Effects of Flooding, Tilting of Stems, and Ethrel Application on Growth, Stem Anatomy and Ethylene Production of Pinus densiflora Seedlings

Flooding of soil, tilting of seedlings, application of ethrelto stems, and combinations of these treatments, variously alteredthe rate of growth and stem anatomy of 2-year-old Pinus densifloraseedlings. Either flooding or tilting increased stem diametergrowth and induced formation of abnormal xylem. Whereas floodingdecreased the rate of dry weight increment of roots and needlesand increased growth of bark tissues, tilting of stems did not.However, tilting decreased the rate of height growth, stimulatedtracheid production, and induced formation of well-developedcompression wood with rounded, thick-walled tracheids, witha high lignin content but without an S3 layer in the tracheidwall. Ethylene appeared to have an important regulatory rolein stimulating growth of bark tissues as shown by thicker barkin flooded seedlings or those treated with ethrel. Ethyleneappeared to have a less important role in regulating formationof compression wood. Flooding increased the ethylene contentsof stems and induced formation of rounded, thick-walled tracheids.However, these tracheids lacked such features of well-developedcompression wood tracheids as a thick S2 layer, high lignincontent, and absence of an S3 layer. Furthermore, applicationof ethrel to vertical stems greatly increased their ethylenecontents but did not induce formation of well-developed compressionwood. Furthermore, ethrel application blocked development ofcertain characteristics of compression wood when applied totilted seedlings. For example an S3 wall layer was absent intracheids of tilted seedlings but present in tracheids of tilted,ethrel-treated seedlings. Also lignification of tracheids wasincreased on the under side of tilted stems, but reduced intilted, ethrel-treated seedlings, further de-emphasizing a directrole of ethylene in the formation of compression wood. Ethreltreatment induced formation of longitudinal resin ducts in thexylem whereas flooding or tilting of stems did not. Key words: Pinus densiflora, xylogenesis, reaction wood, compression wood, lignification, ethrel, ethylene     

ANATOMY OF A LIGNITIZED WOOD FROM SENFTENBERG

A piece of lignitized wood from the Miocene of Senftenberg, Germany, was identified as a type of compression wood, and was studied anatomically, as groundwork, in an investigation on cell wall cellulose and lignin. The wood showed affinity to modern Cryptomeria and Taxodium. In each growth ring the early wood was compacted. By contrast, cellular structure in the late wood had been retained to a high degree. Massive compaction of the early wood most likely occurred during the initial stages of sedimentation prior to mineralization. Numerous cell wall deformations in late wood tracheids apparently originated from compressive forces applied to the wood during sedimentation.     

Occurrence of xylan and mannan polysaccharides and their spatial relationship with other cell wall components in differentiating compression wood tracheids of <Emphasis Type="Italic">Cryptomeria japonica</Emphasis>

Compression wood (CW) tracheids have different cell wall components than normal wood (NW) tracheids. However, temporal and spatial information on cell wall components in CW tracheids is poorly understood. We investigated the distribution of arabino-4-O-methylglucuronoxylans (AGXs) and O-acetyl-galactoglucomannans (GGMs) in differentiating CW tracheids. AGX labeling began to be detected in the corner of the S₁ layer at the early S₁ formation stage. Subsequently, the cell corner middle lamella (ccML) showed strong AGX labeling when intercellular spaces were not fully formed. AGX labeling was uniformly distributed in the S₁ layer, but showed uneven distribution in the S₂ layer. AGX labeling was mainly detected in the inner S₂ layer after the beginning of the helical cavity formation. The outer S₂ layer showed almost no labeling of low substituted AGXs. Only a very small amount of high substituted AGXs was distributed in the outer S₂ layer. These patterns of AGX labeling in the S₂ layer opposed the lignin and β-1-4-galactan distribution in CW tracheids. GGM labeling patterns were almost identical to AGX labeling in the early stages of CW tracheids, and GGM labeling was detected in the entire S₂ layer from the early S₂ formation stage of CW tracheids with some spatial differences in labeling density depending on developmental stage. Compared with NW tracheids, CW tracheids showed significantly different AGX distributions in the secondary cell wall but similar GGM labeling patterns. No significant differences were observed in labeling after delignification of CW tracheids.     

Wood borer performance and wood characteristics of drought‐stressed Scots pine seedlings

Four‐year‐old Scots pine [Pinus sylvestris L. (Pinaceae)] seedlings were exposed to medium and severe drought stress for two consecutive years. The anatomical properties of drought‐stressed Scots pine wood and their impact on the performance of destructive wood boring early instars of Hylotrupes bajulus L. (Coleoptera: Cerambycidae) were studied. Drought stress significantly decreased diameter of earlywood tracheids in both growing years and diameter of latewood tracheids after the second growing season only. Cell lumen area was significantly decreased by both medium and severe drought stress compared to well‐watered controls. In addition, area of cell lumen was significantly smaller in severe drought than in medium drought treatment. The drought stress marginally increased the number of resin canals in the wood, but did not affect the size of resin canals either in wood or bark. The relative growth rate of xylophagous H. bajulus neonatal larvae was not significantly affected by drought stress during the 106‐day feeding period on Scots pine wood blocks. The results show that although water availability was an important factor affecting the development and anatomy of wood cells, observed changes in wood characteristics did not affect the performance of early instars feeding on wood processed from drought‐stressed young Scots pine seedlings.     

Immunolocalization of β-1-4-galactan and its relationship with lignin distribution in developing compression wood of Cryptomeria japonica

Compression wood (CW) contains higher quantities of β-1-4-galactan than does normal wood (NW). However, the physiological roles and ultrastructural distribution of β-1-4-galactan during CW formation are still not well understood. The present work investigated deposition of β-1-4-galactan in differentiating tracheids of Cryptomeria japonica during CW formation using an immunological probe (LM5) combined with immunomicroscopy. Our immunolabeling studies clearly showed that differences in the distribution of β-1-4-galactan between NW (and opposite wood, OW) and CW are initiated during the formation of the S₁ layer. At this stage, CW was strongly labeled in the S₁ layer, whereas no label was observed in the S₁ layer of NW and OW. Immunogold labeling showed that β-1-4-galactan in the S₁ layer of CW tracheids significantly decreased during the formation of the S₂ layer. Most β-1-4-galactan labeling was present in the outer S₂ region in mature CW tracheids, and was absent in the inner S₂ layer that contained helical cavities in the cell wall. In addition, delignified CW tracheids showed significantly more labeling of β-1-4-galactan in the secondary cell wall, suggesting that lignin is likely to mask β-1-4-galactan epitopes. The study clearly showed that β-1-4-galactan in CW was mainly deposited in the outer portion of the secondary cell wall, indicating that its distribution may be spatially consistent with lignin distribution in CW tracheids of Cryptomeria japonica.     

A new method for vulnerability analysis of small xylem areas reveals that compression wood of Norway spruce has lower hydraulic safety than opposite wood

Compression wood is formed at the underside of conifer twigs to keep branches at their equilibrium position. It differs from opposite wood anatomically and subsequently in its mechanical and hydraulic properties. The specific hydraulic conductivity (k_s) and vulnerability to drought‐induced embolism (loss of conductivity versus water potential ψ) in twigs of Norway spruce [Picea abies (L.) Karst.] were studied via cryo‐scanning electron microscope observations, dye experiments and a newly developed ‘Micro‐Sperry’ apparatus. This new technique enabled conductivity measurements in small xylem areas by insertion of syringe cannulas into twig samples. The hydraulic properties were related to anatomical parameters (tracheid diameter, wall thickness). Compression wood exhibited 79% lower k_s than opposite wood corresponding to smaller tracheid diameters. Vulnerability was higher in compression wood despite its narrower tracheids and thicker cell walls. The P₅₀ (ψ at 50% loss of conductivity) was ?3.6 MPa in opposite but only ?3.2 MPa in compression wood. Low hydraulic efficiency and low hydraulic safety indicate that compression wood has primarily a mechanical function.     

G‐fibres in storage roots of Trifolium pratense (Fabaceae): tensile stress generators for contraction

Root contraction has been described for many species within the plant kingdom for over a century, and many suggestions have been made for mechanisms behind these contractions. To move the foliage buds deeper into the soil, the proximal part of the storage root of Trifolium pratense contracts by up to 30%. Anatomical studies have shown undeformed fibres next to strongly deformed tissues. Raman imaging revealed that these fibres are chemically and structurally very similar to poplar (Populus) tension wood fibres, which are known to generate high tensile stresses and bend leaning stems or branches upright. Analogously, an almost pure cellulosic layer is laid down in the lumen of certain root fibres, on a thin lignified secondary cell wall layer. To reveal its stress generation capacities, the thick cellulosic layer, reminiscent of a gelatinous layer (G‐layer) in tension wood, was selectively removed by enzymatic treatment. A substantial change in the dimensions of the isolated wood fibre bundles was observed. This high stress relaxation indicates the presence of high tensile stress for root contraction. These findings indicate a mechanism of root contraction in T. pratense (red clover) actuated via tension wood fibres, which follows the same principle known for poplar tension wood.     

RELATION OF DWARFMISTLETOE (ARCEUTHOBIUM) TO THE XYLEM TISSUE OF CONIFERS. I. ANATOMY OF PARASITE SINKERS AND THEIR CONNECTION WITH HOST XYLEM

Srivastava , L. M., and K. Esau . (U. California, Davis.) Relation of dwarf mistletoe (Arceuthobium) to the xylem tissue of conifers. I. Anatomy of parasite sinkers and their connection with host xylem. Amer. Jour. Bot. 48(2): 159–167. Illus. 1961.—The anatomy of the sinkers of Arceuthobium infecting 7 species of conifers was studied by the use of serial cross, radial, and tangential sections of the host wood. The sinkers were found to be composed of parenchyma cells only, or of parenchyma cells and tracheary elements, including vessel elements. In all species tracheary cells of the sinkers had direct contacts with the host tracheids of axial and radial systems. Typically the sinkers were associated with rays of the host wood. In some species, the centripetal ends of sinkers were wedged in radially among the axial tracheids of the host, but centrifugally such sinkers were usually found associated with rays. In the region of the host cambium the sinker contained parenchyma cells meristematic in appearance and, in 6 out of 7 species, also mature tracheary elements. The oldest of these elements became stretched and ruptured, a circumstance indicating that growth occurred in the part of the sinker embedded in the host cambium. This growth appeared to be coordinated with that of the host cambium, so that the sinker became embedded in the host xylem and phloem. Radial centripetal penetration of sinkers among differentiating axial tracheids of the host possibly occurred to a limited extent.     

Exploring the Ultrastructural Localization and Biosynthesis of β(1,4)-Galactan in Pinus radiata Compression Wood

Softwood species such as pines react to gravitropic stimuli by producing compression wood, which unlike normal wood contains significant amounts of β(1,4)-galactan. Currently, little is known regarding the biosynthesis or physiological function of this polymer or the regulation of its deposition. The subcellular location of β(1,4)-galactan in developing tracheids was investigated in Pinus radiata D. Don using anti-β(1,4)-galactan antibodies to gain insight into its possible physiological role in compression wood. β(1,4)-Galactan was prominent and evenly distributed throughout the S2 layer of developing tracheid cell walls in P. radiata compression wood. In contrast, β(1,4)-galactan was not detected in normal wood. Greatly reduced antibody labeling was observed in fully lignified compression wood tracheids, implying that lignification results in masking of the epitope. To begin to understand the biosynthesis of galactan and its regulation, an assay was developed to monitor the enzyme that elongates the β(1,4)-galactan backbone in pine. A β(1,4)-galactosyltransferase (GalT) activity capable of extending 2-aminopyridine-labeled galacto-oligosaccharides was found to be associated with microsomes. Digestion of the enzymatic products using a β(1,4)-specific endogalactanase confirmed the production of β(1,4)-galactan by this enzyme. This GalT activity was substantially higher in compression wood relative to normal wood. Characterization of the identified pine GalT enzyme activity revealed pH and temperature optima of 7.0 and 20°C, respectively. The β(1,4)-galactan produced by the pine GalT had a higher degree of polymerization than most pectic galactans found in angiosperms. This observation is consistent with the high degree of polymerization of the naturally occurring β(1,4)-galactan in pine.The ability to respond to gravitropic stimuli is important for the survival of most terrestrial plants. Arborescent angiosperm and gymnosperm species generate wood with modified properties, called reaction wood, in response to gravitropic stimuli (Timell, 1969, 1986; Du and Yamamoto, 2007). The formation of reaction wood enables the return of bent stems to a vertical orientation. Interestingly, the location and type of the reaction wood deposited in woody gymnosperms and angiosperms generally differs significantly. Gymnosperms respond to gravitropic stimuli by compression wood formation on the underside of leaning stems (Timell, 1986), and arboreal angiosperms generate reaction wood primarily in the form of tension wood on the upper side of inclined stems (Timell, 1969).Compression wood in conifers differs significantly from normal wood in its anatomical, chemical, and physical properties. Typical anatomical features of severe compression wood are short, rounded, and thick-walled tracheids with a prominent band of lignin in the outer S2 layer of the cell wall as well as spiral checks and the absence of an S3 layer (Timell, 1986). Biochemically, compression wood is characterized by high levels of lignin, rich in condensed p-hydroxyphenyl units, as well as reduced cellulose and galactoglucomannan relative to normal wood (Timell, 1986; Nanayakkara et al., 2005; Yeh et al., 2006). Most striking, though, is that β(1,4)-galactan can constitute more than 10% (w/w) of the cell wall material in severe compression wood but is virtually absent in normal wood (Nanayakkara et al., 2005; Yeh et al., 2006). Recent work suggests that β(1,4)-galactan biosynthesis represents an early step in compression wood formation and confirms that its presence is diagnostic for this wood type (Altaner et al., 2007). However, the molecular signal cascades in conifers that lead to the deposition of β(1,4)-galactan are currently not well understood.Immunological studies in conifer species using the monoclonal anti-β(1,4)-galactan LM5 antibody (Jones et al., 1997) indicate that β(1,4)-galactan in compression wood is located in the S1 and outer S2 layers of mature tracheids but is virtually absent from the primary cell walls (Schmitt et al., 2006; Altaner et al., 2007; Möller and Singh, 2007). Instead of β(1,4)-galactan, most conifers contain small amounts of arabinogalactan, a polysaccharide characterized by a highly branched β(1,3)-galactan backbone (Vikkula et al., 1997; Willför et al., 2002; Laine et al., 2004) in their primary cell walls. The ultrastructural distribution of β(1,4)-galactan in compression wood appears to be largely consistent with highly lignified cell wall layers (Möller and Singh, 2007), which might explain the involvement of β(1,4)-galactan in the formation of lignin-carbohydrate complexes (Mukoyoshi et al., 1981; Minor, 1982; Timell, 1986; Laine et al., 2004).The investigation of β(1,4)-galactan structure in preparations from Pinus sylvestris (Laine et al., 2004) and Pinus radiata (Nanayakkara 2007) revealed a linear polymer. In Pinus densiflora Siebold & Zucc., β(1,4)-galactan was found to be slightly branched at positions C2, C3, and C6 (Mukoyoshi et al., 1981). β(1,4)-Galactan in conifers display a high degree of polymerization (DP), which was originally estimated to be in the range of 200 to 300 (Timell, 1986). More recent studies with P. radiata compression wood found the native polysaccharide to have a DP of approximately 380 (Nanayakkara 2007).β(1,4)-Galactan is a very good biochemical marker for compression wood (Altaner et al., 2007), but its physiological role is currently not well understood. Various functions for β(1,4)-galactan in compression wood have been proposed, such as strengthening of the secondary cell wall, absorption of mechanical stresses, and generation of compressive forces (Möller and Singh, 2007). Furthermore, β(1,4)-galactan is also found in tension wood, with a proposed role in cross-linking cellulose microfibrils (Arend, 2008). However, all of those hypotheses on the molecular function of β(1,4)-galactan in reaction wood await experimental verification.Despite substantial efforts to characterize the biosynthesis of this polymer, β(1,4)-galactan biosynthetic enzymes and their corresponding genes are currently unknown (Peugnet et al., 2001; Geshi et al., 2002, 2004; Abdel-Massih et al., 2003; Kato et al., 2003; Ishii et al., 2004; Konishi et al., 2004, 2007; Gorshkova and Morvan, 2006). However, based on other cell wall polysaccharide biosynthetic enzymes, it is likely that the enzymes involved in the biosynthesis of β(1,4)-galactan are either Golgi localized or pass through the Golgi in transit to the apoplastic space (Reyes and Orellana, 2008).To better understand β(1,4)-galactan synthesis in compression wood formation, we sampled both normal wood and severe compression wood from two 6-year-old P. radiata trees, which displayed stark differences in lignin and carbohydrate content and composition. Using these wood samples, new insights into the subcellular localization of β(1,4)-galactan in pine were generated using confocal laser fluorescence microscopy and transmission electron microscopy. An enzyme assay was developed, based on 2-aminopyridine (2AP)-labeled galacto-oligosaccharides as acceptor molecules, which we used to identify and partially purify a robust, microsome-associated, UDP-Gal-dependent β(1,4)-galactosyltransferase (GalT) activity in compression wood that was virtually undetectable in normal wood. Assays of the partially purified GalT revealed that this enzyme has some properties similar to those of previously characterized pectic GalTs, but a marked difference was observed in the size distribution of the enzymatic products.