Ray Parenchymal Cells Contribute to Lignification of Tracheids in Developing Xylem of Norway Spruce.

A comparative transcriptomic study and a single-cell metabolome analysis were combined to determine whether parenchymal ray cells contribute to the biosynthesis of monolignols in the lignifying xylem of Norway spruce (Picea abies). Ray parenchymal cells may function in the lignification of upright tracheids by supplying monolignols. To test this hypothesis, parenchymal ray cells and upright tracheids were dissected with laser-capture microdissection from tangential cryosections of developing xylem of spruce trees. The transcriptome analysis revealed that among the genes involved in processes typical for vascular tissues, genes encoding cell wall biogenesis-related enzymes were highly expressed in both developing tracheids and ray cells. Interestingly, most of the shikimate and monolignol biosynthesis pathway-related genes were equally expressed in both cell types. Nonetheless, 1,073 differentially expressed genes were detected between developing ray cells and tracheids, among which a set of genes expressed only in ray cells was identified. In situ single cell metabolomics of semi-intact plants by picoliter pressure probe-electrospray ionization-mass spectrometry detected monolignols and their glycoconjugates in both cell types, indicating that the biosynthetic route for monolignols is active in both upright tracheids and parenchymal ray cells. The data strongly support the hypothesis that in developing xylem, ray cells produce monolignols that contribute to lignification of tracheid cell walls.

Cellulose Synthase Stoichiometry in Aspen Differs from Arabidopsis and Norway Spruce.

Cellulose is synthesized at the plasma membrane by cellulose synthase complexes (CSCs) containing cellulose synthases (CESAs). Genetic analysis and CESA isoform quantification indicate that cellulose in the secondary cell walls of Arabidopsis (Arabidopsis thaliana) is synthesized by isoforms CESA4, CESA7, and CESA8 in equimolar amounts. Here, we used quantitative proteomics to investigate whether the CSC model based on Arabidopsis secondary cell wall CESA stoichiometry can be applied to the angiosperm tree aspen (Populus tremula) and the gymnosperm tree Norway spruce (Picea abies). In the developing xylem of aspen, the secondary cell wall CESA stoichiometry was 3:2:1 for PtCESA8a/b:PtCESA4:PtCESA7a/b, while in Norway spruce, the stoichiometry was 1:1:1, as observed previously in Arabidopsis. Furthermore, in aspen tension wood, the secondary cell wall CESA stoichiometry changed to 8:3:1 for PtCESA8a/b:PtCESA4:PtCESA7a/b. PtCESA8b represented 73% of the total secondary cell wall CESA pool, and quantitative polymerase chain reaction analysis of CESA transcripts in cryosectioned tension wood revealed increased PtCESA8b expression during the formation of the cellulose-enriched gelatinous layer, while the transcripts of PtCESA4, PtCESA7a/b, and PtCESA8a decreased. A wide-angle x-ray scattering analysis showed that the shift in CESA stoichiometry in tension wood coincided with an increase in crystalline cellulose microfibril diameter, suggesting that the CSC CESA composition influences microfibril properties. The aspen CESA stoichiometry results raise the possibility of alternative CSC models and suggest that homomeric PtCESA8b complexes are responsible for cellulose biosynthesis in the gelatinous layer in tension wood.

Picea wilsonii transcription factor NAC2 enhanced plant tolerance to abiotic stress and participated in RFCP1-regulated flowering time.

KEY MESSAGE: Picea wilsonii transcription factor PwNAC2 enhanced plant tolerance to salt and drought stress through multiple signaling pathway and interacted with PwRFCP1 to participate in flowering regulation. NAC is one of the largest transcription factor families in plants, however, its role is not yet fully understood. Here, we identified a transcription factor PwNAC2 in Picea wilsonii, which localized in nucleus with transcriptional activity in C-terminal region and can form homodimer by itself. Expression analysis by real-time PCR showed that PwNAC2 was induced by multiple abiotic stresses and phytohormones stimuli. PwRFCP1 (Resemble-FCA-contain-PAT1 domain), an interaction protein of PwNAC2 was screened via yeast two hybrid. Luciferase complementation assay confirmed the interaction in vivo and bimolecular fluorescence complementation assay showed the interaction in nucleus. PwNAC2 overexpression retarded Arabidopsis hypocotyls growth which is closely related to light, whereas promotion of hypocotyls growth by PwRFCP1 is independent on light. Under drought or salt treatment, overexpression of PwNAC2 in Arabidopsis showed more vigorous seed germination and significant tolerance for seedlings by ROS scavenging, reducing of membrane damage, slower water loss and increased stomatal closure. ABA or CBF-pathway marker genes were substantially higher in PwNAC2 transgenic Arabidopsis. Overexpression of PwRFCP1 promotes flowering in transgenic Arabidopsis, whereas PwNAC2 delayed flowering by altering the expression of FT, SOC1 and FLC. In addtioin, PwRFCP1 overexpression plants showed no higher tolerance to stress treatment than Col-0. Collectively, our results indicate that PwNAC2 enhanced plant tolerance to abiotic stress through multiple signaling pathways and participated in PwRFCP1-regulated flowering time.

The Contribution of Carbon and Water in Modulating Wood Formation in Black Spruce Saplings.

Nonstructural carbohydrates (NSCs) play a crucial role in xylem formation and represent, with water, the main constraint to plant growth. We assessed the relationships between xylogenesis and NSCs in order to (1) verify the variance explained by NSCs and (2) determine the influence of intrinsic (tissue supplying carbon) and extrinsic (water availability and temperature) factors. During 2 years, wood formation was monitored in saplings of black spruce (Picea mariana) subjected to a dry period of about 1 month in June and exposed to different temperature treatments in a greenhouse. In parallel, NSC concentrations were determined by extracting the sugar compounds from two tissues (cambium and inner xylem), both potentially supplying carbon for wood formation. A mixed-effect model was used to assess and quantify the potential relationships. Total xylem cells, illustrating meristematic activity, were modeled as a function of water, sucrose, and d-pinitol (conditional r(2) of 0.79). Water availability was ranked as the most important factor explaining total xylem cell production, while the contribution of carbon was lower. Cambium stopped dividing under water deficit, probably to limit the number of cells remaining in differentiation without an adequate amount of water. By contrast, carbon factors were ranked as most important in explaining the variation in living cells (conditional r(2) of 0.49), highlighting the functional needs during xylem development, followed by the tissue supplying the NSCs (cambium) and water availability. This study precisely demonstrates the role of carbon and water in structural growth expressed as meristematic activity and tissue formation.

In Planta Localization of Stilbenes within Picea abies Phloem.

Phenolic stilbene glucosides (astringin, isorhapontin, and piceid) and their aglycons commonly accumulate in the phloem of Norway spruce (Picea abies). However, current knowledge about the localization and accumulation of stilbenes within plant tissues and cells remains limited. Here, we used an innovative combination of novel microanalytical techniques to evaluate stilbenes in a frozen-hydrated condition (i.e. in planta) and a freeze-dried condition across phloem tissues. Semiquantitative time-of-flight secondary ion-mass spectrometry imaging in planta revealed that stilbenes were localized in axial parenchyma cells. Quantitative gas chromatography analysis showed the highest stilbene content in the middle of collapsed phloem with decreases toward the outer phloem. The same trend was detected for soluble sugar and water contents. The specimen water content may affect stilbene composition; the glucoside-to-aglycon ratio decreased slightly with decreases in water content. Phloem chemistry was correlated with three-dimensional structures of phloem as analyzed by microtomography. The outer phloem was characterized by a high volume of empty parenchyma, reduced ray volume, and a large number of axial parenchyma with porous vacuolar contents. Increasing porosity from the inner to the outer phloem was related to decreasing compactness of stilbenes and possible secondary oxidation or polymerization. Our results indicate that aging-dependent changes in phloem may reduce cell functioning, which affects the capacity of the phloem to store water and sugar, and may reduce the defense potential of stilbenes in the axial parenchyma. Our results highlight the power of using a combination of techniques to evaluate tissue- and cell-level mechanisms involved in plant secondary metabolite formation and metabolism.

Xylogenesis: Coniferous Trees of Temperate Forests Are Listening to the Climate Tale during the Growing Season But Only Remember the Last Words!

The complex inner mechanisms that create typical conifer tree-ring structure (i.e. the transition from large, thin-walled earlywood cells to narrow, thick-walled latewood cells) were recently unraveled. However, what physiological or environmental factors drive xylogenesis key processes remain unclear. Here, we aim to quantify the influence of seasonal variations in climatic factors on the spectacular changes in the kinetics of wood cell differentiation and in the resulting tree-ring structure. Wood formation was monitored in three sites over 3 years for three coniferous species (Norway spruce [Picea abies], Scots pine [Pinus sylvestris], and silver fir [Abies alba]). Cell differentiation rates and durations were calculated and related to tracheid final dimensions and corresponding climatic conditions. On the one hand, we found that the kinetics of cell enlargement and the final size of the tracheids were not explained by the seasonal changes in climatic factors. On the other hand, decreasing temperatures strongly constrained cell wall deposition rates during latewood formation. However, the influence of temperature was permanently written into tree-ring structure only for the very last latewood cells, when the collapse of the rate of wall deposition was no longer counterbalanced by the increase of its duration. Our results show that the formation of the typical conifer tree-ring structure, in normal climatic conditions, is only marginally driven by climate, suggesting strong developmental control of xylogenesis. The late breakage of the compensatory mechanism at work in the wall deposition process appears as a clue to understand the capacity of the maximum latewood density to record past temperature conditions.

EXTRA SPINDLE POLES (Separase) controls anisotropic cell expansion in Norway spruce (Picea abies) embryos independently of its role in anaphase progression.

The caspase-related protease separase (EXTRA SPINDLE POLES, ESP) plays a major role in chromatid disjunction and cell expansion in Arabidopsis thaliana. Whether the expansion phenotypes are linked to defects in cell division in Arabidopsis ESP mutants remains elusive. Here we present the identification, cloning and characterization of the gymnosperm Norway spruce (Picea abies, Pa) ESP. We used the P. abies somatic embryo system and a combination of reverse genetics and microscopy to explore the roles of Pa ESP during embryogenesis. Pa ESP was expressed in the proliferating embryonal mass, while it was absent in the suspensor cells. Pa ESP associated with kinetochore microtubules in metaphase and then with anaphase spindle midzone. During cytokinesis, it localized on the phragmoplast microtubules and on the cell plate. Pa ESP deficiency perturbed anisotropic expansion and reduced mitotic divisions in cotyledonary embryos. Furthermore, whilst Pa ESP can rescue the chromatid nondisjunction phenotype of Arabidopsis ESP mutants, it cannot rescue anisotropic cell expansion. Our data demonstrate that the roles of ESP in daughter chromatid separation and cell expansion are conserved between gymnosperms and angiosperms. However, the mechanisms of ESP-mediated regulation of cell expansion seem to be lineage-specific.

Enhanced thermal stability of the thylakoid membranes from spruce. A comparison with selected angiosperms.

Recently, we have found that thermal stability of photosystem II (PSII) photochemistry in spruce needles is higher than in other plants (barley, beech) cultivated under the same temperatures. In this work, temperature dependences of various characteristics of PSII organization were studied in order to obtain complex information on the thermal stability of PSII function and organization in spruce. Temperature dependency of circular dichroism spectra revealed by about 6 °C higher thermal stability of macrodomain organization in spruce thylakoid membranes in comparison with Arabidopsis and barley ones; however, thermal disintegration of light-harvesting complex of PSII did not significantly differ among the species studied. These results thus indicate that thermal stability of PSII macro-organization in spruce thylakoid membranes is enhanced to a similar extent as thermal stability of PSII photochemistry. Clear-native polyacrylamide gel electrophoresis of preheated thylakoids demonstrated that among the separated pigment-protein complexes, only PSII supercomplexes (SCs) revealed considerably higher thermal stability in spruce thylakoids as compared to Arabidopsis and barley ones. Hence we suggest that higher thermal stability of PSII macro-organization of spruce is influenced by the maintenance of PSII SCs in the thylakoid membrane. In addition, we discuss possible effects of different PSII organizations and lipid compositions on the thermal stability of spruce thylakoid membranes.

Function of Sitka spruce stone cells as a physical defence against white pine weevil.

Stone cells are a physical defence of conifers against stem feeding insects such as weevils and bark beetles. In Sitka spruce, abundance of stone cells in the cortex of apical shoot tips is associated with resistance to white pine weevil. However, the mode of action by which stone cells interfere with growth and development of weevil larvae is unknown. We developed a bioassay system for testing potential effects of stone cells, which were isolated from resistant trees, on weevil larvae. Bioassays using artificial diet and controlled amounts of stone cells focused on physical defence. We evaluated the effects of stone cells on establishment of neonate larvae, mandible wear and changes in relative growth rates of third instar larvae. Establishment of neonates and relative growth rates of third instars were significantly reduced by stone cells. Stone cells appeared to be indigestible by weevil larvae. Our results suggest that stone cells affect weevil establishment and development by forming a physical feeding barrier against neonate larvae at the site of oviposition, and by reducing access to nutrients in the cortex of resistant trees, which contain an abundance of stone cells in place of a more nutrient rich tissue in susceptible trees.

Fluorescence-Detected Linear Dichroism of Wood Cell Walls in Juvenile Serbian Spruce: Estimation of Compression Wood Severity.

Fluorescence-detected linear dichroism (FDLD) microscopy provides observation of structural order in a microscopic sample and its expression in numerical terms, enabling both quantitative and qualitative comparison among different samples. We applied FDLD microscopy to compare the distribution and alignment of cellulose fibrils in cell walls of compression wood (CW) and normal wood (NW) on stem cross-sections of juvenile Picea omorika trees. Our data indicate a decrease in cellulose fibril order in CW compared with NW. Radial and tangential walls differ considerably in both NW and CW. In radial walls, cellulose fibril order shows a gradual decrease from NW to severe CW, in line with the increase in CW severity. This indicates that FDLD analysis of cellulose fibril order in radial cell walls is a valuable method for estimation of CW severity.

Uptake of water via branches helps timberline conifers refill embolized xylem in late winter.

Xylem embolism is a limiting factor for woody species worldwide. Conifers at the alpine timberline are exposed to drought and freeze-thaw stress during winter, which induce potentially lethal embolism. Previous studies indicated that timberline trees survive by xylem refilling. In this study on Picea abies, refilling was monitored during winter and spring seasons and analyzed in the laboratory and in situ experiments, based on hydraulic, anatomical, and histochemical methods. Refilling started in late winter, when the soil was frozen and soil water not available for the trees. Xylem embolism caused up to 86.2% ± 3.1% loss of conductivity and was correlated with the ratio of closed pits. Refilling of xylem as well as recovery in shoot conductance started in February and corresponded with starch accumulation in secondary phloem and in the mesophyll of needles, where we also observed increasing aquaporin densities in the phloem and endodermis. This indicates that active, cellular processes play a role for refilling even under winter conditions. As demonstrated by our experiments, water for refilling was thereby taken up via the branches, likely by foliar water uptake. Our results suggest that refilling is based on water shifts to embolized tracheids via intact xylem, phloem, and parenchyma, whereby aquaporins reduce resistances along the symplastic pathway and aspirated pits facilitate isolation of refilling tracheids. Refilling must be taken into account as a key process in plant hydraulics and in estimating future effects of climate change on forests and alpine tree ecosystems.

Wuschel-related homeobox 8/9 is important for proper embryo patterning in the gymnosperm Norway spruce.

Proper embryo development is crucial as that is when the primary body axes are established. In Arabidopsis, AtWOX8 and AtWOX9, members of the Wuschel-related homeobox (WOX) gene family, are critical for embryo development. In Norway spruce, PaWOX8/9, which is expressed in embryos, is the homologue of AtWOX8 and AtWOX9. In this work, it is shown that the transcript abundance of PaWOX8/9 is high during early and late embryogeny and that it decreases when the maturation phase starts. To address the function of PaWOX8/9 during embryo development, RNAi lines were established to down-regulate the transcript level of PaWOX8/9, using both constitutive and inducible promoters. Embryos in the PaWOX8/9 RNAi lines show an aberrant morphology caused by disturbed orientation of the cell division plane at the basal part of the embryonal mass during early and late embryogeny. In addition, the transcript level of several key cell-cycle-regulating genes, for example, PaE2FAB-like and PaCYCLIN B-like, are affected in the PaWOX8/9 RNAi lines. Taken together, our results suggest that PaWOX8/9 may perform an evolutionarily conserved function as a regulator of the establishment of the apical-basal embryo pattern.

Hysteresis in swelling and in sorption of wood tissue.

The swelling and shrinkage of four Picea abies (L. Karst) wood tissue homogeneous samples, of porosity varying between 45% and 78%, is documented with high-resolution synchrotron radiation phase-contrast X-ray tomographic microscopy. We report measurements of the reversible moisture-induced orthotropic swelling/shrinkage strains. Hysteresis is observed when the swelling/shrinkage strain is considered as a function of relative humidity, except for the very high porosity sample. Hysteresis is no longer present when swelling/shrinkage strains are considered versus moisture content, indicating that wood deforms to the same extent whether an amount of moisture is desorbed or adsorbed. Furthermore, swelling anisotropy, in the tangential and radial directions, is found to increase with increasing porosity. The most homogeneous behaviour for a group of cells is found for 30-50 cells, smaller/larger groups having higher orders of variations.

The effect of carbohydrates and osmoticum on storage reserve accumulation and germination of Norway spruce somatic embryos.

Somatic embryogenesis (SE) represents a useful experimental system for studying the regulatory mechanisms of embryo development. In this study, the effect of carbohydrates and osmoticum on storage reserve accumulation and germination of Norway spruce [Picea abies (L.) Karst] somatic embryos were investigated. Using time lapse photography, we monitored development from proliferation of proembryogenic masses (PEMs) to maturation of somatic embryos in two P. abies cell lines cultured on two maturation treatments. A combination of sugar assays, metabolic and proteomic analyses were used to quantify storage reserves in the mature somatic embryos. The maturation treatment containing a nonpermeating osmoticum, polyethylene glycol (PEG, 7.5%) and maltose (3%) as the carbohydrate gave significantly high maturation and low germination frequencies of somatic embryos compared to the treatment with only 3% sucrose. Somatic embryos treated with 3% sucrose contained high levels of sucrose, raffinose and late embryogenesis abundant (LEA) proteins. These compounds are known to be involved in the acquisition of desiccation tolerance during seed development and maturation. In addition the sucrose treatment significantly increased the content of starch in the somatic embryos while the maltose and PEG treatment resulted in somatic embryos with a high content of storage proteins. The high levels of sucrose, raffinose and LEA proteins in the embryos treated with 3% sucrose suggest that sucrose may improve the germination of somatic embryos by promoting the acquisition of desiccation tolerance.

Identification of defensin-encoding genes of Picea glauca: characterization of PgD5, a conserved spruce defensin with strong antifungal activity.

BACKGROUND: Plant defensins represent a major innate immune protein superfamily that displays strong inhibitory effects on filamentous fungi. The total number of plant defensins in a conifer species is unknown since there are no sequenced conifer genomes published, however the genomes of several angiosperm species provide an insight on the diversity of plant defensins. Here we report the identification of five new defensin-encoding genes from the Picea glauca genome and the characterization of two of their gene products, named PgD5 and endopiceasin. RESULTS: Screening of a P. glauca EST database with sequences of known plant defensins identified four genes with homology to the known P. glauca defensin PgD1, which were designated PgD2-5. Whereas in the mature PgD2-4 only 7-9 amino acids differed from PgD1, PgD5 had only 64% sequence identity. PgD5 was amplified from P. glauca genomic DNA by PCR. It codes for a precursor of 77-amino acid that is fully conserved within the Picea genus and has similarity to plant defensins. Recombinant PgD5, produced in Escherichia coli, had a molecular mass of 5.721 kDa, as determined by mass spectrometry. The PgD5 peptide exhibited strong antifungal activity against several phytopathogens without any effect on the morphology of the treated fungal hyphae, but strongly inhibited hyphal elongation. A SYTOX uptake assay suggested that the inhibitory activity of PgD5 could be associated with altering the permeability of the fungal membranes. Another completely unrelated defensin gene was identified in the EST library and named endopiceasin. Its gene codes for a 6-cysteine peptide that shares high similarity with the fungal defensin plectasin. CONCLUSIONS: Screening of a P. glauca EST database resulted in the identification of five new defensin-encoding genes. PgD5 codes for a plant defensin that displays non-morphogenic antifungal activity against the phytopathogens tested, probably by altering membrane permeability. PgD5 has potential for application in the plant biotechnology sector. Endopiceasin appears to derive from an endo- or epiphytic fungal strain rather than from the plant itself.

Localization of phenolics in phloem parenchyma cells of Norway spruce (Picea abies).

Norway spruce (Picea abies) bark contains specialized phloem parenchyma cells that swell and change their contents upon attack by the bark beetle Ips typographus and its microbial associate, the blue stain fungus Ceratocystis polonica. These cells exhibit bright autofluorescence after treatment with standard aldehyde fixatives, and so have been postulated to contain phenolic compounds. Laser microdissection of spruce bark sections combined with cryogenic NMR spectroscopy demonstrated significantly higher concentrations of the stilbene glucoside astringin in phloem parenchyma cells than in adjacent sieve cells. After infection by C. polonica, the flavonoid (+)-catechin also appeared in phloem parenchyma cells and there was a decrease in astringin content compared to cells from uninfected trees. Analysis of whole-bark extracts confirmed the results obtained from the cell extracts and revealed a significant increase in dimeric stilbene glucosides, both astringin and isorhapontin derivatives (piceasides A to H), in fungus-infected versus uninfected bark that might explain the reduction in stilbene monomers. Phloem parenchyma cells thus appear to be a principal site of phenolic accumulation in spruce bark.

Xylem defense wood of Norway spruce compromised by the pathogenic white-rot fungus Heterobasidion parviporum shows a prolonged period of selective decay.

Heterobasidion parviporum, a common pathogenic white-rot fungus in managed Norway spruce forests in northern and central Europe, causes extensive decay columns within stem heartwood of the host tree. Infected trees combat the lateral spread of decay by bordering the heartwood with a fungistatic reaction zone characterized by elevated pH and phenol content. To examine the mode of fungal feeding in the reaction zone of mature Norway spruce trees naturally infected by H. parviporum, we conducted spatial profiling of pectin and hemicellulose composition, and established transcript levels of candidate fungal genes encoding enzymes involved in degradation of the different cell wall components of wood. Colonized inner heartwood showed pectin and hemicellulose concentrations similar to those of healthy heartwood, whereas the carbohydrate profiles of compromised reaction zone, irrespective of the age of fungal activity in the tissue, indicated selective fungal utilization of galacturonic acid, arabinose, xylose and mannose. These data show that the rate of wood decay in the reaction zone is slow. While the up-regulation of genes encoding pectinases and hemicellulases preceded that of the endoglucanase gene during an early phase of fungal interaction with xylem defense, the manganese peroxidase gene showed similar transcript levels during different phases of wood colonization. It seems plausible that the reaction zone components of Norway spruce interfere with both lignin degradation and the associated co-hydrolysis of hemicelluloses and pectin, resulting in a prolonged phase of selective decay.

The endo-1,4-ß-glucanase Korrigan exhibits functional conservation between gymnosperms and angiosperms and is required for proper cell wall formation in gymnosperms.

The evolution of compositional polymers and their complex arrangement and deposition in the cell walls of terrestrial plants included the acquisition of key protein functions. A membrane-bound endoglucanase, termed Korrigan (KOR), has been shown to be required for proper cellulose synthesis. To date, no extensive characterization of the gymnosperm KOR has been undertaken. Characterization of the white spruce (Picea glauca) gene encoding KOR (PgKOR) shows conserved protein features such as polarized targeting signals and residues predicted to be essential for catalytic activity. The rescue of the Arabidopsis thaliana kor1-1 mutant by the expression of PgKOR suggests gene conservation, providing evidence for functional equivalence. Analyses of endogenous KOR expression in white spruce revealed the highest expression in young developing tissues, which corresponds with primary cell wall development. Additionally, RNA interference of the endogenous gymnosperm gene substantially reduced growth and structural glucose content, but had no effect on cellulose ultrastructure. Partial functional conservation of KOR in gymnosperms suggests that its role in cell wall synthesis dates back to 300 million yr ago (Mya), predating angiosperms, which arose 130 Mya, and shows that proteins contributing to proper cellulose deposition are important conserved features of vascular plants.

Causes and correlations in cambium phenology: towards an integrated framework of xylogenesis.

Although habitually considered as a whole, xylogenesis is a complex process of division and maturation of a pool of cells where the relationship between the phenological phases generating such a growth pattern remains essentially unknown. This study investigated the causal relationships in cambium phenology of black spruce [Picea mariana (Mill.) BSP] monitored for 8 years on four sites of the boreal forest of Quebec, Canada. The dependency links connecting the timing of xylem cell differentiation and cell production were defined and the resulting causal model was analysed with d-sep tests and generalized mixed models with repeated measurements, and tested with Fisher's C statistics to determine whether and how causality propagates through the measured variables. The higher correlations were observed between the dates of emergence of the first developing cells and between the ending of the differentiation phases, while the number of cells was significantly correlated with all phenological phases. The model with eight dependency links was statistically valid for explaining the causes and correlations between the dynamics of cambium phenology. Causal modelling suggested that the phenological phases involved in xylogenesis are closely interconnected by complex relationships of cause and effect, with the onset of cell differentiation being the main factor directly or indirectly triggering all successive phases of xylem maturation.

The expression pattern of the Picea glauca Defensin 1 promoter is maintained in Arabidopsis thaliana, indicating the conservation of signalling pathways between angiosperms and gymnosperms.

A 1149 bp genomic fragment corresponding to the 5' non-coding region of the PgD1 (Picea glauca Defensin 1) gene was cloned, characterized, and compared with all Arabidopsis thaliana defensin promoters. The cloned fragment was found to contain several motifs specific to defence or hormonal response, including a motif involved in the methyl jasmonate reponse, a fungal elicitor responsive element, and TC-rich repeat cis-acting element involved in defence and stress responsiveness. A functional analysis of the PgD1 promoter was performed using the uidA (GUS) reporter system in stably transformed Arabidopsis and white spruce plants. The PgD1 promoter was responsive to jasmonic acid (JA), to infection by fungus and to wounding. In transgenic spruce embryos, GUS staining was clearly restricted to the shoot apical meristem. In Arabidopsis, faint GUS coloration was observed in leaves and flowers and a strong blue colour was observed in guard cells and trichomes. Transgenic Arabidopsis plants expressing the PgD1::GUS construct were also infiltrated with the hemibiotrophic pathogen Pseudomonas syringae pv. tomato DC3000. It caused a suppression of defensin expression probably resulting from the antagonistic relationship between the pathogen-stimulated salicylic acid pathway and the jasmonic acid pathway. It is therefore concluded that the PgD1 promoter fragment cloned appears to contain most if not all the elements for proper PgD1 expression and that these elements are also recognized in Arabidopsis despite the phylogenetic and evolutionary differences that separates them.