Publications

Coniferin, the 4-O-β-D-glucopyranoside of trans-coniferyl alcohol, occurs as a dominant metabolite in conifers and has also been reported in woody angiosperms. Coniferin has long been considered as a storage precursor of gymnosperm lignin, and more recently coniferin content of the cambial zone has been suggested as a biochemical indicator for wood formation in conifers. However, coniferin is not present in the cambium except during cambial growth. Moreover, coniferin content rises well before lignification commences in springtime and disappears prior to completion of lignification at the end of the growing season.Appearance and activity of UDP-glucose: coniferyl alcohol glucosyltransferase (CAGT), the enzyme catalysing production of coniferin from coniferyl alcohol, is not specifically linked to lignification but rather specifically associated with the annual cycle of growth and dormancy. Progress in genetic engineering of lignin content and composition has raised questions about the basic concept of lignin biosynthesis, indicating that the metabolic flexibility may be broader than previously thought. The incorporation of labelled coniferin into syringyl units of angiosperm wood as well as interconversions between cinnamyl alcohols and cinnamyl aldehydes have indicated that the turnover of lignin precursors may occur distantly downstream from the phenylpropanoid acid and/ or cinnamyl CoA-ester stages. We found in vitro that CAGT could convert cinnamyl alcohols as well as cinnamaldehydes. Our results support the view that CAGT has a regulatory influence and lignin modulating function exceeding mere substrate supply in support of guaiacyl lignification.

In this investigation, the effects of exogenous indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and 1-aminocyclopropane-1-carboxylic acid (ACC) on anatomical development within cultured segments of <i>Abies balsamea</i> (L.) Mill. were compared, using debudded and defoliated leaders produced in the preceding year as bioassay material. In stem apical regions, IAA promoted radial enlargement of pre-existing cortical resin ducts and attending parenchyma enlargement, whereas IBA promoted cell division and expansion of parenchyma on the outer edge of phloem without altering cortical duct shape. Cortical woody ducts, each partially surrounded by cambium, were observed as a novel but infrequent feature. A single cortical woody duct was spatially associated with each mature leaf as its vascular trace, and they were not encountered elsewhere in the cortex, nor were they induced to form in response to any hormone application. An unknown leaf factor induces the development of cortical woody ducts. Both IAA and IBA promoted cell division in the vascular cambium. The common cellular response at the interface between the latewood boundary and cambial zone was the radial expansion of primary-walled fusiform cambial cell derivatives with little if any ensuing tracheary element (TE) differentiation. Enhanced TE production at basal stem positions occurred when ACC was provided with IAA and/or IBA, and an IAA + IBA + ACC combination produced a basal stem response similar to that in untreated segments having intact leaves. The data support the conclusion that IAA, IBA, and ACC have distinct but complementary roles in the overall regulation of the types of cellular differentiation that contribute to cortex histogenesis and diameter growth of balsam-fir leaders.

Abstract A thin-layer chromatography procedure is described suitable as a limit test for p-chloroacetanilide in paracetamol and phenacetin. The sample is chromatographed on silica gel together with a standard using the solvent mixture cyclohexane: acetone: diisobutylketone: methanol: water (100:80:30:5:1) and detection is by irradiation with ultraviolet light 253.7 mμ followed by examination in light of wavelength 365 mμ. The procedure may be used to limit the p-chloroacetanilide content of tablets containing paracetamol or phenacetin. Other possible impurities in paracetamol and phenacetin are also detected and may be limited by similar procedures.

Under experimental conditions, mature living conifer needles on isolated stem segments induce cells at the junction of the needle trace and the cambium to differentiate into tracheary elements (TEs). The response is an all-or-none event in terms of secondary-wall deposition, but the extent of deposition varies between cells, and hence may serve as a model for developmental plasticity at the cellular level. Attempts to reproduce needle-induced TE differentiation using plant growth regulators such as auxin and cytokinin with or without carbohydrates have been unsuccessful but nevertheless have provided evidence for interactions between the needle-produced tracheid-differentiation factor (tdf) and phytohormones in the regulation of cellular differentiation. Treatment of needle-pair stumps with D-myoinositol 1,4,5-trisphosphate, the second messenger from bovine brain, has induced the tdf response at the needle tracecambium junction; however, unequivocal characterization of the endogenous tdf remains to be achieved. Induction of TE differentiation using pine needle pairs is an attractive experimental approach to understanding regulation of cellular differentiation for a number of reasons: (1) A tissue that normally never differentiates, the vascular cambium, can be induced to differentiate into TEs. (2) The location of differentiation can be accurately predicted, and the response can be compared with that of adjoining, nondifferentiating cells. (3) In vitro research is possible using stem segments in test tubes in the absence of complex media, and interactions between the tdf and other regulatory molecules, or other chemical or physical factors, can readily be investigated. (4) The differentiating tissue is heterotrophic; hence, interpretations about regulation are not confounded by autotrophic considerations. (5) Differentiation occurs in "dormant" cells in the absence of preceding vacuolation, cell division, or cell expansion; therefore, TE differentiation per se can be investigated independently of those processes normally accompanying but not essential for differentiation. (6) Polyribosomes, endoplasmic reticulum, dictyosomes, exocytotic blebs, and other organelles clearly fulfill roles related to TE differentiation and appear worthy of further investigation. (7) The system is well suited for performance of in situ hybridization or immunolocalization research.

A mature bordered pit in secondary xylem of Pinaceae comprises a circular border of secondary-wall material that protrudes into the tracheid lumen and is punctuated by a centralized aperture through which sap flows. The overarching border encloses a pit chamber within which is a “membrane”, or diaphragm, consisting of a central torus and margo strands. Bordered-pit pairs are abundantly present in all woods, and their membranes serve as swinging-diaphragm check valves regulating sap flow between adjoining tracheary elements, simultaneously trapping emboli and particulates in water as it moves from roots to leaves. The cell biology of bordered-pit formation in cambial derivatives of Abies balsamea (L.) Mill. was investigated by light and scanning electron microscopy during early stages of cellular differentiation of cambial derivatives into secondary xylem tracheids. A bordered-pit template (BPT), a bordered-pit organelle (BPO), a bordered-pit basal disk, and additional novel structures were found to be associated with bordered-pit formation. Evidence was found that the membrane does not comprise residual compound middle lamella; rather, the membrane forms de novo as BPO remnants. A cell-biology model and new terminology are introduced to explain how BPTs, BPOs, and basal disks contribute to successive stages in formation of bordered-pit pairs.

Recent studies on the phytohormonal regulation of seasonal cell-division activity in the cambium, primary-wall radial expansion of cambial derivatives, differentiation of xylem cells, and growth of the cortex in forest trees of the north temperate zone are reviewed. Indol-3-ylacetic acid (IAA, auxin) has been characterized by combined gas chromatography-mass spectrometry (GC-MS) in the cambial region of Abies balsamea, Pinus densiflora, Pinus sylvestris and Quercus robur. All of the evidence supports the hypothesis that developing leaves and extending shoots are primary sources of IAA. The rate of ethylene emanation varies among conifer species when adjoining phloem and cambial tissues are incubated in vitro. The cambium from young cuttings of Abies balsamea produces more ethylene than that from older cuttings. Ethylene production by seven-year-old Abies balsamea cambium is substantially increased in vitro when the tissue is provided with exogenous 1-aminocyclopropane-1-carboxylic acid and IAA. In response to elevated ethylene concentrations, cortex growth is accelerated in both hardwood and conifer seedlings. Ethrel (2-chloroethylphosphonic acid) increases ray size and ray-cell number and promotes traumatic resin-canal development in xylem. In Ulmus americana, endogenous ethylene concentrations are inversely correlated with cambial activity. Ethylene decreases vessel diameter in Acer negundo, Acer platanoides and Ulmus americana. Several studies suggest that ethylene has a role in regulating reaction-wood formation in both conifers and hardwoods.

Uridine 5'-diphosphoglucose:coniferyl alcohol glucosyltransferase (CAGT), the enzyme catalyzing synthesis of coniferin from coniferyl alcohol and uridine 5'-diphosphoglucose, was investigated throughout an annual cycle of cambial growth and dormancy in Pinus banksiana Lamb. During dormancy, CAGT activity was not detected in the cambium. CAGT became weakly active in springtime when fusiform cells of the lateral meristem changed from densely protoplasmic to highly vacuolated states, just prior to resumption of cell-division activity. During cambial growth and xylogenesis, CAGT activity in cambial derivatives was greater than that found in the cambial zone. In both cambial zone and developing xylem, seasonally changing CAGT activity paralleled seasonal variation in endogenous coniferin content. CAGT activity disappeared when the cambium entered dormancy in August, prior to completion of lignification in the last differentiating latewood tracheids. In vitro, exogenous coniferin at 0.1 mmol ·L -1 promoted CAGT activity (optimum pH 7.8, temperature 40°C); however, coniferin at &gt;10 mmol ·L -1 inhibited CAGT activity, in agreement with endogenous coniferin content of developing xylem not exceeding that level. The results indicate that the promoter controlling CAGT gene expression may be cambial specific and linked to the overall control of seasonal cambial growth and dormancy.Key words: cambium, coniferin, lignin, phenology, Pinus banksiana, xylogenesis.

Rims of bordered pits form on the primary walls of radially enlarged cambial derivatives prior to the onset of general secondary-wall formation. A recent report (Botany, 2014, 92(7): 495–511) raised the possibility that the chemical composition of the rim might be different from that of the secondary wall. To investigate this, early-stage nonfluorescent and late-stage autofluorescent rims were separated from cambial derivatives of Abies balsamea (L.) Mill. and purified to homogeneity by density-gradient centrifugation. Solid state nuclear magnetic resonance spectroscopy, Raman microspectroscopy, combined gas chromatography – mass spectroscopy, enzyme digestion, and chemical resilience data support the interpretation that cellulose alone is the microfibrillar polysaccharide of nonfluorescent early-stage rims. A lignin is additionally present in late-stage rims, and it evidently bonds with cellulose because rims are extraordinarily resistant to hydrolysis by either enzymes or strong acid.

Thin sections from the three syntypes of Araucarioxylon arizonicum Knowlton, 1889 were re-examined and found to be anatomically distinct. Two of the syntypes are similar and, considering Araucarioxylon ("Araucaroxylon") Kraus, 1870 as nomen superfluum, those two are placed in Pullisilvaxylon gen. n. as nomen novum (lectotype Pullisilvaxylon arizonicum). The third specimen, known as the 'Sherman log', is transferred to Chinleoxylon knowltonii gen. et sp. n. Permineralized woods in three randomly sampled large-diameter logs assumed to be A. arizonicum plesiotypes were found to be anatomically distinct, two being species within Silicisilvaxylon gen. n., the third named Crystalloxylon imprimacrystallum gen. et sp. n. Thus, following descriptions of Arboramosa semicircumtrachea

Gradient gel electrophoretic methods enabled a distinction to be made between coniferyl alcohol oxidase (CAO) of lignifying cell walls and a pI approximately 9 pine "laccase" recently implicated in lignification (Science 1993 260, 672). Following treatment of a partially purified protein mixture from developing xylem of Pinus strobus with 2-[N-morpholine]ethanesulfonic acid (MES) buffer, isoelectric focusing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicated that CAO had been selectively precipitated by MES and thereby purified to electrophoretic homogeneity. Purified CAO was determined to be a cell-wall-bound glycoprotein (38% glycan), M(r) 107,500, pI 7.6, pH and temperature optima 6.3 and 30 degrees C, respectively. By graphite-furnace atomic-absorption analysis, CAO contained one copper atom per protein molecule. Proteins obtained from lignifying cambial derivatives of conifers (family Pinaceae) and from Rhus typhina bark were compared with CAO and the pI approximately 9 pine "laccase" following electrophoresis and Western blotting. For Abies balsamea, Larix laricina, Picea rubens, Pinus banksiana, Pinus taeda, and R. typhina, the isoelectric points of oxidatively active bands were identical to those of purified CAO. In addition, for all species only the pI 7.6 band was immunoreactive with antibodies against periodate-deglycosylated CAO.

Trees were on Earth 394 million years ago (394 Ma) as spore-producing Archaeopteris progymnosperms having largediameter trunks of secondary xylem (morphotype Callixyon) produced by vascular cambium. Plants of smaller stature with primary xylem cores were present in Late Silurian (416 Ma), but they lacked cambium and it remains unclear how and when the first trees evolved. Progymnosperms faded and gymnosperms arose during Middle Carboniferous, and conifers, ginkgos, cycads, tree ferns and cordaites were well established by the Carboniferous–Permian transition (299 Ma). Woods of the earliest conifers were different from those of today, and not until Late Triassic (220 Ma) did any begin producing secondary xylem similar to modern woods, the xylem phenotypes of Cupressaceae and Araucariaceae emerging much earlier than those of Pinaceae and flowering plants. Conifers have persisted and done relatively well despite major extinction events, severe climate change, insectivory, herbivory and microbial activity, all of which were in effect before as well as during the appearance of trees on Earth. Approximately 600 conifer species continue to exist, and the survivors presumably possess the physiological fitness needed to adapt to an ever-changing biosphere. However, this is speculative because their physiology remains less than well understood. Forestry interventions such as planting one species to the exclusion of others have the potential to exacerbate as well as sustain the ongoing existence of our remaining conifers. Key words: bordered pit, cambium, cell biology, cellulose, evolution, lignin, paleobotany, protoplasmic autolysis, secondary growth, wood formation, xylogenesis