Differential induction of cinnamyl alcohol dehydrogenase during defensive lignification in wheat (Triticum aestivum L.): characterisation of the major inducible form

The induction and substrate specificity of cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) was investigated in relation to the deposition of a defensive, syringyl-rich lignin at wound margins in wheat (Triticum aestivum L. cv. Brigadier). Column chromatography of untreated, wounded and elicitor-treated tissues revealed three major CAD forms (CAD-A, -B and -C) of which only CAD-C was responsive to elicitors. Examination of the substrate preference of these fractions indicated p-coumaryl alcohol to be the preferred substrate of CAD-A and CAD-B, whereas sinapyl alcohol was favoured by CAD-C. Activity-stained isoelectric focussing gels revealed in untreated and wounded leaves four CAD isoenzymes with isoelectric points of 4.59 (i), 4.67 (ii), 4.81 (iii), 4.93 (iv). Elicitor treatment generally enhanced the staining of all isoenzymes and resulted in the appearance of two new isoenzymes of 5.22 (v) and pI 5.31 (vi). In activity stained non-denaturing PAGE gels, CAD-C further resolved into two distinct zones of CAD activity. Cinnamyl alcohol dehydrogenase-C was purified to apparent homogeneity and characterisation revealed a 45-kDa subunit peptide which in its native form demonstrated a marked substrate preference for sinapyl alcohol. Overall, the differential induction and substrate preference of CAD-C are consistent with a defensive role during defensive lignification at wound margins in wheat. Received: 20 September 1998 / Accepted: 22 October 1998     

Ectomycorrhizal development on Scots pine (Pinus sylvestris L.) seedlings in different soils

In the present study ectomycorrhizal development of Laccaria bicolor, Rhizopogon luteolus and Suillus bovinus associated with Scots pine (Pinus sylvestris) seedings was studied as affected by primary stand humus, secondary stand humus, podsolic sandy soil or peat in perspex growth chambers. After 9 weeks, ectomycorrhizal development with S. bovinus was significantly greater in peat and primary stand humus than in secondary stand humus or podsolic sandy soil. Ectomycorrhizal development with R. luteolus in secondary stand humus was higher than in primary stand humus. Degree of ectomycorrhizal development of L. bicolor, R. lutuelus and S. bovinus on Scots pine was related to potassium concentration, organic matter content and pH of the soils suggesting that chemical composition of the soils affects ectomycorrhizal development.     

Porphyrin metabolism in chill-stressed seedlings of Scots pine (Pinus sylvestris)

Scots pine ( Pinus sylvestris L.) is generally resistant to chilling temperatures. Porphyrin metabolism under low temperature stress was studied in etiolated seedlings of Scots pine. Low temperatures affect porphyrin accumulation in at least 3 different temperature sensitive sites: 1) the light activated accumulation of 5-aminolevulinic acid, a porphyrin precursor, 2) the metabolism of 5-aminolevulinic acid to form porphyrins and 3) a preferential accumulation of chlorophyll a over chlorophyll b . The temperature sensitivity of pine is compared to maize ( Zea mays L.), a chilling sensitive plant.     

Food reserves of Scots pine (Pinus sylvestris L.)

Summary The amounts of starch, soluble sugars, triacylglycerols, diacylglycerols and free fatty acids were studied in Scots pine (Pinus sylvestris L.) during an annual cycle in current-year needles and in 1-, 2- and 3-year-old needles collected shortly after bud break. Determination of the compounds was performed using specific enzymatic assays, capillary gas chromatography and thin layer chromatography. Newly emerging needles contained relatively large amounts of starch, but only trace amounts of fat. During autumn and winter, fat content rose, while starch content decreased; amounts of both these reserve materials were very high the next spring shortly before bud break and decreased again during shoot elongation. Concentration of intermediates in triacylglycerol biosynthesis (diacylglycerols and free fatty acids), were low in summer and high in winter. The same pattern was observed for fructose and glucose (the predominant soluble sugars), galactose/arabinose and raffinose/melibiose. In contrast, sucrose concentrations were highest in spring and in autumn. Mature needles of different ages collected in May showed significant differences only in their triacylglycerol and starch content. Concentration changes of reserve materials are discussed in relation to season, mobilization and translocation processes, dormancy, frost resistance and the possibility of carbohydrate-fat interconversions.     

Purification and properties of glutamate dehydrogenase in Scots pine (Pinus sylvestris) needles

NADH-dependent glutamate dehydrogenase (GDH. EC 1. 4. 1.2) was isolated from the needles of Scots pine (Pinus sylverstris L.) grown on a rural and on a heavily polluted industrial area, and it was purified about 500 fold. The purification procedure included salt I'ractionation, ion exchange and affinity chromatography. Miehaelis constants for 2-oxoglularale (1.7 mM). for ammonium sultate (19 mM ) and for NADH (42.5 resp. 53 μM) the pH optimum (8.5) the requirements for Ca²⁺ ions, the temperature dependence ofl the enzyme activity (incubation from 0 to 82°C). and the relation between forest region and electrophoretie isoenzyme pattern were determined. The possible role of GDH in the adaptation of plants to ammonia assimilation (detoxification) under stress conditions, particularly with respect to air pollution, is discussed.     

Location,but not defensive genotype,determines ectomycorrhizal community composition in Scots pine (Pinus sylvestris L.) seedlings

1. For successful colonization of host roots, ectomycorrhizal (EM) fungi must overcome host defense systems, and defensive phenotypes have previously been shown to affect the community composition of EM fungi associated with hosts. Secondary metabolites, such as terpenes, form a core part of these defense systems, but it is not yet understood whether variation in these constitutive defenses can result in variation in the colonization of hosts by specific fungal species.
2. We planted seedlings from twelve maternal families of Scots pine (Pinus sylvestris) of known terpene genotype reciprocally in the field in each of six sites. After 3 months, we characterized the mycorrhizal fungal community of each seedling using a combination of morphological categorization and molecular barcoding, and assessed the terpene chemodiversity for a subset of the seedlings. We examined whether parental genotype or terpene chemodiversity affected the diversity or composition of a seedling''s mycorrhizal community.
3. While we found that terpene chemodiversity was highly heritable, we found no evidence that parental defensive genotype or a seedling''s terpene chemodiversity affected associations with EM fungi. Instead, we found that the location of seedlings, both within and among sites, was the only determinant of the diversity and makeup of EM communities.
4. These results show that while EM community composition varies within Scotland at both large and small scales, variation in constitutive defensive compounds does not determine the EM communities of closely cohabiting pine seedlings. Patchy distributions of EM fungi at small scales may render any genetic variation in associations with different species unrealizable in field conditions. The case for selection on traits mediating associations with specific fungal species may thus be overstated, at least in seedlings.

     

Photoreactivation of the UV light effects on growth of Scots pine (Pinus sylvestris L.) seedlings

Summary Ultraviolet-B light (UV-B) and ultraviolet-A light (UV-A) at higher doses exert a strong inhibitory (toxic) effect on axis growth in Scots pine (Pinus sylvestris L.) seedlings. This effect is unrelated to control of growth rate by phytochrome. Rather, after a toxic UV dose growth of the pine seedling no longer responded to phytochrome. Both, the effect of UV-B as well as the inhibiting effect of UV-A could be photoreactivated by blue light (B). The action of UV-A was 2 fold: (i) it exerted a toxic effect which could be photoreactivated by B, and (ii) applied after UV-B it photoreactivated to some extent the toxic UV-B effect. Obviously, the UV-A range causes a toxic effect, and at the same time is capable of photoreactivating the toxic UV effect. At higher doses the toxic effect prevails.     

Purification and characterization of isoforms of cinnamyl alcohol dehydrogenase fromEucalyptus xylem

Two distinct isoforms of cinnamyl alcohol dehydrogenase, CAD 1 and CAD 2, have been purified to homogeneity from xylem-enriched fractions ofEucalyptus gunii Hook and partially characterized. They differ greatly in terms of both physical and biochemical properties, and can be separated by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B. The native molecular weight of of CAD 1 is 38 kDa as determined by gel-filtration chromatography on Superose 6, and this isoform is likely to be a monomer since it yields a polypeptide of 35 kDa upon sodium dodecyl sulfatepolyacrylamide gel electrophoresis. It has a low substrate affinity for coniferyl andp-coumaryl alcohols and their corresponding aldehydes. No activity with sinapyl aldehyde and alcohol was detected. The more abundant isoform is CAD 2, which has a native molecular weight of 83 kDa and is a dinier composed of two subunits of slightly different molecular weights (42–43 kDa). These subunits show identical peptide patterns after digestion with N-chlorosuccinimide. The isoform, CAD 2, has a high substrate affinity for all the substrates tested. The two isoforms are immunologically distinct as polyclonal antibodies raised against CAD 2 do not cross-react with CAD 1. The characterization of two forms of CAD exhibiting such marked differences indicates their involvement in specific pathways of monolignol utilisation.Abbreviations CAD cinnamyl alcohol dehydrogenase - DTT dithiothreitol - NCS N chlorosuccinimide - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work was supported by the European Economic Community project AGRE 0021 (OPLIGE) in the scope of the ECLAIR PROGRAMME. The authors whis to thank Drs. L. Davin and N. Lewis (Washington State University) for kindly providing synthesized substrates, Dr. Annie Boudet for excellent technical assistance, and Dr. M. Campbell for fruitful discussions (Université Paul Sabatier, Toulouse, France). We would also like to thank Dr. M. M. Cordonnier-Pratt and Dr. L. Pratt (University of Georgia, Athens, USA) for helpful advice and antibody production.     

Gene silencing of cinnamyl alcohol dehydrogenase in Pinus radiata callus cultures

Xylem-derived Pinus radiata cell cultures, which can be induced to differentiate tracheary elements (TEs), were transformed with an RNAi construct designed to silence cinnamyl alcohol dehydrogenase (CAD), an enzyme involved in the biosynthesis of monolignols. Quantitative enzymatic CAD measurements revealed reduced CAD activity levels in most transclones generated. TEs from transclones with approximately 20% residual CAD activity did not release elevated levels of vanillin, which was derived from coniferyl-aldehyde through a mild alkali treatment. However, the activation of the phenylpropanoid pathway in transclones with approximately 20% residual CAD activity through the application of non-physiological concentrations of sucrose and l-phenylalanine produced phenotypic changes. The accumulation of metabolites such as dihydroconiferyl-alcohol (DHCA), which also accumulates in the P. taeda CAD mutant cad-n1, was observed. These results indicate that a substantial reduction in CAD activity is necessary for this enzyme to become a rate-limiting step in lignin biosynthesis in conifers such as P. radiata and confirm that transformable P. radiata callus cultures can be useful to investigate the function of xylogenesis-related genes in conifers.     

Genetic analysis of cinnamyl alcohol dehydrogenase in loblolly pine: single gene inheritance, molecular characterization and evolution

The gene encoding the monolignol biosynthetic enzyme cinnamyl alcohol dehydrogenase (CAD, E.C. 1.1.1.195) can be expressed in response to different developmental and environmental cues. Control of Cad gene expression could involve either differential regulation of more than one Cad gene or, alternatively combinatorial regulation of a single Cad gene. In loblolly pine (Pinus taeda L.), we found several electrophoretic variants (allozymes) of CAD and a high level of heterozygosity (h_e=0.46). Analysis of inheritance patterns of pine CAD allozymes gave segregation ratios that were consistent with Mendelian expectations for a single functional gene. The identity of the full-length Cad cDNA sequence was confirmed by alignment with peptide sequences obtained from purified active enzyme and by extensive similarity to Cad sequences from other species. Southern blot analysis of genomic DNA using the Cad cDNA as a hybridization probe gave simple patterns, consistent with our interpretation that pine Cad is a single-copy gene. Phylogenetic analysis and evolution rate estimates showed that Cad sequences are diverging less rapidly in the gymnosperms than in the angiosperms. The Cad mRNA was present in both lignifying tissues and a non lignifying tissue (the megagametophyte) of pine. The presence of a single gene suggests that different regulatory mechanisms for a single Cad gene, rather than differential regulation of several genes, can account for its expression in response to different cues.     

Ribosomal RNA genes in Scots pine (Pinus sylvestris L.): Chromosomal organization and structure

The nuclear 18S, 5.8S and 25S rRNA genes exist as thousands of rDNA repeats in the Scots pine genome. The number and location of rDNA loci (nucleolus organizers, NORs) were studied by cytological methods, and a restriction map from the coding region of the Scots pine rDNA repeat was constructed using digoxigenin-labeled flax rDNA as a probe. Based on the maximum number of nucleoli and chromosomal secondary constrictions, Scots pine has at least eight NORs in its haploid genome. The size of the Scots pine rDNA repeat unit is approximately 27 kb, two- or threefold larger than the typical angiosperm rDNA unit, but similar in size to other characterized conifer rDNA repeats. The intergenic spacer region (IGS) of the rDNA repeat unit in Scots pine is longer than 20 kb, and the transcribed spacer regions surrounding the 5.8S gene (ITS1 and ITS2) span a region of 2.9 kb. Restriction analysis revealed that although the coding regions of rDNA repeats are homogeneous, heterogeneity exists in the intergenic spacer region between individuals, as well as among the rDNA repeats within individuals.     

Abscisic acid in shoots and roots of Scots pine (Pinus sylvestris L.) seedlings grown in controlled long-day and short-day environments

Scots pine (Pinus sylvestris L.) seedlings grown in nutrient solution in controlled-environment chambers were used. The effects of a shortday (SD, early autumn) treatment on growth and the content of free and alkaline hydrolysable abscisic acid (ABA) in shoots and roots were investigated. The weekly relative growth rates of seedlings grown continuously under long-day (LD, summer) conditions were stable at approx. 0.08 g g^–1 d^–1 between weeks four and eight from germination. Weekly relative growth rates of seedlings transferred to SD conditions decreased rapidly to a then stable level of approx. 0.04 g g^–1 d⁰¹. Shoot elongation ceased within two weeks of SD treatment. The content of both free and alkaline hydrolysable ABA was approx. 40–50% higher in shoots of seedlings grown for five weeks in LD plus one week in SD than in shoots of seedlings grown for five or six weeks in LD. Two additional weeks of SD did not change the free ABA content. Three weeks in simulated late autumn (SD but decreased temperatures) and three weeks in simulated winter (lower light intensity and temperature) further increased the content of free ABA in the shoots. A transfer back to LD conditions reduced the ABA content to a level equal to the level found during the first LD period. The recovery of radioactive ABA at certain times after application ofr[³H] ABA was the same in shoots and roots of LD-grown and SD-treated seedlings.Abbreviations ABA abscisic acid - LD long day(s) - RGR₇ weekly relative growth rates - SD short day(s)     

Sensitivity to Cd or Zn of host and symbiont of ectomycorrhizal Pinus sylvestris L. (Scots pine) seedlings

Pinus sylvestris seedlings infected with either the ectomycorrhizal (ECM) fungus Paxillus involutus or Suillus variegatus were exposed to a range of Cd or Zn concentrations. This was done to investigate the relationship between the sensitivity of ECM fungi and their host plants over a wide range of concentrations. P. involutus ameliorated the toxicity of Cd and Zn to P. sylvestris with respect to root length, despite significant inhibition of ECM infection levels by Cd (Cd EC₅₀ [effective concentration which inhibits ECM infection by 50%] values were: P. involutus 3.7 μg g^-1 Cd; S. variegatus 2.3 μg g^-1 Cd). ECM infection by P. involutus also decreased Cd and Zn transport to the plant shoots at potentially toxic concentrations and also influenced the proportion of Zn transported to the roots and shoots, with a higher proportion retained in the roots of the seedlings. ECM infection did increase host biomass production, but this was not affected by the presence of Cd or Zn. Root and shoot biomass production by P. sylvestris, in both the presence and absence of ECM fungi, was unaffected by Cd and Zn at all concentrations tested. This revised version was published online in August 2006 with corrections to the Cover Date.     

Fourier-transform infrared and Raman spectroscopic evidence for the incorporation of cinnamaldehydes into the lignin of transgenic tobacco (Nicotiana tabacum L.) plants with reduced expression of cinnamyl alcohol dehydrogenase

Xylem from stems of genetically manipulated tobacco plants which had had cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) activity down-regulated to a greater or lesser degree (clones 37 and 49, respectively) by the insertion of antisense CAD cDNA had similar, or slightly higher, lignin contents than xylem from wild-type plants. Fourier-transform infrared (FT-IR) microspectroscopy indicated that down-regulation of CAD had resulted in the incorporation of moieties with conjugated carbonyl groups into lignin and that the overall extent of cross-linking, particularly of guaiacyl (4-hydroxy-3-methoxyphenyl) rings, in the lignin had altered. The FT-Raman spectra of manipulated xylem exhibited maxima consistent with the presence of elevated levels of aldehydic groups conjugated to a carbon-carbon double bond and a guaiacyl ring. These maxima were particularly intense in the spectra of xylem from clone 37, the xylem of which exhibits a uniform red coloration, and their absolute frequencies matched those of coniferaldehyde. Furthermore, xylem from clone 37 was found to have a higher content of carbonyl groups than that of clone 49 or the wild-type (clone 37: clone 49: wild-type; 2.4:1.6:1.0) as measured by a degradative chemical method. This is the first report of the combined use of FT-IR and FT-Raman spectroscopies to study lignin structure in situ. These analyses provide strong evidence for the incorporation of cinnamaldehyde groups into the lignin of transgenic plants with down-regulated CAD expression. In addition, these non-destructive analyses also suggest that the plants transformed with antisense CAD, in particular clone 37, may contain lignin that is less condensed (cross-linked) than that of the wild-type. Received: 27 May 1996 / Accepted: 30 July 1996     

Climate-tree-growth relationships of Scots pine stands (Pinus sylvestris L.) exposed to soil dryness

 Dendroclimatological techniques were used to assess the impact of climatic factors on radial tree growth (total ring-width and latewood-width) of stunted Scots pine trees (Pinus sylvestris L.) exposed to soil dryness and nutrient deficiency on a dolomite substrate. The response of eight scattered populations representing various habitats, yet influenced by the same regional climate was investigated. Total ring-width and latewood-width were dated, standardized and several chronology statistics, which estimate the chronology signal strength and the potential climate signal in the series, were determined. Dendroclimatic analysis comprised evaluation of event/pointer years as well as response function elements. Response function analysis indicates that at most sites wide rings are significantly associated with high precipitation in April to June and cool conditions in May of the current year, and high precipitation in August to September of the preceding year. Latewood chronologies show a lower climate signal at all sites. Limited water availability causes a homogeneous relationship of radial tree growth to climate at all habitats, though site characteristics (slope magnitude, slope aspect, soil depth, vegetation cover) differ substantially. Cluster analysis of negative event/pointer years suggests that within the study area stands react to extreme climatic events depending on susceptibility to soil dryness, which is primarily determined by site topography. Received: 5 January 1998 / Accepted: 22 April 1998     

Effects of frost hardening, dehardening and freezing stress on in vivo chlorophyll fluorescence of seedlings of Scots pine (Pinus sylvestris L.)

Abstract. The kinetics of in vivo chlorophyll fluorescence of photosystem II (PS II) was measured at room temperature and 77 K during frost hardening of seedlings of Scots pine (Pinus sylvestris L.), and after exposure of frost-hardened shoots to sub-freezing temperatures. A more pronounced decrease in variable fluorescence yield for the upper exposed than for the lower shaded surface of the needles suggested that some photoinhibition occurred during prolonged frost hardening at 50 μmol photons m^?2 s^?1 and 4°C. Reversible inhibition of photosynthesis after exposure to sub-freezing temperatures was initially manifested as an increase of steady-state energy-dependent fluorescence quenching (q_E) and a reduction in the rate of O₂ evolution. Further inhibition after treatment at still lower temperatures caused a progressive decline of steady-state photochemical quenching (q_Q) and the rate of O₂ evolution, whereas q_E remained high. This implies an inactivation of enzymes in the photosynthetic carbon reduction cycle decreasing the consumption of ATP and NADPH, which is likely to cause an increase of membrane energization and a reduction of the primary electron acceptor (Q_A) of PS II. Alternatively, the changes in q_Q and q_E might be attributed to an inhibition of photophosphorylation. Severe, irreversible damage to photosynthesis resulted in a suppression of q_E and of variable fluorescence yield, probably because the photochemical efficiency of PS II was impaired. Changes in the fast fluorescence kinetics at room temperature after severe freezing damage were interpreted as an inhibition of the electron flow from Q_A to the plastoquinone pool. It is suggested that irreversible freezing injury to needles of frost-hardened P. sylvestris causes damage to the Q_B,-protein.     

Daily dynamics in xylem cell radial growth of Scots pine (Pinus sylvestris L.)

Daily dynamics of radial cell expansion during wood formation within the stems of 25-year-old Scots pine trees (Pinus sylvestris L.), growing in field conditions, were studied. The samples of forming wood layers were extracted 4 times per day for 3 days. Possible variations in the growth on different sides of the stem, duration of cell development in radial cell expansion phase and dynamics of cell growth in this phase were taken into account. The perimeters of tracheid cross-sections as a reflection of primary cell wall growth were the criterion of growth in a radial direction. For the evaluation of growing cell perimeters a special system for digital processing and image analysis of tracheid cross-sections of the forming wood was used. Growth rate for certain time intervals was estimated by the change in the relation of the perimeter of each observed cell in each of ten tracheid rows in each of 12 trees to the perimeter of the xylem cell of the same row before the expansion. Temporal differences in average values of the relations were estimated by Analyses of Variance. The existence of daily dynamics of Scots pine xylem cell radial growth has been proved. Intensive growth of pine tracheids has been shown to occur at any time of the day and to depend on the temperature regime of the day and the night as well as water supply of stem tissues. Moreover, reliable differences (P = 0.95) in the increment of cell walls during tracheid radial expansion have been found. Pulsing changes of the water potentials both of the cell and the apoplast, as the reason for the fluctuations of radial cell growth rate, were discussed.