种子的扩散距离和存活率.于1998年6月19～20日,7月3日和10月23日共在24个样点释放1440粒山杏种子.几乎所有释放的种子在10d内被鼠类取走.夏天释放的种子比秋天释放的种子消失的速度快.大多数种子的扩散距离在20m以内,小于鼠类的活动距离.鼠类吃掉种子的速度很快,但当种子变得稀少时,种子存活率有所提高.山杏种子6、7月份的每日存活率小于其它月份的每日存活率.     

Changes in behaviour associated with pair formation in the polychaete Harmothoë imbricata (L.)

Spacing between individuals in populations of Harmothoë imbricata has been investigated both on the shore and in the laboratory. Males tend to occur closer together than females, and the mean male‐male individual distance measured was less than the mean distance between females; male—female distances for immature worms were intermediate. When worms mature they pair: the male mounts the female and lies across her dorsal surface. There is evidence that after spawning the members of a pair separate. Contact responses between worms have been investigated in the laboratory. Most encounters between immature worms lead to separation, as a result of one or both worms moving rapidly away from the other or fighting; females show more marked avoidance behaviour than males. The majority of male‐male and female‐female encounters between mature worms also lead to separation but in male‐female encounters the male usually mounts the female. A male which has mounted a female becomes highly aggressive and will attack intruding males but not females.     

Structural mapping of Kelch13 mutations associated with artemisinin resistance in malaria

Mutations in Plasmodium falciparum gene kelch13 (pfkelch13) are strongly and causally associated with resistance to anti-malarial drug artemisinin, but their effects on PfKelch13 structure and function remain unclear. Utilizing the publicly available three-dimensional structure of PfKech13 (PDB ID: 4yy8), we find that most of the mutations in its propeller domain occur in two spatial clusters. Of these, one cluster is enriched in surface exposed residues which may drive PfKelch13-centered protein interactions, and the second cluster mostly contains residues which are buried and whose mutations may destabilize PfKelch13 structure. The most prevalent resistant mutations C580Y and Y493H are distal from the above two clusters. The C580Y mutation creates sterically unfavourable contacts while Y493H possibly alters the hydrophobic core of the propeller domain. These analyses will facilitate further experimental studies aimed at understanding how mutations in pfkelch13 lead to artemisinin resistance.     

Inheritance of Organelle DNA in Rye (Secale cereale L.) with Triticale (×Triticale Thch.) Hybrids

The mode of inheritance of chloroplast and mitochondrial DNA (mtDNA) in rye × triticale intergeneric hybrids has been studied with the use of specific PCR markers for loci 18S/5S and 3rbcL in organelle DNA. In rye × triticale BC₁, mtDNA copies of two types, paternal and maternal, have been found; in BC₂ plants, only paternal mtDNA and chloroplast DNA (cpDNA) have been detected. Mechanisms determining the inheritance and/or differential amplification of organelles of a specific type are discussed.     

Radiation-induced mutations from accessory buds of sweet cherry, Prunus avium L. cv ‘Bing’

 Dormant scions of ‘Bing’ were exposed to 1–2.5 krad of gamma radiation in order to induce useful mutations. The main buds were excised and the scions grafted to allow the growth of accessory buds into primary (V1) shoots. The frequency and types of mutations on secondary (V2) populations are described. In a population of 3324 V2 shoots, the overall mutation frequency was 6.4%: 4.2% partial, 1.6% total and 0.3% growth-reduced mutants were identified. The experiment was repeated using 3 krad- and 4 krad-fractionated doses in water. Differences in mutation frequency at 3 krad and 4 krad were not significant. Of 2562 surviving V2 shoots derived from the irradiation of accessory buds of both standard and V1 shoots, the overall mutation frequency was 3.3%: 1.7% were partial-leaf mutants, 1.0% were total-leaf mutants, and 0.54% were growth-reduced mutants. For maximum mutation rate with adequate survival we suggest acute irradiation of accessory buds in air at dosages approximating LD₅₀ (2.75–3 krad). A larger mutant sector was present in V₁ shoots derived from accessory buds than those from main buds as revealed by the higher number of total mutant repeats in the families. Received: 21 August 1997 / Accepted: 17 November 1997     

Expression of the pea (Pisum sativum L.) α-tubulin gene TubA1 is correlated with cell division activity

Microtubules are thought to be major determinants of plant morphogenesis, through effects on planes of cell division and on directions of differential cell expansion. In differentiation and redifferentiation processes, tubulin expression may prove a useful early indicator of cell activity. We examined the expression and localization of the pea -tubulin gene TubA1 in situ and in transgenic alfalfa (Medicago sativa) to explore its use as a probe for plant development, and as a test case for correct developmental expression between two legume species commonly compared for studies of symbiosis with Rhizobium. The TubA1 mRNA was more abundant in root tips and immature leaves than in other tissues of pea. The promoter of TubA1 was fused to -glucuronidase (GUS) to analyze -tubulin expression in transgenic alfalfa. Transient assays indicated that the TubA1 gene is transcribed at moderate levels compared to the cauliflower mosaic virus (CaMV) 35S promoter. Histochemical staining for GUS activity confirmed a correlation between TubA1 expression and cell division in nodules, roots and leaves. TubA1 promoter activity was first detected in the inner cortex of the root between 18 h and 24 h after spot inoculation with Rhizobium meliloti. Expression of a c-myc epitope fused to the carboxy-terminus of TubA1 resulted in an incorporation into the microtubular cytoskeleton, demonstrating the effectiveness of at least one epitope tag in creating functional tubulin fusions.     

Antibacterial activity of 3,3′-dihydroxycurcumin (DHC) is associated with membrane perturbation

Curcumin is a plant diphenylheptanoid and has been investigated for its antibacterial activity. However, the therapeutic uses of this compound are limited due to its chemical instability. In this work, we evaluated the antimicrobial activity of diphenylheptanoids derived from curcumin against Gram-positive and Gram-negative bacteria, and also against Mycobacterium tuberculosis in terms of MIC (Minimum Inhibitory Concentration) and MBC (Minimum Bactericidal Concentration) values. 3,3′-Dihydroxycurcumin (DHC) displayed activity against Enterococcus faecalis, Staphylococcus aureus and M. tuberculosis, demonstrating MIC values of 78 and 156 µg/mL. In addition, DHC was more stable than curcumin in acetate buffer (pH 5.0) and phosphate buffer (pH 7.4) for 24 h at 37 °C. We proposed that membrane and the cell division protein FtsZ could be the targets for DHC due to that fact that curcumin exhibits this mode of antibacterial action. Fluorescence microscopy of Bacillus subtilis stained with SYTO9 and propidium iodide fluorophores indicated that DHC has the ability to perturb the bacterial membrane. On the other hand, DHC showed a weak inhibition of the GTPase activity of B. subtilis FtsZ. Toxicity assay using human cells indicated that DHC has moderate capacity to reduce viability of liver cells (HepG2 line) and lung cells (MRC-5 and A549 lines) when compared with doxorubicin. Alkaline comet assay indicated that DHC was not able to induce DNA damage in A549 cell line. These results indicated that DHC is promising compound with antibacterial and antitubercular activities.     

Expression and ��-glucan binding properties of Scots pine (Pinus sylvestris L.) antimicrobial protein (Sp-AMP)

Scots pine (Pinus sylvestris) secretes a number of small, highly-related, disulfide-rich proteins (Sp-AMPs) in response to challenges with fungal pathogens such as Heterobasidion annosum, although their biological role has been unknown. Here, we examined the expression patterns of these genes, as well as the structure and function of the encoded proteins. Northern blots and quantitative real time PCR showed increased levels of expression that are sustained during the interactions of host trees with pathogens, but not non-pathogens, consistent with a function in conifer tree defenses. Furthermore, the genes were up-regulated after treatment with salicylic acid and an ethylene precursor, 1-aminocyclopropane-1-carboxylic-acid, but neither methyl jasmonate nor H(2)O(2) induced expression, indicating that Sp-AMP gene expression is independent of the jasmonic acid signaling pathways. The cDNA encoding one of the proteins was cloned and expressed in Pichia pastoris. The purified protein had antifungal activity against H. annosum, and caused morphological changes in its hyphae and spores. It was directly shown to bind soluble and insoluble β-(1,3)-glucans, specifically and with high affinity. Furthermore, addition of exogenous glucan is linked to higher levels of Sp-AMP expression in the conifer. Homology modeling and sequence comparisons suggest that a conserved patch on the surface of the globular Sp-AMP is a carbohydrate-binding site that can accommodate approximately four sugar units. We conclude that these proteins belong to a new family of antimicrobial proteins (PR-19) that are likely to act by binding the glucans that are a major component of fungal cell walls.