Differential anatomical response of Norway spruce stem tissues to sterile and fungus infected inoculations

The anatomical defense responses in stems of Norway spruce (Picea abies) clones of different resistance to pathogenic fungi were characterized over time and distance from small mechanical wounds or wounds inoculated with the root rot fungus Heterobasidion annosum. Common responses for both treatments included division of ray parenchyma and other cells in the cambial zone, accumulation of phenolic inclusions in ray parenchyma cells, activation of phloem parenchyma (PP) cells, and formation of traumatic resin ducts (TDs) in the xylem. TD formation occurred synchronously from a tangential layer of cells, or symplasmic domain, within the zone of xylem mother cells. TD induction is triggered by a signal, which propagates a developmental wave in the axial direction at about 2.5 cm per day. TDs are formed at least 30 cm above single inoculations within 16–36 days after inoculation. The size and number of TDs is attenuated further away from the inoculation site, indicating a dose-dependent activity leading to TD development. Compared to sterile wounding, fungal inoculation gave rise to more and larger TDs in all clones, and multiple rows of TDs in weak clones. Fungal inoculation also induced the formation of more new PP cells, increasing the number of PP cells in the phloem in the year of inoculation up to 100%. TD and PP cell formation was greater in susceptible compared to resistant clones and after fungal versus sterile inoculation. Potential mechanisms responsible for this variable response are discussed.     

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.     

Wound-induced traumatic resin duct development in stems of Norway spruce (Pinaceae): anatomy and cytochemical traits

Wounding of Norway spruce by inoculation with sterile agar, or agar containing the pathogenic fungus Ceratocystis polonica, induced traumatic resin duct formation in the stem. Visible anatomical responses occurred in the cambium 6-9 d post-inoculation. Near the inoculation site cellular proliferation, polyphenolic accumulation, and lignification were induced as a wound reaction to seal the damaged area. Five centimetres from the inoculation site cells in the cambial zone swelled and divided to form clusters. By 18 d post-inoculation, these cells began to differentiate into resin duct epithelial cells surrounding incipient schizogenous lumens. Mature axial traumatic ducts appeared by 36 d as a row of ducts in the xylem centripetal to the cambium. The ducts formed an interconnected network continuous with radial resin ducts. Parenchyma cells surrounding the ducts accumulated polyphenols that disappeared as the cells differentiated into tracheids. These polyphenols appeared to contain fewer sugar residues compared to those accumulating in the secondary phloem, as indicated by the periodic acid-Schiff's staining. The epithelial cells did not accumulate polyphenols but contained immunologically detectable phenylalanine ammonia lyase (EC 4.3.1.5), indicating synthesis of phenolics as a possible resin component. These findings may represent a defense mechanism in Norway spruce against the pathogenic fungus Ceratocystis polonica.     

Induced responses in stilbenes and terpenes in fertilized Norway spruce after inoculation with blue-stain fungus, Ceratocystis polonica

In conifers, attacks by bark beetles and associated pathogenic fungi cause an induced wound response, which is characterized by accumulation of antifungal compounds and morphological changes that aid wound healing. In this article the stilbene and terpene concentrations of Norway spruce phloem were monitored as symptoms of induced wound responses in relation to changed nutrient conditions caused by fertilization. Plots of mature Norway spruce were fertilized with N, P or NPK. One year after fertilization the trees were artificially infected with Ceratocystis polonica, a pathogenic fungus associated with the bark beetle Ips typographus. The response of stilbenes to fungal inoculation was mainly qualitative. The concentration of stilbene glycosides in the phloem decreased, and in the immediate vicinity of the site of fungal inoculation, stilbene glycosides were less frequent than in mechanically wounded or unwounded phloem. Corresponding stilbene aglycones were most frequent inside the reaction lesion. The concentration of total stilbene aglycones near the inoculation site was significantly lower in N-fertilized trees than in unfertilized trees. Fungal inoculation caused a strong quantitative response in terpenes. The total terpene concentration of the phloem increased significantly, to almost 100 times greater near the inoculation site compared to the constitutive values. N fertilization significantly reduced the total terpene and total stilbene aglycone concentrations near the inoculation sites. Thus, N fertilization may reduce the ability of Norway spruce to defend itself against fungal pathogens.     

Ethylene in induced conifer defense: cDNA cloning, protein expression, and cellular and subcellular localization of 1-aminocyclopropane-1-carboxylate oxidase in resin duct and phenolic parenchyma cells

Members of the Pinaceae family have complex chemical defense strategies. Conifer defenses associated with specialized cell types of the bark involve constitutive and inducible accumulation of phenolic compounds in polyphenolic phloem parenchyma cells and oleoresin terpenoids in resin ducts. These defenses can protect trees against insect herbivory and fungal colonization. The phytohormone ethylene has been shown to induce the same anatomical and cellular defense responses that occur following insect feeding, mechanical wounding, or fungal inoculation in Douglas fir (Pseudotsuga menziesii) stems (Hudgins and Franceschi in Plant Physiol 135:2134–2149, 2004). However, very little is known about the genes involved in ethylene formation in conifer defense or about the temporal and spatial patterns of their protein expression. The enzyme 1-aminocyclopropane-1-carboxylate oxidase (ACO) catalyzes the final step in ethylene biosynthesis. We cloned full-length and near full-length ACO cDNAs from three conifer species, Sitka spruce (Picea sitchensis), white spruce (P. glauca), and Douglas fir, each with high similarity to Arabidopsis thaliana ACO proteins. Using an Arabidopsis anti-ACO antibody we determined that ACO is constitutively expressed in Douglas fir stem tissues and is up-regulated by mechanical wounding, consistent with the wound-induced increase of ethylene levels. Immunolocalization showed cytosolic ACO is predominantly present in specialized cell types of the wound-induced bark, specifically in epithelial cells of terpenoid-producing cortical resin ducts, in polyphenolic phloem parenchyma cells, and in ray parenchyma cells.J.W. Hudgins and Steven G. Ralph contributed equally to this work.     

Traumatic oil glands induced by pruning in the wound-associated phloem of Eucalyptus globulus: chemistry and histology

The natural occurrence of oil glands in various organs such as bark and leaves is well established as a characteristic of Eucalyptus, but this is the first reported case of traumatic oil glands induced in response to wounding. The new phloem enveloping the wound, which had developed within the 2 years following branch pruning in 5-year-old Eucalyptus globulus Labill., was morphologically distinct from healthy stem phloem. Histological examinations revealed this wound-associated phloem to be largely composed of secretory cavities similar in appearance to oil glands. Subsequent analysis of the wound-associated phloem extracts by GC-MS confirmed the presence of volatile terpenes and phenols. The total extracted oil content determined for wound-associated phloem extracts was significantly higher (>4 times) than for healthy stem phloem extracts. A comparison of the relative abundances of ten individual terpenoids from wound-associated phloem and healthy phloem revealed a number of significant differences in terpene composition. Implications of the role of terpenes as inducible secondary metabolites in tree wound responses are discussed.     

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.     

Quantitative comparison of chemical, biological and mechanical induction of secondary compounds in Pinus pinaster seedlings

Chemical elicitors and mechanical treatments simulating real insect herbivory have been increasingly used to study induced defensive responses in woody plants. However, simultaneous quantitative comparisons of plant chemical defences elicited by real and simulated herbivory have received little attention. In this paper we compared the effects of real herbivory, simulated herbivory using two chemical elicitors, and mechanical damage treatments on the quantitative secondary chemistry of Pinus pinaster juveniles (namely on non-volatile resin in the stem and total phenolics in the needles). The real herbivory involved phloem wounding by Hylobius abietis and defoliation by Brachyderes lusitanicus (two pine weevils); the chemical elicitors to simulate herbivory induction were 40 mM methyl jasmonate (MJ) and 20 μM benzothiadiazole (BTH); and the mechanical treatments included phloem wounding and needle clipping. We also performed an additional experiment for assessing at what extent insect extracts could increase plant responses over mechanical damage. Chemical induction with MJ, mechanical wounding and real phloem herbivory by H. abietis all produced quantitatively similar results, increasing the concentration of resin in the stem and total phenolics in the needles by equivalent magnitudes. Exogenous application of BTH increased the concentration of phenolic compounds in pine needles, but did not show the same effect on stem resin. Contrastingly, we did not find significant changes in the concentration of resin in the stem or phenolics in the needles after needle clipping and B. lusitanicus feeding. Mechanical damage followed by the application of extracts from the insects B. lusitanicus and H. abietis on the injured tissues did not increase the responses in comparison to mechanical damage alone. The fact that strong induced responses elicited by phloem wounding insects are equally elicited by phloem injuries suggests that defences in pine trees are raised with low specificity regarding biotic enemies. Results from this paper support future methodological approaches using chemical elicitors and mechanical damage as simulated herbivory treatments for the experimental induction of conifer defences.     

Accumulation of essential oils in relation to root differentiation in Angelica archangelica L

The accumulation of essential oils in Angelica archangelica subsp. archangelica roots at different developmental stages was investigated through histochemical and chemical analyses. Roots less than 1 mm in diameter showed a primary diarch structure and two primary secretory ducts in the pericycle. These ducts were ephemeral and probably became dysfunctional early on. Oil accumulation was observed only in the secondary secretory ducts formed by cambium activity and located in the secondary phloem. Gas chromatographic analyses revealed that only taproots exceeding 5 mm in diameter contained a high concentration of alpha- and beta-phellandrene, which appreciably influence the oil's aroma.     

Application of methyl jasmonate on Picea abies (Pinaceae) stems induces defense-related responses in phloem and xylem

Application of 100 mmol/L methyl jasmonate (MJ) to the intact bark of 30-yr-old Norway spruce induced anatomical reactions related to defense. Within 30 d, a single MJ treatment induced swelling of existing polyphenolic parenchyma cells (PP cells) and an increase in their phenolic contents and formation of additional PP cells and of traumatic resin ducts (TDs) at the cambial zone. These changes occurred up to 7 cm away from the application zone. Treatment enhanced resin flow and increased resistance to the blue-stain fungus, Ceratocystis polonica. Methyl jasmonate application to the oldest internode of 2-yr-old saplings also induced TD formation, and, more surprisingly, TDs were formed in the untreated internode. Traumatic ducts were not formed in branches, ruling out an effect of volatile MJ on the upper internode. Methyl jasmonate application never gave rise to a hypersensitive response, cell death, tissue necrosis, or wound periderm, indicating the amount of MJ transported across the periderm was very low relative to the application concentration. This is the first report of a single compound giving rise to major cellular features related to acquired resistance and previously shown to be induced by wounding, fungal infection, and bark beetles in Norway spruce.     

Ultrastructural evidence for a dual function of the phloem and programmed cell death in the floral nectary of Digitalis purpurea

BACKGROUND AND AIMS: The floral nectary of Digitalis purpurea is a transitory organ with stomatal exudation of nectar. In this type of nectary, the nectar is thought to be transported to the exterior via intercellular ducts that traverse the nectariferous tissue. The latter is also traversed by a ramified system of phloem strands from which prenectar sugar is most probably unloaded. The aims of this study were to provide some of the basic information needed to evaluate the possible mechanism involved in nectar secretion and to discover the fate of the nectary. METHODS: The ultrastructure of the nectary was investigated at different stages of development by analysis of a series of ultrathin (7 x 10(-8) m) sections 7 x 10(-7) m apart from one another. Proportions of the cells typical of the nectary were documented by 3D-reconstruction and morphometry. KEY RESULTS: The phloem consisted of variably shaped sieve elements and companion cells which, as a rule, were more voluminous than the sieve elements. Direct contact between the phloem strands and intercellular ducts was observed. In contrast to the phloem, which remained structurally intact beyond the secretory phase, the nectariferous tissue exhibited degenerative changes reminiscent of programmed cell death (PCD), which started as early as the onset of secretion and progressed in a cascade-like fashion until final cell death occurred in the exhausted nectary. Hallmarks of PCD were: increased vacuolation; increase in electron opacity of individual cells; progressive incorporation of plasmatic components into the vacuole reminiscent of autophagy; degradation of plastids starting with hydrolysis of starch; deformation of the nucleus and gradual disappearance of chromatin; loss of tonoplast integrity and subsequent autolysis of the rest of cellular debris. Degeneration of the cells occurred against a background of increasing cell size. CONCLUSIONS: The cytological and anatomical evidence presented here, and calculations of the solute fluxes necessary for accumulation of starch and for the production of nectar support the view that: (a) in the foxgloves' nectary, apoplastic phloem unloading dominates, at least during exudation of nectar; (b) the obsolete nectary may be dismantled by PCD; and (c) at least the products of late nectary degradation are loaded via the apoplast into the unchanged phloem and exported to sinks elsewhere in the plant for reallocation.     

Resin secretory canals in <Emphasis Type="Italic">Protium heptaphyllum</Emphasis> (Aubl.) Marchand. (Burseraceae): a tridimensional branched and anastomosed system

Protium heptaphyllum is a Burseraceae species known by the production of aromatic resin with medicinal, economic, and ecological values. Information on the development, architecture, and lifetime of the secretory system are crucial to understand the resin production and contribute to a more sustainable tapping regime. We investigated the histology and ultrastructure of the secretory canals under a developmental point of view. Stem samples were analyzed under light and transmission electron microscopy by conventional and cytochemical methods. Secretory canals, originated from procambium and cambium, occurred immersed in the primary and secondary phloem. Mature canals have a secretory epithelium and a wide lumen where the exudate is accumulated. A sheath of parenchyma cells with meristematic features surrounds the epithelium. The canals originate by schizogenesis and develop by schyzolysigenesis. Canals active in secretion occurred since the shoot apex and near the cambium. In the dilation zone of the secondary phloem, secretory canals exhibit sclerified epithelial and sheath cells and are inactive in secretion. Secreting epithelial cells have subcellular apparatus consistent with oleoresin, polysaccharides, and enzymes secretion. Pectinase and cellulase were cytochemically detected in developing canals and are involved in cell wall changes associated to canal growth and release of exudate. In P. heptaphyllum, the secretory system has a complex structure resultant from longitudinal growth, lateral ramification, and fusion of the adjacent canals, in addition to intrusive growth of both epithelial and sheath cells. Although some anatomical results are already known, ultrastructural data represent the novelty of this work. Our findings can contribute to the establishment of more efficient and sustainable techniques for resin extraction in this species.     

Phloem parenchyma cells are involved in local and distant defense responses to fungal inoculation or bark-beetle attack in Norway spruce (Pinaceae)

The anatomical response of Norway spruce bark polyphenolic parenchyma cells (PP cells) to inoculation with the phytopathogenic fungus Ceratocystis polonica and attack by its bark-beetle vector Ips typographus was examined. Fungal inoculation on the periderm surface had no effect, while inoculation just below the periderm or halfway into the phloem (mid-phloem) generated detectable responses within 3 wk. The responses included increase in PP cell size and in periodic acid-Schiff's staining of PP cell phenolics, wound periderm initiation from PP cells, and cambial zone traumatic resin duct formation. Fungi were not seen in samples 3 wk after subperiderm or mid-phloem inoculation, but were found in some samples 6 and 9 wk after mid-phloem inoculation. In contrast, inoculations into the cambium resulted in partial (3 wk) or complete (6 and 9 wk) fungal colonization and death of tissue in the infected area. This indicates that PP cells have defenses capable of inhibiting fungal growth. Samples taken near bark-beetle galleries had similar anatomical responses as inoculated samples, validating the inoculation approach to studying defense responses in spruce. These results show that PP cells represent not only a constitutive defense system, but are also involved in local and remote inducible defenses against fungal and beetle attack.     

臭椿茎中分泌道的发育及其组织化学研究

利用植物解剖学方法研究臭椿茎和叶柄中分泌道的结构、分布和发育过程.结果表明:臭椿茎和叶柄中的分泌道分布于髓的周缘,次生木质部中无分泌道.分泌道是由一层分泌细胞围绕分泌腔而构成,分泌细胞外有1～2层鞘细胞.分泌道以裂生方式形成,其发育过程可分为3个阶段:原始细胞阶段、形成阶段和成熟阶段.在原始细胞阶段,一群原始细胞具浓厚细胞质,细胞核清晰可见;形成阶段,原始细胞的中央细胞间细胞壁中层降解,细胞壁分离,形成腔隙,随着分泌细胞数量的增加,分泌腔体积扩大;成熟阶段的分泌道具有12～16个分泌细胞,1～2层鞘细胞,分泌腔直径为30～50μm.组织化学研究表明,分泌细胞及分泌道内含物中含大量的萜类、多糖和脂类物质.机械创伤能够诱导次生木质部中产生创伤分泌道.臭椿茎中的分泌道和创伤性分泌道在抵御生物和非生物胁迫中起重要作用.     

Defense response of pine stem phloem to wounding and treatment with mycelial extracts from <Emphasis Type="Italic">Ceratocystis laricicola</Emphasis>

Ophiostomatoid fungi colonize the conducting tissues of conifer stems, the phloem and the xylem. These pathogenic fungi penetrate into the stem through injuries made by xylophagous insects vectoring these pathogens. In this study the response of the phloem of Scotch pine (Pinus sylvestris L.) to wounding (treatment 1) was compared with the response to wounding combined with application of high-molecular-weight compounds isolated from the mycelium of the ophiostomatoid fungus Ceratocystis laricicola Redfern & Minter (treatment 2). Both treatments induced the appearance of necrosis in the inner bark, the formation of periderm separating living and dead tissues, and formation of the callus alongside the wound perimeter. In addition, the bark accumulated lignin, bound proanthocyanidins, and resins, with a parallel decrease in the content of free proanthocyanidins, low-molecular-weight carbohydrates, and non-lignin components of the cell wall (P > 0.95). The size of necrotic spots, as well as changes in the content of most substances, were significantly higher in the treatment 2 than in the treatment 1 (P > 0.95). The accumulation of lignin in cell walls of phloem sieve cells was delayed in the treatment 2 as compared with that in the treatment 1. This suggested that the mycelial extract temporarily inhibited lignification at the early stage of the wound response. This disturbance of the cell wall protective transformation led to the hypothesis that the fungal suppressors retard the repair of inner bark injured by insects, thereby favoring the invasion of conifer tissues by ophiostomatoid fungi.     

Concentrations of lignin and wall-bound ferulic acid after wounding in the phloem of Chamaecyparis obtusa

The changes of lignin and wall-bound ferulic acid induced by wounding were quantitatively and histochemically investigated in the phloem of Chamaecyparis obtusa. Histochemical staining of lignin was first observed in the necrotic region of the phloem 7 days after wounding and developed in 14 days. Increases of the wall-bound ferulic acid and lignin concentrations were detected in the necrotic tissue at 7 and 14 days, respectively. The concentrations continued to increase until 28 days. The lignin concentration of the callus tissue was observed to be lower than that of the healthy tissue at 14 days, and reached a similar level after 28 days. No quantitative changes of lignin and wall-bound ferulic acid were observed in other tissues. The results indicated that lignin synthesis could be maintained after the phloem cells were discolored and seemed to be necrotic. Distribution and timing of the wall-bound ferulic acid and lignin accumulation suggested that the increased wall-bound ferulic acid was involved in the lignin synthesis after wounding.     

Resin ducts of Pinus hatepensis Mill.–Their structure, development and pattern of arrangement

The primary resin ducts in the axis of plants of Pinus halepensis Mill, consist of two separate systems the pattern of which is correlated with the vascular systems of the organs in which they appear. These systems are: (1) ducts of the roots and the hypocotyl; (2) ducts of all the branches and juvenile leaves or scaleS. Both systems are produced by the apical meristemS. In the needles there is a third system of primary resin ducts situated in the mesophyll. These ducts are produced only to a small extent by the apical meristem of the needle and mainly by its intercalary meristem. In addition to these primary ducts of the needle, which form a separate system for each needle, at the base of the needle there may be ducts of secondary origin which are situated within the vein. These are continuous with secondary ducts of the brachyblast axis.
The secondary ducts constitute one system in the secondary xylem and phloem of the roots, branches and needle bases. They are formed by the cambium. In the xylem there are vertical and radial ducts which together form co-planar radial networkS. Each radial duct starts from a vertical duct. The first location of the stimulus for the formation of the two types of ducts is discussed. In the phloem there are only radial ducts, continuous with the radial ducts of the xylem. The cavities of the radial phloem and xylem ducts are not continuous, as there are no intercellular spaces in the region of the cambium.
The innermost vertical ducts of the secondary xylem form a kind of transitional type, in respect of their response to internal and external factors, between the primary resin ducts and the bulk of the secondary resin ducts.     

Vegetative anatomy of Pachycortnus (Anacardiaceae)

Pachycormus discolor , an arborescent desert perennial endemic to Baja California, has small, pinnately compound, hypostomatic, bifacial leaves produced on short shoots and photosynthetic stem phelloderm covered by exfoliating translucent phellem. Tightly packed laminal palisade cells are filled with tannins and lack chloroplasts. Spongy mesophyll is the major photosynthetic tissue. Leaves possess unicellular trichomes with secondary walls and uniseriate trichomes with glandular heads. Schizogenous resin ducts occur in primary phloem of stems, leaves and roots as well as all living tissues of the bark. Developmental studies reveal that initiation and differentiation of foliar primordia resembles that of other dicotyledons except that tannin cells and secretory ducts arise precociously. Primary vasculature is an open sympodial system with three principal traces diverging toward each foliar primordium. The wood is highly specialized and comprises mostly unlignified cells packed with starch grains. Thick bark is mainly produced as annual layers of secondary phloem marked by a ring of secretory ducts each surrounded by tannin cells. The possible adaptive significance of these unusual anatomical features is discussed.