Coniferyl alcohol metabolism in conifers -- I. Glucosidic turnover of cinnamyl aldehydes by UDPG: coniferyl alcohol glucosyltransferase from pine cambium.

UDPG: coniferyl alcohol glucosyltransferase (CAGT; EC 2.4.1.111) isolated from cambial tissues of Pinus strobus was able to convert cinnamyl aldehydes as well as dihydroconiferyl alcohol into their corresponding 4-O-beta-D-glucosides in vitro. Cinnamyl aldehydes were glucosylated with comparable efficiency to coniferyl alcohol, the physiological substrate for CAGT. Seasonal patterns of CAGT activity for aldehydes were similar to those of coniferyl alcohol. Formation of cinnamyl aldehyde and additional monolignol glucosides indicates that precursor flux and availability for lignification is likely greater than previously recognized.     

Structural Changes in the Vascular Cambium of Pinus strobus L. during an Annual Cycle

The changes in the ultrastructure of cambial cells of easternwhite pine (Pinus strobus L.) during an annual cycle are observedand recorded as are relationships of cambial cells during dormancyand at resumption of cambial activity. Cambial activity wasresumed late in March or early in April, when a few cells dividedpericlinally. Cambial activity reached a maximum during thelatter part of May with 15 to 20 undifferentiated cells present.In July it declined markedly, and the number of undifferentiatedcells equalled that of the dormant period. The xylem and phloemtissue cells produced late in the annual cycle overwinteredat varying developmental stages. In October cambial cells structurallyresembled dormant cells. The number of dormant cells in easternwhite pine cambium varied from 6 to 10. Active cells were characterizedby a large central vacuole, by an abundance of all cell organelles,and by thin cell walls. Dormant cells were characterized bynumerous small vacuoles, by structurally and quantitativelymodified cell organelles, and by relatively thick cell walls.     

Enzymes of glyoxylate in conifers

The high level of lipids in seeds of some species of conifers suggested that the glyoxylate cycle might have a role in conifer seed metabolism.

Six species (Pinus pinea, Pinus pinaster, Pinus canariensis, Pinus strobus, Abies alba, and Cupressus sempervirens) were investigated for their lipid content and malate synthase and isocitrate lyase level. The fatty acid composition of the triglyceride fraction was also investigated. The correlation between lipid content of germinating seed with the presence of the cycle was confirmed. The enzymes of the glyoxylate cycle were not detected in Cupressus sempervirens where the lipid content is very low.

     

Antifungal activity of the essential oils from some species of the genus Pinus

The chemical composition of the essential oils from the needles of Pinus ponderosa (north american pine), P. resinosa (red pine) and P. strobus (eastern white pine) has been determined by GC/MS (FID). The essential oils from P. resinosa and P. ponderosa in comparison to P. strobus have been characterized by the higher content of beta-pinene (42.4%, 45.7% and 7.9% respectively). On the other hand, a-pinene (17.7%) and germacrene D (12.2%) were dominant compounds of P strobus. Moreover the essential oil from P. resinosa was more rich in myrcene-15.9%. Estragole and delta-3-carene, each one in amount ca 8% were identified only in P. ponderosa. The content of essential oils in the needles slightly varied--0.65%--P. resinosa, 0.4%--P strobus, 0.3%--P. ponderosa. The antifungal activity has been investigated towards Fusarium culmorum, F solani and F. poae. The strongest activity was observed for the essential oil from P. ponderosa, which fully inhibited the growth of fungi at the following concentrations--F. culmorum, F. solani at 2% and F. poae at 5%.     

Proteomic comparison of needles from blister rust-resistant and susceptible Pinus strobus seedlings reveals upregulation of putative disease resistance proteins

In order to characterize a hypersensitive-like reaction in selected Pinus strobus seedlings to Cronartium ribicola, a proteomic comparison of needles from resistant and susceptible seedlings was undertaken using two-dimensional gel electrophoresis (2-DE). The results revealed 19 polypeptides specific to resistant seedlings and seven of these specific to infected resistant seedlings. There were 13 polypeptides up-regulated (> or = 3-fold increase) in resistant family P327 in comparison to needle tissue from susceptible and mock-inoculated seedlings. Electrospray ionization liquid chromatography and tandem mass spectrometry was used to sequence 11 proteins from the 2-DE gels. Sequences obtained from electrospray ionization liquid chromatography and tandem mass spectrometry were used for MS-BLAST and Pro-ID database searches allowing identification with a 95 to 99% confidence level. Six proteins were determined to be homologs of proteins with known roles in disease resistance, five were determined to be homologs of members of the leucine-rich repeat (LRR) superfamily, and one was a homolog of heat shock protein 90, a protein that serves as a cofactor for certain LRR proteins. This is the first report of members of the LRR family with functional homologs in Pinus strobus and of a molecular basis for white pine blister rust resistance in Pinus strobus.     

Coniferyl alcohol, sinapyl alcohol and scopoletin in tobacco callus tissue during growth of a subculture

Coniferyl and sinapyl alcohols were isolated, identified and quantitatively determined as unbound (or weakly bound) phenylpropanoids in neutral hot-water extracts of Nicotiana tabacum L. callus tissue. This is the first identification of these alcohols in cultured tobacco callus. Scopoletin was also detected in these extracts, and it was the most abundant of these three phenylpropanoids with concentrations that ranged from 50–119 μg/g dry wt. Coniferyl alcohol (17–34 μg/g dry wt.) and sinapyl alcohol (23–35 μg/g dry wt.) were present in nearly equimolar concentration ratios and at levels which were about half those determined for unbound (or weakly bound) scopoletin. The amount of scopoletin extracted increased about 10 times when 1 M HCl-50% methanol - 0.3% ascorbic acid was used as the extractant. This indicated that most scopoletin moieties were strongly bound, perhaps by acid-hydrolyzable linkage. Coniferyl alcohol and sinapyl alcohol were not found in the acid extracts, presumably because they were acid-labile. In general, the concentration of each endogenous unbound (or weakly bound) phenylpropanoid appeared to remain relatively constant throughout the growth phase of the subculture. The only exceptions to this were the relatively higher concentrations of scopoletin and coniferyl alcohol present during the initial 0–2 weeks of subculture.     

Assessing genetic diversity and structure of fragmented populations of eastern white pine (Pinus strobus) and western white pine (P. monticola) for conservation management

Aims Many pine populations in Canada have fragmented distributions resulting from the effects of glaciations, overharvesting and white pine blister rust infections. Forest fragmentation can modify gene flow and reduce genetic diversity. Selective logging can reduce the density of trees, thereby altering mating patterns and increasing inbreeding. The hypothesis of the present study is that forest fragmentation will not increase inbreeding and will have no effect on genetic diversity parameters in the Canadian Pinus moniticola and P. strobus populations targeted because of (i) the long life span of the pine species, (ii) outbreeding and self-incompatibility of P. monticola and P. strobus and (iii) wind pollination resulting in high gene flow among populations. We studied the genetic diversity of P. strobus across its range in Canada, and we completed a detailed analysis of the genetic structure of P. monticola populations from western Canada using microsatellites genetic markers.Methods Seed samples from 10 P. monticola populations and 10 P. strobus populations were collected from western and eastern Canada, respectively. The mother trees included in seed lots were representative of each stand. Genomic DNA extracted from each sample was amplified with microsatellite primers. The intra- and interpopulation genetic diversity parameters were assessed using Popgene and Genepop softwares and the genetic distances among populations within each species using the PowerMarker software.Important findings Pinus monticola and P. strobus exhibited moderate to high genetic diversity. Also, both species showed low levels of inbreeding despite the geographic isolation and small stand size. Gene flow estimates were high and population differentiation values were relatively low for these fragmented forest sites.     

The Cambial Activity and on Which the Effects of Exogenous IAA in the Stem of Pinus sylvestris L.

The dormant cambial zone consisted of 5–6 cell layers in the main stem of Pinus sylvestris L. trees that were ca. I00 years old. Time of cambial reactivation was comparable at one (bottom) and 8 (top) meters above the ground. In spring, when the cambium reactivated, the number of cambial cells slightly increased and phloem cells were formed. The production of xylem cells followed 3–4 weeks later. The formation of xylem cells decreased, whereas that of phloem cells increased between late June and early July. Cambial reaction in 1-year-old cuttings that were debudded and treated apically with IAA in lanolin was similar to that in the ca. 100-year-old main stem. However, in debudded cuttings treated with plain lanolin, the number of cells in the carnbial zone decreased during the first week of culture, and only a few phloem cells were formed. Later, the fusiform cambial cells of the cambial zone were divided transversely and lost their typical morphology. It is proposed that some factor(s) from roots may stimulate the initiation of cambial cell division, because when the cambium reactivated, the number of cambial cells slightly increased in the ca. 100-year-old main stem, but decreased in the 1-year-old cuttings.     

Coniferyl alcohol reactivity at the air/water interface

In order to investigate the sensitivity of the lignin monomer coupling reactions to the environment physicochemical conditions, coniferyl alcohol (CA) was polymerised at the air/water interface. Characterisation of the interface during the reaction by surface pressure measurement and ellipsometry demonstrates that the reaction occurs near or at the interface. Coupling products were analysed by HPLC and compared to reaction products obtained in the case of polymerisation in solution. Relative proportions of beta-beta and beta-O-4 dehydrodimers were found to increase in air/water interface experiment.     

Rapid expansion of microsatellite sequences in pines

Microsatellite persistence time and evolutionary change was studied among five species of pines, which included a pair of closely related species (Pinus sylvestris and Pinus resinosa) in the subgenus Pinus, their relative Pinus radiata, and another closely related species pair (Pinus strobus and Pinus lambertiana) in the subgenus Strobus. The effective population sizes of these species are known to have ranged from the very small bottlenecks of P. resinosa to vast populations of P. sylvestris. This background allowed us to place the microsatellite evolution in a well-defined phylogenetic setting. Of 30 loci originating from P. strobus and P. radiata, we were able to consistently amplify 4 in most of the these pine species. These priming sites had been conserved for over 100 Myr. The four microsatellites were sequenced in the five species. Flanking sequences were compared to establish that the loci were orthologous. All microsatellites had persisted in these species, despite very different population sizes. We found a recent microsatellite duplication: a closely related pair of loci in P. strobus, where the other four species had just one locus. On two independent occasions, the repeat area of this same microsatellite (locus RPS 105a/b) had grown from a very low repeat number to 15 or 17 in the last 10-25 Myr. Other parts of the same compound microsatellite had remained virtually unchanged. Locus PR 4.6 is known to be polymorphic in both P. radiata and P. sylvestris, but the polymorphism in the two species is due to different motifs. The very large pine genomes are highly repetitive, and microsatellite loci also occur as gene families.     

Coniferyl alcohol oxidase — a catechol oxidase?

The physico-chemical properties of coniferyl alcohol oxidase (CAO), a copper containing glycoprotein spatiotemporally associated with lignification in conifers, is reported here. By electron paramagnetic resonance spectroscopy, only type 3 copper was indicated in CAO. CAO oxidizes several laccase substrates; however, it is not a blue-copper protein and monoclonal antibodies against both native and deglycosylated CAO did not recognize any of several laccases. The N-terminal sequence of CAO, H₂N-X E L A Y S P P Y X P S, was non-homologous with known enzymes. Transparent copper, tetrameric structure, aminoacid composition, phenylhydrazine and tropolone inhibition, and SDS enhancement of CAO activity indicate that CAO is an o-diphenol oxidase.     

Maturation of somatic embryos of Pinus strobus is promoted by a high concentration of gellan gum

Application of somatic embryogenesis to Pinus strobus clonal propagation and genetic improvement was hampered by the difficulty in achieving synchronous maturation of a large number of somatic embryos that would germinate and produce plants. Media containing abscisic acid (80 μ M ) and osmotic agents such as sucrose, polyethylene glycol and/or dextran did not sustain development of mature somatic embryos from plated embryonal masses. This indicated that factors other than osmotic agents might be involved in sustaining development of Pinus strobus somatic embryos to maturity. It was subsequently found that media lacking osmotica but containing a high concentration of gellan gum (1%) induced significant improvement in the development of mature somatic embryos in the presence of 80 or 120 μ M abscisic acid. This positive effect was independent of the genotype and all four tested lines displayed similar responses. Media containing gellan gum at concentrations from 0.4 to 1.2% formed gels that varied in their strength. Gel strength was proportional to the concentration of gellan gum in the specific medium but varied depending on the medium formulation. Gel strength increased with the duration of storage of the culture medium by 46% (SD 14) after 14 days of storage. Preliminary results showed that embryos matured on high gellan gum media displayed improved germination frequencies. These results indicate that in Pinus strobus the water status and possibly other medium characteristics that are influenced by increased concentration of gelling agent have stimulatory effects on maturation of somatic embryos.     

Dirigent-mediated podophyllotoxin biosynthesis in Linum flavum and Podophyllum peltatum

Given the importance of the antitumor/antiviral lignans, podophyllotoxin and 5-methoxypodophyllotoxin, as biotechnological targets, their biosynthetic pathways were investigated in Podophyllum peltatum and Linum flavum. Entry into their pathways was established to occur via dirigent mediated coupling of E-coniferyl alcohol to afford (+)-pinoresinol; the encoding gene was cloned and the recombinant protein subsequently obtained. Radiolabeled substrate studies using partially purified enzyme preparations next revealed (+)-pinoresinol was enantiospecifically converted sequentially into (+)-lariciresinol and (-)-secoisolariciresinol via the action of an NADPH-dependent bifunctional pinoresinol/lariciresinol reductase. The resulting (-)-secoisolariciresinol was enantiospecifically dehydrogenated into (-)-matairesinol, as evidenced through the conversion of both radio- and stable isotopically labeled secoisolariciresinol into matairesinol, this being catalyzed by the NAD-dependent secoisolariciresinol dehydrogenase. (-)-Matairesinol was further hydroxylated to afford 7'-hydroxymatairesinol, this being efficiently metabolized into 5-methoxypodophyllotoxin. Thus much of the overall biosynthetic pathway to podophyllotoxin has been established, that is, from the dirigent mediated coupling of E-coniferyl alcohol to the subsequent conversions leading to 7'-hydroxymatairesinol.     

SEASONAL SECONDARY GROWTH IN NEEDLE LEAVES OF PINUS STROBUS AND PINUS BRUTIA

Mature needles and elongating current year's needles of Pinus strobus growing in Massachusetts and P. brutia growing in Israel were collected monthly or bimonthly for seasonal analysis of leaf cambial activity. Mature needles produced secondary phloem but no xylem, and, regardless of the season, had a cambial zone from 2 to 3 cell layers wide. In the current year's needles maturation was basipetal and the procambium differentiated into primary xylem, primary phloem, and the phloem-producing vascular cambium before needle maturity. One- and 2-year-old needles of Pinus strobus produced slightly over 4 cell layers of phloem between April 15 and September 1 of 1983, with a peak production rate of about 2 cell layers per month in May and early June. One-year-old needles of P. brutia produced about 6 phloem cell layers in 1983, with phloem being produced throughout the year except in midsummer. This was contrasted by fall and winter dormancy in needles of P. strobus.     

Patterns of protein synthesis in the cambial region of Scots pine shoots during reactivation

Changes in protein synthesis in cambial region cells were monitored in 1-year-old cuttings of Scots pine ( Pinus sylvestris L.) collected in November, when the cambium was dormant, and subjected to environmental conditions that promoted or inhibited cambial growth. The proteins were labelled in vivo with L-[³⁵S]-methionine and separated using 2-dimensional polyacrylamide gel electrophoresis. In budded cuttings cultured under environmental conditions favoring cambial reactivation, there was a reproducible quantitative change in 55 proteins (33 induced and 22 repressed), a less certain increase or decrease in 40 proteins, and no apparent change in about 150 proteins. Under the same conditions, 8 proteins were induced and 6 others were repressed in debudded cuttings treated apically with 1 mg indole-3-acetic acid (IAA) in 1 g lanolin, in which cambial reactivation occurred, compared with debudded cuttings treated with plain lanolin in which the cambium did not reactivate. Three of the proteins induced in the IAA-reated cuttings only appeared after cambial cell division and derivative differentiation actually began, and the same proteins were found in budded cuttings after their cambium had become reactivated. In contrast, protein expression in cuttings exposed to environmental conditions that prevented cambial reactivation was similar at the beginning and end of the experimental period. These results indicate that the cambium was in the quiescence stage of dormancy at the start of the experiment, that quiescent cambial region cells can synthesize proteins as soon as exposed to environmental conditions favoring reactivation, and that only 3 of the approximately 250 proteins detected were specifically involved in cambial growth     

Cambial activity and annual rhythm of xylem production of Pinus kesiya Royle ex. Gordon (Pinaceae) in relation to phenology and climatic factors growing in sub-tropical wet forest of North East India

The interrelationship between phenological events, climatic factors, periodicity of cambial activity and seasonal production of xylem was examined in Pinus kesiya Royle ex. Gordon growing in sub-tropical wet forest of Meghalaya state, India. Reactivation of dormant cambium occurs after sprouting of new needles during the middle of February. Since the formation of reproductive cones takes place simultaneously with vegetative bud break and needle formation, cone formation could also lead to the enhancement of cambial activity. The activity of cambium and xylem production decline gradually towards November and cease from end of December to end of January. There was no correlation between needle fall and cambial activity. Due to the production of three flushes of new needles and branches in a year the tree never becomes completely leafless. It was evident from correlation and regression analysis that the annual course of average temperature plays an important role for the reactivation of vascular cambium after dormancy. The differentiation of xylem elements correlated with mean temperature in the first place and secondly with precipitation. Increase in length of fusiform initials and their derivatives could be correlated with relative humidity, precipitation and mean maximum temperature. Dormancy was imposed by low temperature and less precipitation. The data are discussed in the light of cambial activity, xylem production and phenological events.     

Comparative anatomy and ultrastructure of active and dormant vascular cambium in rhizome ofBotrychium ternatum

Vascular cambium ofBotrychium ternatum rhizome varied according to age, position and season was studied by light and electron microscopy. Cambium at the 6th internode (6-year-old cambium) had the greatest number of active cambial cells in August and September, thus it was in the most active stage. The active cells were characterized by the presence of a large vacuole, few storage materials such as starch grains within plastids or lipid droplets, a thin tangential wall; and various cell organelles in the thin peripheral layer of cytoplasm. When the 6-year-old cambium reached its dormant season after November, the dormant cells were filled with numerous storage materials and had few cell organelles. Our observations suggested that the initiation and cessation of cambial activity may be correlated with the annual life cycle of this plant: the vegetative and reproductive leaves began to emerge in June and July, respectively, and the sporophyll withered in November after the spore dispersal. Most cambial cells at the 10th internode, which remained in a dormant state throughout the year, were filled with numerous storage materials. Our results indicated that the activity of vascular cambium in the 10th internode was determinate.     

THE NATURE OF PLASMODESMATA IN NORMAL (LIVING) PLANT TISSUE

Techniques have been devised for the demonstration and study of plasmodesmata in an unaltered state in plant tissues. Text figures and data are presented for plasmodesmata in the cambium and certain other tissues of Pinus strobus, parenchyma tissues of Lycopersicum esculentum, and ray and wood parenchyma tissue of Sequoia sempervirens. In all cases, the plasmodesmata were of similar morphology, with a diameter of approximately 0.2μ. Comparative studies are presented showing the effects of various swelling agents which were used in past studies, and of dehydration which is a normal concomitant of most histological procedures. The present paper provides an important base line for the comparison of the appearance of plasmodesmata in living tissue to the findings of recent studies with the electron microscope.