The influence of Ceratocystis polonica inoculation and methyl jasmonate application on terpene chemistry of Norway spruce,Picea abies

Constitutive and inducible terpene production is involved in conifer resistance against bark beetles and their associated fungi. In this study 72 Norway spruce (Picea abies) were randomly assigned to methyl jasmonate (MJ) application, inoculation with the bluestain fungus Ceratocystis polonica, or no-treatment control. We investigated terpene levels in the stem bark of the trees before treatment, 30 days and one year after treatment using GC–MS and two-dimensional GC (2D-GC) with a chiral column, and monitored landing and attack rates of the spruce bark beetle, Ips typographus, on the trees by sticky traps and visual inspection. Thirty days after fungal inoculation the absolute amount and relative proportion of (+)-3-carene, sabinene, and terpinolene increased and (+)-α-pinene decreased. Spraying the stems with MJ tended to generally increase the concentration of most major terpenes with minor alteration to their relative proportions, but significant increases were only observed for (?)-β-pinene and (?)-limonene. Fungal inoculation significantly increased the enantiomeric ratio of (?)-α-pinene and (?)-limonene 1 month after treatment, whereas MJ only increased that of (?)-limonene. One year after treatment, both MJ and fungal inoculation increased the concentration of most terpenes relative to undisturbed control trees, with significant changes in (?)-β-pinene, (?)-β-phellandrene and some other compounds. Terpene levels did not change in untreated stem sections after treatment, and chemical induction by MJ and C. polonica thus seemed to be restricted to the treated stem section. The enantiomeric ratio of (?)-α-pinene was significantly higher and the relative proportions of (?)-limonene were significantly lower in trees that were attractive to bark beetles compared to unattractive trees. One month after fungal inoculation, the total amount of diterpenes was significantly higher in putative resistant trees with shorter lesion lengths than in putative susceptible trees with longer lesions. Thus, terpene composition in the stem bark may be related to resistance of Norway spruce against I. typographus and C. polonica.     

Host resistance elicited by methyl jasmonate reduces emission of aggregation pheromones by the spruce bark beetle, Ips typographus

We treated Norway spruce (Picea abies) stems with methyl jasmonate (MeJA) to determine possible quantitative and qualitative effects of induced tree defenses on pheromone emission by the spruce bark beetle Ips typographus. We measured the amounts of 2-methyl-3-buten-2-ol and (S)-cis-verbenol, the two main components of the beetle's aggregation pheromone, released from beetle entrance holes, along with phloem terpene content and beetle performance in MeJA-treated and untreated Norway spruce logs. As expected, phloem terpene levels were higher and beetle tunnel length was shorter (an indication of poor performance) in MeJA-treated logs relative to untreated logs. Parallel to the higher phloem terpene content and poorer beetle performance, beetles in MeJA-treated logs released significantly less 2-methyl-3-buten-2-ol and (S)-cis-verbenol, and the ratio between the two pheromone components was significantly altered. These results suggest that host resistance elicited by MeJA application reduces pheromone emission by I. typographus and alters the critical ratio between the two main pheromone components needed to elicit aggregation. The results also provide a mechanistic explanation for the reduced performance and attractivity observed in earlier studies when bark beetles colonize trees with elicited host defenses, and extend our understanding of the ecological functions of conifer resistance against bark beetles.     

Methyl jasmonate induces traumatic resin ducts,terpenoid resin biosynthesis,and terpenoid accumulation in developing xylem of Norway spruce stems

Norway spruce (Picea abies L. Karst) produces an oleoresin characterized by a diverse array of terpenoids, monoterpenoids, sesquiterpenoids, and diterpene resin acids that can protect conifers against potential herbivores and pathogens. Oleoresin accumulates constitutively in resin ducts in the cortex and phloem (bark) of Norway spruce stems. De novo formation of traumatic resin ducts (TDs) is observed in the developing secondary xylem (wood) after insect attack, fungal elicitation, and mechanical wounding. Here, we characterize the methyl jasmonate-induced formation of TDs in Norway spruce by microscopy, chemical analyses of resin composition, and assays of terpenoid biosynthetic enzymes. The response involves tissue-specific differentiation of TDs, terpenoid accumulation, and induction of enzyme activities of both prenyltransferases and terpene synthases in the developing xylem, a tissue that constitutively lacks axial resin ducts in spruce. The induction of a complex defense response in Norway spruce by methyl jasmonate application provides new avenues to evaluate the role of resin defenses for protection of conifers against destructive pests such as white pine weevils (Pissodes strobi), bark beetles (Coleoptera, Scolytidae), and insect-associated tree pathogens.     

Induction of terpene biosynthesis in dinoflagellate symbionts of Caribbean gorgonians

This report describes a series of experiments designed to determine if terpene biosynthesis is inducible in two families of marine terpenes, pseudopterosins from the gorgonian coral Pseudopterogorgia elisabethae and fuscol from Eunicea fusca. Since we have recently shown that terpene biosynthesis is not under the control of the invertebrate host, but rather occurs within a dinoflagellate preparation, we examined the terpene content of the dinoflagellate symbiont following a decrease in UV/vis radiation as well as in response to the addition of methyl jasmonate, salicylic acid and gibberellic acid. We demonstrated that pseudopterosin and fuscol biosynthesis can be markedly increased through the addition of the plant bioactive substances. We also demonstrated that, while the terpene content of P. elisabethae increases in response to decreased UV/vis light, this is due primarily to an increase in the concentration of the dinoflagellate rather than simply an induction of terpene biosynthesis.     

季节变化对聚果榕-榕小蜂互利共生系统生长与繁殖的影响

榕属植物及其传粉昆虫榕小蜂是自然界协同进化的经典模型,榕果内雌花资源如何分配一直是备受关注的问题。为验证季节变化对榕树-榕小蜂互利共生系统生长与繁殖的影响,该研究以西双版纳地区的聚果榕(Ficus racemosa)为材料,分析了季节变化对榕果大小、自然进蜂量以及榕树-榕小蜂繁殖的影响,并利用人工控制性放蜂实验和模型拟合,探讨榕果最适进蜂量及不同季节进蜂量对雌花资源分配的影响。结果表明:季节对榕果直径有显著影响,雨季的榕果直径显著小于干热季和雾凉季;不同季节的自然进蜂量也有显著差别,苞片口对调节进蜂数量有重要作用;季节对榕树-榕小蜂繁殖分配也有影响,雾凉季产生的种子数量和榕小蜂数量均最多;同时人工控制实验和二次抛物线模型拟合结果表明,母代雌蜂数量与种子及榕小蜂后代数量均呈抛物线关系,雌蜂数量过多或过少都对榕树-榕小蜂的繁殖不利,自然进蜂量与拟合的最优进蜂量基本一致。研究结果说明榕果进化出了适应西双版纳地区季节变化的繁殖策略。     

Exogenous application of methyl jasmonate elicits defenses in Norway spruce (Picea abies) and reduces host colonization by the bark beetle Ips typographus

The terpenoid and phenolic constituents of conifers have been implicated in protecting trees from infestation by bark beetles and phytopathogenic fungi, but it has been difficult to prove these defensive roles under natural conditions. We used methyl jasmonate, a well-known inducer of plant defense responses, to manipulate the biochemistry and anatomy of mature Picea abies (Norway spruce) trees and to test their resistance to attack by Ips typographus (the spruce bark beetle). Bark sections of P. abies treated with methyl jasmonate had significantly less I. typographus colonization than bark sections in the controls and exhibited shorter parental galleries and fewer eggs had been deposited. The numbers of beetles that emerged and mean dry weight per beetle were also significantly lower in methyl jasmonate-treated bark. In addition, fewer beetles were attracted to conspecifics tunneling in methyl jasmonate-treated bark. Stem sections of P. abies treated with methyl jasmonate had an increased number of traumatic resin ducts and a higher concentration of terpenes than untreated sections, whereas the concentration of soluble phenolics did not differ between treatments. The increased amount of terpenoid resin present in methyl jasmonate-treated bark could be directly responsible for the observed decrease in I. typographus colonization and reproduction.     

Up-regulation of soyasaponin biosynthesis by methyl jasmonate in cultured cells of Glycyrrhiza glabra

Exogenously applied methyl jasmonate (MeJA) stimulated soyasaponin biosynthesis in cultured cells of Glycyrrhiza glabra (common licorice). mRNA level and enzyme activity of beta-amyrin synthase (bAS), an oxidosqualene cyclase (OSC) situated at the branching point for oleanane-type triterpene saponin biosynthesis, were up-regulated by MeJA, whereas those of cycloartenol synthase, an OSC involved in sterol biosynthesis, were relatively constant. Two mRNAs of squalene synthase (SQS), an enzyme common to both triterpene and sterol biosyntheses, were also up-regulated by MeJA. In addition, enzyme activity of UDP-glucuronic acid: soyasapogenol B glucuronosyltransferase, an enzyme situated at a later step of soyasaponin biosynthesis, was also up-regulated by MeJA. Accumulations of bAS and two SQS mRNAs were not transient but lasted for 7 d after exposure to MeJA, resulting in the high-level accumulation (more than 2% of dry weight cells) of soyasaponins in cultured licorice cells. In contrast, bAS and SQS mRNAs were coordinately down-regulated by yeast extract, and mRNA accumulation of polyketide reductase, an enzyme involved in 5-deoxyflavonoid biosynthesis in cultured licorice cells, was induced transiently by yeast extract and MeJA, respectively.     

Induced terpene accumulation in Norway spruce inhibits bark beetle colonization in a dose-dependent manner

Background

Tree-killing bark beetles (Coleoptera, Scolytinae) are among the most economically and ecologically important forest pests in the northern hemisphere. Induction of terpenoid-based oleoresin has long been considered important in conifer defense against bark beetles, but it has been difficult to demonstrate a direct correlation between terpene levels and resistance to bark beetle colonization.

Methods

To test for inhibitory effects of induced terpenes on colonization by the spruce bark beetle Ips typographus (L.) we inoculated 20 mature Norway spruce Picea abies (L.) Karsten trees with a virulent fungus associated with the beetle, Ceratocystis polonica (Siem.) C. Moreau, and investigated induced terpene levels and beetle colonization in the bark.

Results

Fungal inoculation induced very strong and highly variable terpene accumulation 35 days after inoculation. Trees with high induced terpene levels (n = 7) had only 4.9% as many beetle attacks (5.1 vs. 103.5 attacks m⁻²) and 2.6% as much gallery length (0.029 m m⁻² vs. 1.11 m m⁻²) as trees with low terpene levels (n = 6). There was a highly significant rank correlation between terpene levels at day 35 and beetle colonization in individual trees. The relationship between induced terpene levels and beetle colonization was not linear but thresholded: above a low threshold concentration of ∼100 mg terpene g⁻¹ dry phloem trees suffered only moderate beetle colonization, and above a high threshold of ∼200 mg terpene g⁻¹ dry phloem trees were virtually unattacked.

Conclusion/Significance

This is the first study demonstrating a dose-dependent relationship between induced terpenes and tree resistance to bark beetle colonization under field conditions, indicating that terpene induction may be instrumental in tree resistance. This knowledge could be useful for developing management strategies that decrease the impact of tree-killing bark beetles.     

Phytogenic biosynthesis and emission of methyl acetate

Acetylation of plant metabolites fundamentally changes their volatility, solubility and activity as semiochemicals. Here we present a new technique termed dynamic ¹³C‐pulse chasing to track the fate of C_1–3 carbon atoms of pyruvate into the biosynthesis and emission of methyl acetate (MA) and CO₂. ¹³C‐labelling of MA and CO₂ branch emissions respond within minutes to changes in ¹³C‐positionally labelled pyruvate solutions fed through the transpiration stream. Strong ¹³C‐labelling of MA emissions occurred only under pyruvate‐2‐¹³C and pyruvate‐2,3‐¹³C feeding, but not pyruvate‐1‐¹³C feeding. In contrast, strong ¹³CO₂ emissions were only observed under pyruvate‐1‐¹³C feeding. These results demonstrate that MA (and other volatile and non‐volatile metabolites) derive from the C_2,3 atoms of pyruvate while the C₁ atom undergoes decarboxylation. The latter is a non‐mitochondrial source of CO₂ in the light generally not considered in studies of CO₂ sources and sinks. Within a tropical rainforest mesocosm, we also observed atmospheric concentrations of MA up to 0.6 ppbv that tracked light and temperature conditions. Moreover, signals partially attributed to MA were observed in ambient air within and above a tropical rainforest in the Amazon. Our study highlights the potential importance of acetyl coenzyme A (CoA) biosynthesis as a source of acetate esters and CO₂ to the atmosphere.     

Impact of methyl jasmonate on squalene biosynthesis in microalga Schizochytrium mangrovei

Squalene is an effective antioxidant and a potential chemopreventive agent. In this work, the effect of methyl jasmonate (MJA) on squalene biosynthesis in microalga Schizochytrium mangrovei was investigated. The maximum squalene content (1.17 ± 0.06 mg/g cell dry weight, DW) reached during the next 3 h after MJA treatment (0.1 mM) at 48 h of cultivation, which was 60% higher than that of control. The activity of squalene synthase (SS) increased 2-fold over control at this point. The maximum cholesterol content of 0.45 ± 0.03 mg/g DW was reached at hour 51 when MJA concentration was 0.4 mM, whereas the squalene content was lower at this point. The observations suggested that the increased squalene content was resulted from an increased activity of SS. MJA could be used to regulate the key enzymes in squalene biosynthetic pathway for the increased production of this compound in thraustochytrids. This research also provided novel information on the stimulation effect of methyl jasmonate on the biosynthesis of essential intermediate involved in the primary metabolism in microorganism.     

Stimulation of Rg3 ginsenoside biosynthesis in ginseng hairy roots elicited by methyl jasmonate

It has been recognized that ginsenoside Rg3 is not naturally produced in ginseng although this ginsenoside can accumulate in red ginseng as the result of a thermal process. In order to determine whether or not Rg3 is synthesized in ginseng, hairy roots were treated with methyl jasmonate (MJ). From HPLC analysis, no peak for Rg3 was observed in the controls. However, Rg3 did accumulate in hairy roots that were MJ-treated for 7?days. Rg3 content was 0.42?mg/g (dry weight). To gain more insight into the effects of MJ on UDP-glucosyltransferase (UGT) activity, we attempted to evaluate ginsenoside Rg3 biosynthesis by UGT. A new peak for putative Rg3 was observed, which was confirmed by LC-MS/MS analysis. Our findings indicate that the proteins extracted from our hairy root lines can catalyze Rg3 from Rh2. This suggests that our ginseng hairy root lines possess Rg3 biosynthesis capacity.     

Ethylene biosynthesis in Amaranthus caudatus seeds in response to methyl jasmonate

Methyl jasmonate (JA-Me) at 10^–3 M completely inhibited Amaranthus caudatus seed germination. Exogenous ethylene could totally reverse this inhibition. The inhibitor of ethylene action, 2,5-norbornadiene (NBD), increased the sensitivity of seeds to JA-Me. Methyl jasmonate inhibited ethylene production and also decreased both 1-aminocyclopropane-1-carboxylic acid (ACC) and malonyl ACC (MACC) content. Likewise, ACC oxidase activity in vivo was decreased by jasmonate. Similarly ACC oxidase activity in vitro isolated from seeds incubated in the presence of JA-Me was lower than that isolated from untreated seeds.The inhibitory JA-Me action on seed germination seems to be mainly associated with the inhibition of ethylene biosynthesis. Both inhibition of ACC synthase and ACC oxidase activity and/or synthesis can be involved.     

Wood diameter indicates diurnal and long-term patterns of xylem water potential in Norway spruce

The stem diameter of adult Norway spruce trees was measured to see whether changes in xylem water potential lead to detectable radial deformation of the wood. The dendrometers used in these experiments measured only the dimensional changes of the woody cylinder (sap- and heartwood). Wood diameter was measured close to the ground and just below the living crown. After correction for thermal expansion of dendrometers and wood, diurnal variation of wood diameter ranged between 50 and 180 µm. Psychrometric measurements showed that xylem water potential varied in parallel to wood diameter. Diameter changes were always more pronounced at the higher stem position and exhibited a clear diurnal pattern. During the day, wood diameter decreased with increasing vapor pressure deficit and transpiration rate and with decreasing twig water potential. At night, the wood re-expanded but did not always reach the dimension of the previous day. Pre-dawn wood diameter decreased during periods of soil drought, a process which rapidly stopped and reversed after rain events. On several days, oscillation in wood diameter was observed during the mid-day hours. The oscillation had a period of approximately 50 min and showed a phase shift between different stem heights. All observed patterns of wood shrinkage and expansion were consistent with the hypothesis that xylem water tension leads to an elastic contraction of xylem conduits. The results demonstrate that xylem diameter is more suitable than whole-stem diameter for monitoring changes in xylem water potential.     

棉蚜和茉莉酸甲酯诱导棉花挥发物组分分析

采用固相微萃取和气质联用技术,对健康棉叶、蚜害棉叶、蚜害棉叶-棉蚜复合物、茉莉酸甲酯处理棉叶以及棉蚜蜜露和棉蚜自身挥发物成分进行了提取和组分分析。共检测出27种挥发性化学物质。健康棉叶、蚜害棉叶、蚜害棉叶-棉蚜复合物、茉莉酸甲酯处理棉叶挥发物中,均含α-蒎烯、β-蒎烯、β-石竹烯、十五烷、十六烷、十七烷、-Cyclohexen-1-ol,1-(1,5-dimethyl-4-hexenyl)-4-methyl-7种组份。蚜害棉叶或蚜害棉叶-棉蚜复合物中检测到α-水芹烯、α-石竹烯、α-法尼烯、2,6,10-三甲基十五烷、(E,Z)-2,6-二甲基-2,4,6-辛三烯、2,6,10,14-四甲基十五烷、蒽、二酚基丙烷8种组份;3-蒈烯、(E)-β-法尼烯只在蚜害棉叶及茉莉酸甲酯处理棉叶中被检测出。在棉蚜及其蜜露中都能检查到壬醛、癸醛、十四烷、十五烷、十六烷、十七烷;而1-甲氧基-4-[(Z)-1-丙烯基]苯、(E)-β-法尼烯、菲只在棉蚜蜜露中被检查到;6,10-二甲基-5,9-十一双烯-2-酮只在棉蚜中被检测到。研究结果为进一步开展棉花、棉蚜挥发物对天敌昆虫的吸引作用研究奠定了基础。     

Morphological and stomatal responses of Norway spruce foliage to irradiance within a canopy depending on shoot age

Morphological and stomatal responses of Norway spruce (Picea abies) foliage to light availability were studied in respect to shoot age. Needle minor diameter (D(1), anatomical width), major diameter (D(2), anatomical thickness), dry weight (M), and tissue density index (I(D)) increased, and needle flatness (Fl) and specific leaf area (SLA) decreased with foliage age, while shade foliage demonstrated higher morphological plasticity as compared to sun foliage. Needle minor diameter, dry weight, and the ratio of total to projected leaf area increased, and needle flatness and specific leaf area decreased with daily average photosynthetic photon flux density (Q(D)). The current-year foliage exhibited the highest variation with irradiance, while the morphological plasticity decreased with needle ageing. The morphological characteristics of needles were independent of irradiance if Q(D) was above 300 μmol m(-2) s(-1). D(1) was the only linear needle characteristic which significantly changed with light availability within a canopy, and thus determined needle flatness, SLA, as well as the ratio of total to projected leaf area (TLA/PLA). Needle flatness was a characteristic responding most sensitively to the photosynthetic photon flux density, R(2) was 0.68, 0.44, and 0.49 for the current-year, 1-year-old, and 2-year-old foliage, respectively. TLA/PLA ranged from 2.2 to 4.0 depending on D(1). Variation in SLA in response to light availability can be attributed to changes both in needle shape and tissue density. Stomatal responses to photosynthetic photon flux density (Q(P)) depended on foliage type (sun or shade) and age. Sun needles demonstrated higher daily maximum leaf conductances to water vapour compared to shade needles. The shade needles responded more sensitively to changes in Q(P) at dawn and sunset than the sun needles, while older needles of both foliage types exhibited faster stomatal responses. The light-saturation of leaf conductance (g(L)) was achieved by 20 μmol m(-2) s(-1) for shade foliage, and approximately by 50 μmol m(-2) s(-1) for sun foliage. As a rule, g(L) changed in response to irradiance faster in the evening, i.e. at decreasing irradiance. Stomata were not usually completely closed in the dark before sunrise and after sunset, the phenomenon being more pronounced in older shoots and sun needles. Nightly water losses from spruce foliage are attributable primarily to older shoots, and are related to age-dependent changes in stomatal responsiveness.