Characterization of lipoxygenase isoforms in olive callus cultures

Lipoxygenase activity is critical for the development of flavours and aromas in olive oils. We have partly purified isoforms of molecular mass 95 kDa that have activity against linoleic or alpha-linolenic acids by a simple procedure from olive callus cultures.     

Lipoxygenase pathway in olive callus cultures (Olea europaea)

Stimulation of the lipoxygenase pathway in olive fruit initiates a cascade of reactions that begins with the regio- and stereospecific di-oxygenation of polyunsaturated fatty acids containing a cis, cis-1,4 pentadiene moiety. Later products of the pathway include volatiles that influence the organoleptic properties of harvested olive oil. In this study, we have investigated lipoxygenase activity in olive callus cultures, and found that there is evidence of several isoforms of the enzyme with different pH optima and substrate specificities. Endogenous lipoxygenase activity was detected throughout the growth cycle of olive callus, particularly during the log phase of growth, suggesting that olive lipoxygenases are intimately involved in growth. The most prominent lipoxygenase activity in tissue cultures was found to be soluble but significant activities were detected in the plastid fraction. In addition, hydroperoxide lyase (HPL) activity was measured in the calli; both 13- and 9-HPL activities were found which were particulate.     

Lipid Biosynthesis in Olive Cultures

Differentiation in olive callus cultures was induced by changingthe plant growth regulator content, particularly 2,4-dichlorophenoxyaceticacid, in the growth media at 25°C. These cultures have beenmaintained for an extended period with low polyploid nucleilevels. Analysis of olive callus cultures indicated that theacyl lipid composition varied according to the state of differentiationand to incubation temperature. Heterotrophic olive callus wascharacterized by its ability to accumulate triacylglycerol richin oleate, a situation comparable to developing olive fruit.In fact, oleate-rich triacylglycerol was enhanced in heterotrophiccallus cultured at 35°C. Greening calli, however, were noticeablyfound to possess typical chloroplastic lipids, indicating thepresence of intracellular chloroplastic structures, which wasconfirmed by electron microscopy. These cultures also exhibitedacyl compositions with increased amounts of linoleate and     

A lipoxygenase with dual positional specificity is expressed in olives (Olea europaea L.) during ripening

Plant lipoxygenases (LOXs) are a class of widespread dioxygenases catalysing the hydroperoxidation of polyunsaturated fatty acids. Although multiple isoforms of LOX have been detected in a wide range of plants, their physiological roles remain to be clarified. With the aim to clarify the occurrence of LOXs in olives and their contribution to the elaboration of the olive oil aroma, we cloned and characterized the first cDNA of the LOX isoform which is expressed during olive development. The open reading frame encodes a polypeptide of 864 amino acids. This olive LOX is a type-1 LOX which shows a high degree of identity at the peptide level towards hazelnut (77.3%), tobacco (76.3%) and almond (75.5%) LOXs. The recombinant enzyme shows a dual positional specificity, as it forms both 9- and 13-hydroperoxide of linoleic acid in a 2:1 ratio, and would be defined as 9/13-LOX. Although a LOX activity was detected throughout the olive development, the 9/13-LOX is mainly expressed at late developmental stages. Our data suggest that there are at least two Lox genes expressed in black olives, and that the 9/13-LOX is associated with the ripening and senescence processes. However, due to its dual positional specificity and its expression pattern, its contribution to the elaboration of the olive oil aroma might be considered.     

Antimicrobial activity and inhibition of aflatoxin B1 formation by olive plant tissue constituents

Ethanolic extracts of olive callus tissues, added at 0.5 or 1.0% to media on which Aspergillus flavus was grown, inhibited aflatoxin production by 90% without inhibiting the fungal growth. The extract was found to contain mainly caffeic acid and, to a lesser extent, catechin and coumarins. The fungicidal and bactericidal activity of caffeic acid, catechin, coumarin and p-, o- or m-coumaric acid were tested and only caffeic acid and o-coumaric acid inhibited aflatoxin production. The inhibitory effect had no correlation with the growth of the fungus. Only coumarin at 10 mmol/1 totally inhibited fungal growth. Of the phenolic constituents of callus tissues tested, catechin and caffeic acid (10 mmol/l) showed bactericidal activity towards Pseudomonas aeruginosa and Staphylococcus aureus.     

Antimicrobial activity and inhibition of aflatoxin B1 formation by olive plant tissue constituents

Ethanolic extracts of olive callus tissues, added at 0.5 or 1.0% to media on which Aspergillus flavus was grown, inhibited aflatoxin production by 90% without inhibiting the fungal growth. The extract was found to contain mainly caffeic acid and, to a lesser extent, catechin and coumarins. The fungicidal and bactericidal activity of caffeic acid, catechin, coumarin and p-, o- or m-coumaric acid were tested and only caffeic acid and o-coumaric acid inhibited aflatoxin production. The inhibitory effect had no correlation with the growth of the fungus. Only coumarin at 10 mmol/1 totally inhibited fungal growth. Of the phenolic constituents of callus tissues tested, catechin and caffeic acid (10 mmol/1) showed bactericidal activity towards Pseudomonas aeruginosa and Staphylococcus aureus.     

The effect of salt stress on lipid peroxidation and antioxidative enzymes in callus of two <Emphasis Type="Italic">Acanthophyllum</Emphasis> species

The changes in lipid peroxidation and the involvement of the antioxidant system in relation to salt stress tolerance were investigated in the callus of Acanthophyllum glandulosum and Acanthophyllum sordidum. The callus was subjected to NaCl stress (50–200 mM) for 40 d. The callus of A. glandulosum was less sensitive to NaCl stress than that of A. sordidum. Increasing concentrations of NaCl from 50 to 200 mM correlated to increased proline content in A. glandulosum. Total protein content was higher in extracts of A. glandulosum than in extracts of A. sordidum under both control and salinity treatments. Compared with A. sordidum, lipid peroxidation and H₂O₂ content were lower and the activities of superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), ascorbate peroxidase, and glutathione reductase were higher in A. glandulosum under salt stress. Activity staining of antioxidant enzymes separated by native polyacrylamide gel electrophoresis (PAGE) revealed that callus of A. sordidum had five Fe-SOD isoforms and one Mn-SOD isoform, all of which were reduced by salinity. In A. glandulosum, two Mn-SOD, three Fe-SOD, and one Cu/Zn-SOD isoforms were detected. Out of these six SOD isoforms, expression of the Mn-SOD and Fe-SOD isoforms was enhanced at 100 mM and higher NaCl concentrations. Two and six GPX isoforms were detected in A. sordidum and A. glandulosum, respectively. Expression of the single CAT isoform in A. sordidum was preferentially reduced by salinity. In A. glandulosum, the two CAT isoforms showed differential down regulation under NaCl stress, with the CAT2 isoform detected only under control condition. These results suggest that A. glandulosum callus is better protected against salinity-induced oxidative damage by maintaining higher activities of antioxidant enzymes than the callus of A. sordidum.     

杉木未成熟胚胚性愈伤组织诱导影响因素探析

该研究从基因型、6-BA浓度、外植体接种方式和合子胚发育阶段等方面,分析杉木未成熟胚胚性愈伤组织诱导的影响因素。结果表明:基因型、6-BA浓度、外植体接种方式和合子胚发育阶段均对胚性愈伤组织诱导频率有不同程度影响。6种基因型中,有3种基因型诱导出胚性愈伤组织,其中基因型S18胚性愈伤组织诱导频率最高,为11.7%。6-BA浓度在1.0~1.5 mg·L~(-1)范围内时,基因型S18的胚性组织诱导频率较高。以在去皮种子的一端切开一个小口的接种方式为最优,将合子胚剥出的方式易造成合子胚褐化死亡,将未剥皮的种子切开一个小口后直接接入培养基的方式不利于愈伤组织生成。适合胚性愈伤组织诱导的合子胚发育阶段为受精至胚器官分化阶段,合子胚进入成熟阶段后不利于胚性愈伤组织诱导,合子胚易生长成完整植株。     

Induction of β-1,3-glucanase in callus cultures <Emphasis Type="Italic">in vitro</Emphasis>

Sodium salicylate (NaSA) increased induction of both intracellular and extracellular beta-1,3-glucanases in calluses of campion and duckweed. NaSA concentrations from 30 to 100 mM were optimal for induction of intracellular glucanase in the campion callus, and for induction of extracellular glucanase the optimal concentration varied from 5 to 100 mM. The glucanase activity in the duckweed callus was lower than in the campion callus, and co-cultivation of the campion callus with Trichoderma harzianum mycelium increased the production of intracellular and extracellular beta-1,3-glucanases and polygalacturonase in the callus. Biosynthesis by T. harzianum of glucanases, extracellular polygalacturonase and xylanase, and of intracellular galactosidase was increased. The co-cultivation was accompanied by increased activity of intracellular acidic isoform of glucanase Glu-3 secreted by the callus cells into the medium, whereas NaSA activated in the callus culture the extracellular acidic isoform Glu-1 and extracellular basic isoform Glu-5. These data indicate the induction of these isoforms and the specificity of protective response of plant cells to different factors.     

Pectin Methylesterase Isoforms in Tomato (Lycopersicon esculentum) Tissues (Effects of Expression of a Pectin Methylesterase Antisense Gene)

We have identified two major groups of pectin methylesterase (PME, EC 3.1.1.11) isoforms in various tissues of tomatoes (Lycopersicon esculentum). These two groups exhibited differential immuno-cross-reactivity with polyclonal antibodies raised against tomato fruit PME or flax callus PME and differences in their accumulation patterns in tissues of wild-type and transgenic tomato plants expressing a PME antisense gene. The group I isoforms with isoelectric points (pls) of 8.2, 8.4, and 8.5 are specific to fruit tissue, where they are the major forms of PME activity. The group II PME isoforms, with pl values of 9 and above, are observed in both vegetative and fruit tissues. The group I isoforms cross-react with polyclonal antibodies raised to a PME isoform purified from fruit, whereas the group II isoforms cross-react with antibodies to a PME purified from flax callus. Expression of a fruit-specific PME anti-sense gene impairs accumulation of the group I PME isoforms, with no apparent effect on the accumulation of the group II PME isoforms. The absence of any noticeable effects on growth and development of transgenic plants suggests that the group I PME isoforms are not involved in plant growth and development and may play a role under special circumstances such as cell separation during fruit ripening.     

Separation,characterization and catalytic properties of Lip2 isoforms from Candida sp. 99-125

Lipase (EC.3.1.1.3) from Candida sp. 99-125 was separated into four isoforms (isoform A, isoform B, isoform C, and isoform D) by two steps of ion exchange chromatography. As analyzed on SDS- and non-denaturing PAGE, the four isoforms were homogenous and had the same molecular weight of approximate 38 kDa. MALDI-TOF peptide mass fingerprinting maps and circular dichroism spectra showed the isoforms had similar peptide patterns belonging to the same protein encoded by the YLlip2 gene and different secondary structures. The isoforms had a little distinct optimum temperature in the range of 20–35 °C, and the same optimum pH (8.0). They remained to be active in methanol, ethanol and ethylene glycol at the concentration of 10% and 20% (v/v) and acetone at the concentration of 10% (v/v), and sensitive to EDTA. Triton X-100, Sodium cholate and CHAPS slightly increased their activities. The metal ion Ca²⁺ and Mg²⁺ had mild effect on lipase activity. The isoforms showed a preference for long chain fatty acid triglyceride (triolein and olive). The lipase purified by one step of ion exchange chromatography and isoforms were less active than crude enzyme to catalyze cetyl alcohol and oleic acid in n-hexane, whereas the presence of small concentration of added water dramatically activated crude lipase but less the purified preparations.     

Vegetative growth retardation,improved rooting and viability of olive cuttings in response to application of growth retardants

Spray and soil treatments of paclobutrazol and uniconazole were applied to young and mature olive plants and olive cuttings. Two clear phases, were found in the growth response of olive shoots to growth retardants: an early phase, which retards and even inhibits growth considerably; and a later phase, during which the shoots are released from the retardation and start to elongate rapidly. A somewhat slower response of the plants to soil application than to spray application of growth retardants was noticed. Paclobutrazol enhanced the rooting of cv. Manzanillo cuttings, whether applied to the mother plants or to the cuttings themselves. Indole-3-butyric acid (IBA) was needed in both cases. Sprouting was shown to reduce rooting. Paclobutrazol significantly inhibited sprouting and increased the meristematic activity in the base of the cuttings. Rooting of the hard-to-root cv. Kalamata was not enhanced by the treatments although callus formation was induced and viability was prolonged.     

Levels of free and conjugated indole-3-acetic acid in ethylene-treated leaves and callus of olive

Changes in levels of free and conjugated indole-acetic acid (IAA) resulting from ethylene treatment were examined in leaves and callus of olive ( Olea europea var. Manzanillo) by electron capture gas chromotography and by isotope dilution analysis. In both olive leaves and olive tissue cultures, the level of free IAA did not change much after the exposure to ethylene, but the levels of bound IAA increased almost three times in comparison with untreated controls. This is the first report of changes in endogenous, bound auxin accompanying ethylene treatment.     

Isolation,culture and division of olive (Olea europaea L.) protoplasts

Protoplasts from Olea europaea L. have been compared in terms of their yield, viability, cell division and callus differentiation. Viable protoplasts were isolated from in vitro cultured leaves and cotyledons by an overnight incubation in an enzyme solution containing 1–1.5% driselase and 0.5M sucrose. This method allowed high yield of purified protoplasts, which floated and formed a dark green band at the meniscus, after centrifugation. Purified protoplasts were diluted to 3×10⁴ protoplasts·ml^–1 in culture medium. After cell wall regeneration, protoplasts gradually increased their volumes under appropriate conditions. The first divisions occurred during the second week in culture. Division efficiency ranged from 5.2 to 9.8% after 20 days in culture. Two weeks later visible microcolonies developed only from cotyledon protoplasts. After 6 weeks in culture, the microcalli were transferred to a solidified culture medium with 0.6% agarose, which induced active callus growth.Abbreviations OM olive proliferation medium, Rugini 1984 - Omg OM for the germination of olive embryos - OMr=OM for root induction - OMp=OM for protoplasts - OMc=OM for callus - BN Bourgin and Nitsch medium 1967 - IBA indol-3-butyric acid - NAA naphthalene acetic acid - 2,4-D dichlorophenoxyacetic acid.     

Isolation and Preliminary Characterization of ATPase from Olive Calli Grown at Different Auxin/Cytokinin Ratio

ATPase activity was studied in calli from olive (Olea europaea L.) petioles cultured in media modified in their auxin/cytokinin ratio in order to induce different morphogenetic responses. Addition of 0.54 μM α-naphthaleneacetic acid (NAA) or 14 μM zeatin (ZEA) did not induce any morphogenesis in calli and proton pump activity in vivo was very low, while calli produced roots at 27 or 11 μM NAA + 0.28 μM ZEA and possessed clearly detectable proton pump activity. ATPase activity associated with microsomes isolated by differential centrifugation from different callus cultures had the same pH optimum and similar sensitivity toward nitrate and azide. However, microsomes isolated from non-morphogenetic calli had higher specific ATPase activity which was very poorly (6 %) inhibited by vanadate. Also, the fractionation of these microsomes on a continuous sucrose gradient showed two peaks of ATPase activity, the more pronounced one co-purifying with the Golg i marker enzyme, Triton-stimulated UDPase activity, suggesting thus the presence of very high ATPase activity in Golgi secretory vesicles. On the contrary, ATPase activity of microsomes from calli producing roots was more sensitive to vanadate (30 - 40 % inhibition). Furthermore, the component of ATPase activity attributable to Golgi secretory vesicles was less abundant. This revised version was published online in July 2006 with corrections to the Cover Date.     

Comparison of fatty acid patterns in plant parts and respective callus cultures of Cucumis melo

Root, hypocotyl, cotyledon, stem and leaf of Cucumis melo var. utilissimus seedlings were used for callus induction. Comparison was made between these parts, between callus tissues originating from all the parts and between each part and its callus, with respect to the fatty acid composition of total lipids. In all the parts there was a greater proportion of unsaturated fatty acids, the predominant fatty acid in root, stem and leaf being linolenic acid whilst in the cotyledon linoleic predominated. In the hypocotyl these two acids were present in equal amounts. In callus cultures the proportion of saturated acids was greater and the predominant fatty acid was palmitic. The major unsaturated fatty acid in callus cultures was linolenic. The analysis showed that callus tissue and its respective plant part had different fatty acid patterns and that all the callus cultures had very similar patterns irrespective of their origin.     

基于响应面设计茎段形成层培养猕猴桃幼苗的优化研究

为降低猕猴桃组培快繁中的污染率,提高其繁殖效率,该文以猕猴桃的幼嫩茎段为外植体,采用两步培养法进行茎段形成层的愈伤及成苗诱导研究,并利用响应面设计软件对NAA浓度、6-BA浓度、低渗处理时间进行了各条件的优化,同时通过组织切片确定愈伤的来源及幼苗的形成方式。结果表明:(1)培养过程中撕除茎段周皮能显著降低污染率,用200～400 mg·L^-1的PVP处理猕猴桃茎段可有效防止去皮茎段的褐化。(2)愈伤诱导的最佳条件为预培养28.3 h、NAA 4.45 mg·L^-1、6-BA 0.28 mg·L^-1,而幼苗形成的最佳条件为预培养26.4 h、NAA 4.84 mg·L^-1、6-BA 0.42 mg·L^-1。这表明形成层愈伤诱导需较长低渗处理时间和较高生长素,而成苗诱导则需较高生长素、激动素及较短的低渗处理时间。(3)组织切片观察结果表明猕猴桃愈伤组织源于形成层干细胞的分裂,且幼苗株源于胚状体的发育。综上结果表明,通过除去猕猴桃嫩茎周皮,外加抗氧化、低渗处理,可有效降低猕猴桃组培快繁中的污染率,提高繁殖系数和胚状体发生率,为猕猴桃种苗的规模化生产提供技术支撑。     

培养条件对三七愈伤组织生长和皂苷积累的影响

以MS为基础培养基,改变激素配比、氮源和光照等因素,以分光光度法和HPLC法分析三七愈伤组织培养过程中皂苷含量的变化。结果表明:培养条件对三七愈伤组织中皂苷积累有一定影响,激素配比对愈伤组织中皂苷含量的影响最大,在0.5 mg·L-12,4-D+1.0 mg·L-16-BA组合下,培养物中总皂苷含量最多,达到4.72%±0.29%;在总氮量为60 mmol·L-1条件下,45 mmol·L-1KNO3+7.5 mmol·L-1NH4NO3(NO3-/NH4+=7∶1)时,愈伤组织皂苷含量最多,达到4.71%±0.17%;分别在1 000 lx和500 lx光强下每天光照12 h的愈伤组织,皂苷含量均低于黑暗培养的愈伤组织,三者皂苷含量分别为1.94%±0.31%、2.38%±0.12%和3.57%±0.27%,光照引起愈伤组织表面变绿及细胞分化,可能是抑制愈伤组织中皂苷合成与积累的主要原因;HPLC检测发现,三七愈伤组织和根中均含有Rg1、Re、Rb1及Rd四种皂苷,但栽培三七根含有R1皂苷,而三七愈伤组织中未检测到R1,其原因需要进一步研究。该研究结果为未来愈伤组织培养成为部分代替人工栽培生产三七天然产物的潜在途径提供了研究基础。     

火百合花丝组织培养器官形成的细胞组织学研究

以火百合花丝节段为外植体,接种在附加6-BA 1.0 mg/L和NAA 0.2 mg/L的MS培养基上,诱导出愈伤组织及器官。经细胞组织学观察表明,细胞启动、愈伤组织形成及器官再生皆发生于外植体形态学下端的切口边缘及内方,而形态学上端的细胞自始至终未启动。器官发生的途径是通过愈伤组织间接产生,有的愈伤组织团单独形成芽或根,而有的愈伤组织团则同时分别从表面形成芽,内部形成根,通过维管组织连接成完整的植株。     

Studies on genetic transformation of olive (<Emphasis Type="Italic">Olea europaea</Emphasis> L.) somatic embryos: I. Evaluation of different aminoglycoside antibiotics for <Emphasis Type="Italic">nptII</Emphasis> selection; II. Transient transformation via particle bombardment

As a first step to the establishment of a genetic transformation protocol for olive somatic embryos obtained from the seeds of cv. ‘Picual’, the efficiencies of different aminoglycoside antibiotics as selective agents to be used with the nptII marker gene, and the particle bombardment technique for transient transformation have been evaluated. Among the three antibiotics tested, paromomycin and kanamycin showed a similar inhibitory effect and, at 200 mg l⁻¹, both of them impaired callus growth after 8 weeks of culture. However, when isolated embryos were cultured in the presence of these antibiotics, a 20% of the embryos still remained viable at 400 mg l⁻¹. Neomycin was discarded as a selective agent since it showed only a moderate toxic effect. Contrary to solid medium, when olive callus was cultured in liquid medium supplemented with different paromomycin concentrations for 3 weeks, the callus growth was impaired at the lowest antibiotic concentration, 3 mg l⁻¹. Best conditions for transient transformation of olive callus using PDS-1000/He system were a 6 cm target distance and a 900 psi bombardment pressure. pCGU∆1 plasmid, containing the gus gene under the control of sunflower ubiquitin promoter yielded a significantly higher number of gus expression areas per bombarded explant than pGUSINT or pJGUS5 plasmids, where the gus gene is driven by CaMV35S promoter or CaMV35S with enhancer, respectively. Almost 45% of bombarded explants showed gus expression 12 weeks after bombardment.