Endophytic Colonisation of Opium Poppy, Papaver somniferum, by an Entomopathogenic Beauveria bassiana Strain

Beauveria bassiana strain EABb 04/01-Tip isolated from stem-borer larvae of Timaspis papaveris (Hymenoptera: Cynipidae), a serious pest of opium poppy in Spain, was shown to be able to become established endophytically in this pharmaceutical crop. Microbiological, molecular and light and electron microscopic methods were used to study fungal colonisation and to describe its mode of penetration. After inoculation with a foliar spray of conidia, microbiological methods showed 100% of plants examined 24, 48, 72 and 144 h after treatment to be colonised endophytically by the fungus, although the percentage of previously surface sterilised leaf pieces showing fungal growth was 100% at 24 and 48 h, and 80 and 75% at 72 and 144 h after treatment, respectively. The fungus was also observed in leaf pieces obtained from newly formed leaves, indicating that it could spread from treated leaves to leaves formed after fungal application. For molecular studies, a polymerase chain reaction (PCR) protocol was used to amplify the ITS1-5.8S-ITS2 regions of the rDNA of the plant and the fungus. This procedure allowed the detection of the fungus on the surface of the leaves and also endophytically, but only at 72 h after treatment. A nucleotide BLAST search revealed that the ITS1-5.8S-ITS2 sequence of strain EABb 04/01-Tip showed 100% homology with a similar sequence from Cordyceps bassiana. SEM images revealed that although numerous conidia were observed on the leaf surface, few germinated and penetrated. Intracellular colonisation by B. bassiana was not observed, but hyphae were detected growing into the xylem vessels. The fungus was found to colonise 40.5 ± 4.3% of seedlings (with two cotyledons and the two first real leaves) from seeds dressed with a fungal spore suspension. These results may have implications in the biological control of T. papaveris, including the possible systemic protection of the plant against this cynipid.     

Quantitative studies on the mating system of opium poppy (Papaver somniferum L.)

Summary Nearly 400 individuals at two locations and over a number of years were crossed and subsequently scored for selfing versus outcrossing in eight monohybrid populations of opium poppy (Papaver somniferum). Two different marker loci, petal colour (R/r) and capsule size (B/b) were used to determine the male gametes that had effected fertilizations in F₂ recessives (rr and bb). The estimates of the outcrossing parameter were found to vary with year, location and for the marker locus used ( range: 0.0988–0.3704). Study of two dihybrid crosses involving the two loci simultaneously, further confirmed that outcrossing at the R/r locus was significantly greater than that at the B/b locus. The nature of the outcrossing was, in general, nonrandom. Selfmg predominated in this species; however, there was a high frequency of natural outcrossing for generating variations in P. somniferum.CIMAP publication No. 1086     

Anther culture with different genotypes of opium poppy (Papaver somniferum L.): effect of cold treatment

This study concerns anther culture and the production of microspore-derived calluses and plants of the opium poppy (Papaver somniferum L.). It was confirmed that growth regulators were necessary for microspore callus production. Cold treatment (7 d at 7°C) of the buds prior to culture lead to a twofold increase in the frequency of responsive anthers and in the number of calluses per 100 anthers plated. Callus was produced from cultured anthers of several genotypes, covering a wide genetic background. Step by step removal of growth regulators from the culture medium promoted organogenesis and plant regeneration. Most regenerated plants were diploid. The overall process of microspore embryogenesis closely resembled that described in previous reports on somatic callus production and plant regeneration from poppy hypocotyls in vitro.     

Purification and Biochemical Characterization of Asclepain c I from the Latex of Asclepias curassavica L.

In this work we report the isolation, purification and characterization of a new protease from latex of Asclepias curassavica L. Crude extract (CE) was obtained by gathering latex on 0.1 M citric-phosphate buffer with EDTA and cysteine with subsequent ultracentrifugation. Proteolytic assays were made on casein or azocasein as substrates. Caseinolytic activity was completely inhibited by E-64. Stability at different temperatures, optimum pH and ionic strength were evaluated by measuring the residual caseinolytic activity at different times after the incubation. CE showed the highest caseinolytic activity at pH 8.5 in the presence of 12 mM cysteine. CE was purified by cation exchange chromatography (FPLC). Two active fractions, homogeneous by SDS-PAGE, were isolated. The major purified protease (asclepain cI) showed a molecular mass of 23.2 kDa by mass spectrometry and a pI higher than 9.3. The N-terminal sequence showed a high similarity with those of other plant cysteine proteinases. When assayed on N-alpha-CBZ-aminoacid-p-nitrophenyl esters, the enzyme showed higher preference for the glutamine derivative. Determinations of kinetic parameter (km and Kcat) were performed with PFLNA.     

Effects of NaCl and Proline on Polyphenol Oxidase Activity in Bean Seedlings

In this work, the effects of NaCl (0, 50, 100, and 150 mM), proline (0, 5 and 10 mM) and NaCl + proline in combinations on activity of polyphenol oxidase (PPO; E.C. 1.10.3.1) and soluble protein content have been investigated in the root, stem and leaf tissues of bean (Phaseolus vulgaris L.) seedlings grown in embryo culture. PPO activities were higher in all the tissues treated with NaCl, proline and NaCl + proline combinations those that of the control tissues. The protein content was very high in tissues exposed to proline and NaCl + proline combination, but NaCl alone decreased protein contents in root and leaf tissues. The results suggest that proline may play a role as an enzyme-stabilizing agent in salt stress.     

Head group specificity of phospholipase D isoenzymes from poppy seedlings (<Emphasis Type="Italic">Papaver somniferum</Emphasis> L.)

The biocatalytical potential of two new phospholipase D (PLD) isoenzymes from poppy seedlings (Papaver somniferum L.), PLD-A and PLD-B, was examined by comparing their activities in phospholipid transformation. Both enzymes showed the same ratio in rates of hydrolysis [phosphatidylcholine (PC):phosphatidylglycerol (PG):phosphatidylserine:phosphatidylinositol=1:0.5:0.3:0.1] and were inactive towards phosphatidylethanolamine (PE). PLD-A did not catalyze head group exchange whereas PLD-B showed a high transphosphatidylation potential in the conversion of PC into PG and PE. This enzyme also catalyzed the transesterification of octadecylphosphocholine into octadecylphosphoglycerol or octadecylphosphoethanolamine.     

Identification of Indian Landraces of Opium Poppy Papaver somniferum Resistant to Damping-off and Downy Mildew Fungal Diseases

A set of 32 landraces (traditional local cultivars) of poppy Papaver somniferum originating in the Indian states of Rajasthan, Madhya Pradesh, Uttar Pradesh and Tamilnadu, and four Indian commercial varieties were screened over four cropping seasons for their reaction to downy mildew disease caused by Peronospora arborescens and damping-off disease caused by Pythium dissotocum under both field and artificial inoculation conditions. The landrace 1018 was found to be resistant to damping-off disease and the landraces 1014 and N3 were resistant to downy mildew disease. A yield trial conducted over two seasons showed that the damping-off disease-resistant landrace 1018 was superior to all the commercial varieties in seed, morphine and/or codeine yield. The experiments provided further evidence that there is considerable genetic variability between the landraces.     

The Role of Peroxidase and Polyphenol Oxidase Isozymes in Wheat Resistance to Alternaria triticina

Polyphenol oxidase activity was higher in resistant wheat cultivar ACC-8226 than in susceptible cultivar MP-845 in control sets and after inoculation of Alternaria triticina. However, similar polyphenol oxidase isozyme pattern was found in control and inoculated sets of both the cultivars, but the band intensity was higher after inoculation. Three and four peroxidase isozymes were found in ACC-8226 and MP-845, respectively. An extra peroxidase isozyme band was observed in both the cultivars after inoculation. The results suggest an active role of peroxidase and polyphenol oxidase in defence mechanism of wheat plants.     

丹参多酚氧化酶基因的克隆及表达分析

前期研究发现多酚氧化酶(PPO)能正向调控丹酚酸B合成,该研究运用RACE技术,从丹参毛状根中克隆到多酚氧化酶基因(SmPPO,GenBank登录号为KF712274)全长序列,其cDNA全长1 930bp,开放阅读框为1 770bp,编码589个氨基酸。将SmPPO与管状花目其它4个物种进行氨基酸序列比对,在N端类囊体转移结构域中发现都存在2个N-豆蔻酰化位点。在丹参毛状根培养液中加入不同诱导因子,利用实时荧光定量PCR检测,发现该基因在酵母提取物处理中表达量显著上调,但在银离子、抗坏血酸和L-半胱氨酸处理中表达受到明显抑制。运用HPLC技术同步检测毛状根中丹酚酸B含量,显示出与基因表达相同的变化趋势。研究表明,丹参中多酚氧化酶基因(SmPPO)对丹酚酸B的合成具有正向调控作用。     

Phylogenetic studies in Papaver section Oxytona: cytogenetics of the species and interspecific hybrids

Summary Chromosome behaviour at meiosis was studied in the F₁, F₂, and backcross generations, in the three species of Papaver section Oxytona, and in artificially induced autopolyploids of P. bracteatum. Close homology was found between the genome of P. bracteatum and that of the two polyploid species, P. orientale and P. pseudo-orientale, suggesting that the P. bracteatum genome is present in both polyploid species. A genetic mechanism controlling bivalent pairing in the polyploid species is suggested. Further study is needed for finding out the breeding potential of interspecific hybridization in section Oxytona.Contribution no. 1569-E, 1985 series from the Agricultural Research Organization, The Volcani Center, Bet Dagan 50 250. Israel     

Purification and Characterization of a New Plant Endopeptidase Isolated from Latex of Asclepias fruticosa L. (Asclepiadaceae)

Asclepias fruticosa L. is a small shrub containing latex with proteolytic activity. The crude extract (latex diluted 1:250 and ultracentrifuged) contained 276 g of protein/mL and the proteolytic activity reached 1.2 caseinolytic U/mL. This enzyme preparation was very stable even after 2 hours at 45°C, but was quickly inactivated after 5 minutes at 80°C. Chromatographic purification was achieved by FPLC using a cation exchanger (SP-Sepharose FF). Thus, a unique proteolitically active fraction could be isolated, being homogeneous by bidimensional electrophoresis and mass spectrometry (M_r = 23,652). The optimum pH range was achieved at 8.5–10.5. The enzyme activity was completely inhibited by specific cysteine peptidases inhibitors. Isoelectric focusing followed by zymogram showed the enzyme had a pI greater than 9.3. The N-terminus sequence (LPDSVDWREKGVVFPIRNQGK) shows a great deal of similarity to those of the other cysteine endopeptidases isolated from latices of Asclepiadaceae even when a high degree of homology could be observed with other plant cysteine endopeptidases.     

1,2-Dehydroreticuline synthase, the branch point enzyme opening the morphinan biosynthetic pathway

A synthase which oxidizes (S)-reticuline to 1,2-dehydroreticuline has been found to occur in seedlings of opium poppy (Papaver somniferum L.). Due to its instability, this enzyme could only be partly purified (ca. 5-fold enrichment). Partial characterization at this stage of purification showed that it does not need a redox cofactor and accepts both (S)-reticuline and (S)-norreticuline as substrates. [1-(2)H, (13)C]-(R,S)-reticuline was enzymatically converted into [1-(13)C]-dehydroreticuline, which has been identified by mass spectrometry. Release of the hydrogen atom in position C-1 of the isoquinoline alkaloid during the oxidative conversion, was exploited as a sensitive assay system for this enzyme. The enzyme has a pH optimum of 8.75, a temperature optimum of 37 degrees C and the apparent K(M) value for the substrate reticuline was shown to be 117 microM. Moreover it could be demonstrated by sucrose density gradient centrifugation that the enzyme is located in vesicles of varying size. In combination with the previously discovered strictly stereoselective and NADPH dependent 1,2-dehydroreticuline reductase the detection of this enzyme, the 1,2-dehydroreticuline synthase, provides the necessary inversion of configuration and completes the pathway from two molecules of L-tyrosine via (S)-norcoclaurine to (R)-reticuline in opium poppy involving a total number of 11 enzymes.     

A note on the genomic consequences of regular bivalent formation and continued fertility in triploids

The genomic evolution of triploid plants with regular bivalent formation is discussed. The conclusion is reached that although all the progeny of an originally triploid individual will be triploid numerically, only part of the progeny will be triploid genomically. The consequences of this for triploid identification by means of chromosome morphology and isozyme numbers is discussed.     

棘托竹荪子实体多酚氧化酶特性及其抑制剂的研究

以邻苯二酚为底物采用分光光度法对棘托竹荪(Dictyophora echinovolvata)子实体多酚氧化酶的酶学特性和不同抑制剂对多酚氧化酶活性的影响进行了研究。结果表明,棘托竹荪子实体多酚氧化酶的最适反应pH为8.0,最适反应温度为45℃,高温短时处理能显著抑制多酚氧化酶的活性;L-半胱氨酸(L-Cys)、氯化钠(NaCl)、维生素C(Vc)和柠檬酸(C6H8O7)等对多酚氧化酶均有抑制作用。经正交实验筛选的抑制剂组合(5.0 g/L L-Cys、20.0 g/L NaCl、1.5 g/L Vc、10.0 g/L C6H8O7)可以完全抑制多酚氧化酶的活性。