Transcriptome analysis of female and male flower buds of Idesia polycarpa Maxim. var. vestita Diels

Background: Idesia polycarpa Maxim. var. vestita Diels, a dioecious plant, is widely used for biodiesel due to the high oil content of its fruits. However, it is hard to distinguish its sex in the seedling stage, which makes breeding and production problematic as only the female tree can produce fruits, and the mechanisms underlying sex determination and differentiation remain unknown due to the lack of available genomic and transcriptomic information. To begin addressing this issue, we performed the transcriptome analysis of its female and male flower. Results: 28,668,977 and 22,227,992 clean reads were obtained from the female and male cDNA libraries, respectively. After quality checks and de novo assembly, a total of 84,213 unigenes with an average length of 1179 bp were generated and 65,972 unigenes (78.34%) could be matched in at least one of the NR, NT, Swiss-Prot, COG, KEGG and GO databases. Functional annotation of the unigenes uncovered diverse biological functions and processes, including reproduction and developmental process, which may play roles in sex determination and differentiation. The Kyoto Encyclopedia of Genes and Genomes pathway analysis showed many unigenes annotated as metabolic pathways, biosynthesis of secondary metabolites pathways, plant­ pathogen interaction, and plant hormone signal transduction. Moreover, 29,953 simple sequence repeats were identified using the microsatellite software. Conclusion: This work provides the first detailed transcriptome analysis of female and male flower of I. polycarpa and lays foundations for future studies on the molecular mechanisms underlying flower bud development of I. polycarpa.

Organismal responses to habitat change: herbivore performance, climate and leaf traits in regenerating tropical dry forests.

The ecological effects of large-scale climate change have received much attention, but the effects of the more acute form of climate change that results from local habitat alteration have been less explored. When forest is fragmented, cut, thinned, cleared or otherwise altered in structure, local climates and microclimates change. Such changes can affect herbivores both directly (e.g. through changes in body temperature) and indirectly (e.g. through changes in host plant traits). We advance an eco-physiological framework to understand the effects of changing forests on herbivorous insects. We hypothesize that if tropical forest caterpillars are climate and resource specialists, then they should have reduced performance outside of mature forest conditions. We tested this hypothesis with a field experiment contrasting the performance of Rothschildia lebeau (Saturniidae) caterpillars feeding on the host plant Casearia nitida (Salicaceae) in two different aged and structured tropical dry forests in Area de Conservación Guanacaste, Costa Rica. Compared to more mature closed-canopy forest, in younger secondary forest we found that: (1) ambient conditions were hotter, drier and more variable; (2) caterpillar growth and development were reduced; and (3) leaves were tougher, thicker and drier. Furthermore, caterpillar growth and survival were negatively correlated with these leaf traits, suggesting indirect host-mediated effects of climate on herbivores. Based on the available evidence, and relative to mature forest, we conclude that reduced herbivore performance in young secondary forest could have been driven by changes in climate, leaf traits (which were likely climate induced) or both. However, additional studies will be needed to provide more direct evidence of cause-and-effect and to disentangle the relative influence of these factors on herbivore performance in this system.

Contrasting responses in the growth and energy utilization properties of sympatric Populus and Salix to different altitudes: implications for sexual dimorphism in Salicaceae.

An interesting ecological and evolutionary puzzle arises from the observations of male-biased sex ratios in genus Populus, whereas in the taxonomically related Salix, females are generally more dominant. In the present study, we combined results from a field investigation into the sex ratios of the Salicaceous species along an altitudinal gradient on Gongga Mountain, and a pot experiment by monitoring growth and energy utilization properties to elucidate the mechanisms governing sexual dimorphism. At middle altitudes 2000 and 2300 m, the sex ratios were consistent with a 1:1 equilibrium in sympatric Populus purdomii and Salix magnifica. However, at the lower and higher ends of the altitudinal gradient, skewed sex ratios were observed. For example, the male:female ratios were 1.33 and 2.36 in P. purdomii at 1700 and 2600 m respectively; for S. magnifica the ratio was 0.62 at 2600 m. At 2300 m, the pot-grown seedlings of both species exhibited the highest biomass accumulation and total leaf area, simultaneously with the balanced sex ratios in the field. At 3300 m, the specific leaf area in male P. purdomii was 23.9% higher than that of females, which may be the morphological cause for the observed 19.3% higher nitrogen allocation to Rubisco, and 20.6% lower allocation to cell walls. As such, male P. purdomii showed a 32.9% higher foliar photosynthetic capacity, concomitant with a 12.0% lower construction cost. These properties resulted in higher photosynthetic nitrogen- and energy-use efficiencies, and shorter payback time (24.4 vs 40.1 days), the time span that a leaf must photosynthesize to amortize the carbon investment. Our results thus suggested that male P. purdomii evolved a quicker energy-return strategy. Consequently, these superior energy gain-cost related traits and the higher total leaf area contributed to the higher growth rate and tolerance in stress-prone environments, which might, in part, shed new light on the male-biased sex ratios in Populus. However, no significant sexual difference was observed in S. magnifica for all the above parameters, thereby implying that the female-biased sex ratios in Salix cannot be explained in terms of the energy-use properties studied here.

Phenological cues drive an apparent trade-off between freezing tolerance and growth in the family Salicaceae.

With increasing concern about the ecological consequences of global climate change, there has been renewed interest in understanding the processes that determine species range limits. We tested a long-hypothesized trade-off between freezing tolerance and growth rate that is often used to explain species range limits. We grew 24 willow and poplar species (family Salicaceae) collected from across North America in a greenhouse common garden under two climate treatments. Maximum entropy models were used to describe species distributions and to estimate species-specific climate parameters. A range of traits related to freezing tolerance, including senescence, budburst, and susceptibility to different temperature minima during and after acclimation were measured. As predicted, species from colder climates exhibited higher freezing tolerance and slower growth rates than species from warmer climates under certain environmental conditions. However, the average relative growth rate (millimeters per meter per day) of northern species markedly increased when a subset of species was grown under a long summer day length (20.5 h), indicating that genetically based day-length cues are required for growth regulation in these species. We conclude that the observed relationship between freezing tolerance and growth rate is not driven by differences in species' intrinsic growth capacity but by differences in the environmental cues that trigger growth. We propose that the coordinated evolution of freezing tolerance and growth phenology could be important in circumscribing willow and poplar range limits and may have important implications for species' current and future distributions.

Are vegetative reproduction capacities the cause of widespread invasion of Eurasian Salicaceae in Patagonian river landscapes?

In recent decades, invasive willows and poplars (Salicaceae) have built dense floodplain forests along most of the rivers in Patagonia, Argentina. These invasion processes may affect Salix humboldtiana as the only native floodplain tree species in this region. It is assumed, that the property to reproduce vegetatively can play an important role in the establishment of invasive species in their new range. Thus, in order to contribute to a better understanding of willow and poplar invasions in riparian systems and to assess the potential impacts on S. humboldtiana the vegetative reproduction capacities of native and invasive Salicaceae were analysed. In a greenhouse experiment, we studied cutting survival and growth performance of the three most dominant invasive Salicaceae of the Patagonian Río Negro region (two Salix hybrids and Populus spec.), as well as S. humboldtiana, taking into account three different moisture and two different soil conditions. In a subsequent experiment, the shoot and root biomass of cuttings from the former experiment were removed and the bare cuttings were replanted to test their ability to re-sprout. The two invasive willow hybrids performed much better than S. humboldtiana and Populus spec. under all treatment combinations and tended to re-sprout more successfully after repeated biomass loss. Taking into account the ecology of vegetative and generative recruits of floodplain willows, the results indicate that the more vigorous vegetative reproduction capacity can be a crucial property for the success of invasive willow hybrids in Patagonia being a potential threat for S. humboldtiana.

Estaquia caulinar de guaçatonga (Casearia sylvestris Swartz) nas quatro estações do ano, com aplicação de diferentes concentrações de AIB / Stem cuttings of "guaçatonga" (Casearia sylvestris Swartz) in the four seasons of the year with the use of different IBA concentrations

Casearia sylvestris Swartz (Salicaceae) ou guaçatonga é uma árvore nativa do México, da América Central, e da América do Sul, com grande importância ecológica, farmacológica, e comercial. No entanto, como a maioria das espécies nativas de interesse medicinal no Brasil, a guaçatonga não é cultivada comercialmente, sendo obtida por extrativismo. O presente trabalho foi conduzido com o objetivo de testar um protocolo de propagação vegetativa de guaçatonga por meio da estaquia, visando identificar qual a melhor estação do ano para o enraizamento de estacas e avaliar o efeito da utilização do regulador vegetal ácido indolbutírico (AIB). No outono, inverno e primavera de 2007, e no verão de 2008, estacas caulinares semilenhosas de 12-14 cm de comprimento e com duas folhas foram preparadas e tratadas com AIB (0, 1000, 2000 e 3000 mg L-1), em solução alcoólica, através da imersão rápida por 10 segundos da base das estacas, e foram plantadas em tubetes contendo substrato Plantmax HT® em casa-de-vegetação sob nebulização intermitente. O delineamento experimental utilizado foi o inteiramente casualizado, com 4 repetições, 4 tratamentos, e 16 estacas por parcela. Todos os experimentos foram avaliados após 90 dias, sendo que para dois deles (primavera 2007 e verão 2008) prolongou-se o tempo de permanência em casa-de-vegetação para melhor desenvolvimento das raízes. Foram avaliados os parâmetros: porcentagem de estacas enraizadas, porcentagem de estacas vivas (com calos e sem raízes, sem calos e sem raízes), porcentagem de folhas retidas, porcentagem de estacas mortas, número de raízes, comprimento das três maiores raízes (cm), e média da massa seca das raízes (mg). Não ocorreu enraizamento nas estacas retiradas no outono e no inverno. Com as estacas retiradas na primavera obteve-se 39,1% de enraizamento. Estacas coletadas no verão não responderam como o esperado, apresentando, após 240 dias, 6,3% de enraizamento no tratamento com 3000 mg L-1 de AIB. O AIB até 3000 mg L-1 não estimulou o enraizamento de estacas de guaçatonga e a melhor estação do ano para a estaquia é a primavera.

Casearia sylvestris Swartz (Salicaceae), or "guaçatonga", is a tree native to Mexico and Central and South America, with great ecological, pharmacological and commercial relevance. Similarly to most native species of medicinal interest in Brazil, "guaçatonga" is not commercially cultivated and is obtained by means of extraction. The aim of this study was to test a protocol for the vegetative propagation of "guaçatonga" by means of stem cutting, identifying the best season for stem rooting and assessing the effect of using the growth regulator indolebutyric acid (IBA). In the fall, winter and spring 2007 and summer 2008, semi-hardwood cuttings with 12-14 cm length and two leaves were prepared and treated with IBA (0, 1000, 2000 and 3000 mg L-1), in alcohol solution, by rapidly immersing for 10 seconds the base of cuttings and planting them in tubes containing Plantmax HT® in greenhouse under intermittent nebulization. Experimental design was completely randomized with 4 replicates, 4 treatments and 16 cuttings per plot. All experiments were evaluated after 90 days, and for two of them the time of maintenance in greenhouse was prolonged in order to improve root development. The following parameters were evaluated: percentage of rooted cuttings, percentage of live cuttings (with callus and without roots, without callus and without roots), percentage of retained leaves, percentage of dead cuttings, number of roots, length of the largest roots (cm) and mean dry mass of roots (mg). There was no rooting on cuttings collected during the fall and the winter. For cuttings collected in the spring, 39.1% rooting was obtained. Cuttings collected in the summer did not show the expected results, presenting after 240 days 6.3% rooting when treated with 3000 mg L-1 IBA. Up to 3000 mg L-1, IBA did not stimulate the rooting of "guaçatonga" cuttings and the best season for cutting is spring.

Growth, physiological and molecular traits in Salicaceae trees investigated for phytoremediation of heavy metals and organics.

Worldwide, there are many large areas moderately contaminated with heavy metals and/or organics that have not been remediated due to the high cost and technical drawbacks of currently available technologies. Methods with a good potential for coping with these limitations are emerging from phytoremediation techniques, using, for example, specific amendments and/or plants selected from various candidates proven in several investigations to be reasonably efficient in extracting heavy metals from soil or water, or in co-metabolizing organics with bacteria flourishing or inoculated in their rhizospheres. Populus and Salix spp., two genera belonging to the Salicaceae family, include genotypes that can be considered among the candidates for this phytoremediation approach. This review shows the recent improvements in analytical tools based on the identification of useful genetic diversity associated with classical growth, physiological and biochemical traits, and the importance of plant genotype selection for enhancing phytoremediation efficiency. Particularly interesting are studies on the application of the phytoremediation of heavy metals and of chlorinated organics, in which microorganisms selected for their degradation capabilities were bioaugmented in the rhizosphere of Salicaceae planted at a high density for biomass and bioenergy production.

Integrated analysis of tropical trees growth: a multivariate approach.

BACKGROUND AND AIMS: One of the problems analysing cause-effect relationships of growth and environmental factors is that a single factor could be correlated with other ones directly influencing growth. One attempt to understand tropical trees' growth cause-effect relationships is integrating research about anatomical, physiological and environmental factors that influence growth in order to develop mathematical models. The relevance is to understand the nature of the process of growth and to model this as a function of the environment. METHODS: The relationships of Aphananthe monoica, Pleuranthodendron lindenii and Psychotria costivenia radial growth and phenology with environmental factors (local climate, vertical strata microclimate and physical and chemical soil variables) were evaluated from April 2000 to September 2001. The association among these groups of variables was determined by generalized canonical correlation analysis (GCCA), which considers the probable associations of three or more data groups and the selection of the most important variables for each data group. KEY RESULTS: The GCCA allowed determination of a general model of relationships among tree phenology and radial growth with climate, microclimate and soil factors. A strong influence of climate in phenology and radial growth existed. Leaf initiation and cambial activity periods were associated with maximum temperature and day length, and vascular tissue differentiation with soil moisture and rainfall. The analyses of individual species detected different relationships for the three species. CONCLUSIONS: The analyses of the individual species suggest that each one takes advantage in a different way of the environment in which they are growing, allowing them to coexist.

Age-related shifts in leaf chemistry of clonal aspen (Populus tremuloides).

Developmental changes in plant structure and function can influence both mammalian and arthropod feeding preferences for many woody plant species. This study documents age-related changes that occur in the leaf chemistry of trembling aspen (Populus tremuloides Michx., Salicaceae) and discusses implications for the herbivore community and ecosystem processes. We collected leaves from replicate ramets from six age classes (1-25+ yr) in each of seven aspen clones growing in south central Wisconsin, USA. Chemical analyses were conducted to determine concentrations of condensed tannins, phenolic glycosides (salicortin and tremulacin), nitrogen, starch, and soluble sugars. Each variable differed significantly among clones and among age classes. On average, condensed tannin concentrations doubled in the first five years and then remained fairly constant among older age classes. Combined phenolic glycoside (salicortin + tremulacin) concentrations were high in the youngest ramets (ca. 19%) and decreased sharply with age. Developmental changes in tannin, salicortin, and tremulacin concentrations exceeded those of nitrogen and carbohydrates. Developmental shifts of this magnitude, and the age-related tradeoff that occurs between condensed tannins and phenolic glycosides, are likely to have significant influence on the herbivore community of aspen and may influence leaf litter decomposition and nutrient cycling.

Shoot life span in relation to successional status in deciduous broad-leaved tree species in a temperate forest.

In trees, leaf life span is closely related to successional status. Although leaves are attached to shoots, shoot life span has been insufficiently studied in the context of ecological systems. Interspecific variation in shoot survivorship was investigated over 27 months in 15 temperate hardwood tree species. Relationships between shoot architecture and shoot survival were also investigated. Shoot life span was shortest in early successional species, and longest in late successional species, in each of the families Betulaceae and Fagaceae. In Salicaceae, all of which were early successional species, shoot life span was longer in mountainous than in riparian species. Early successional or riparian species distributed longer shoots densely, even in proximal positions on mother shoots, resulting in mutual shading and consequent early and massive shoot shedding. By contrast, late successional or mountainous species concentrated shoots in distal positions, allowing shoots to receive equally favorable light, resulting in a longer life span. These results reveal close relationships between shoot life span and environmental resource availability or successional status and suggest a causal relationship between shoot shedding and shoot architecture.

Herbivore attack in Casearia nitida influenced by plant ontogenetic variation in foliage quality and plant architecture.

Traits influencing plant quality as food and/or shelter for herbivores may change during plant ontogeny, and as a consequence, influence the amount of herbivory that plants receive as they develop. In this study, differences in herbivore density and herbivory were evaluated for two ontogenetic stages of the tropical tree Casearia nitida. To assess plant ontogenetic differences in foliage quality as food for herbivores, nutritional and defensive traits were evaluated in saplings and reproductive trees. Predatory arthropods were quantified and the foraging preferences of a parasitoid wasp of the genus Zacremnops were assessed. In addition, survival rates of lepidopteran herbivores (Geometridae) were evaluated experimentally. Herbivore density was three times higher and herbivory was 66% greater in saplings than in reproductive trees. Accordingly, concentrations of total foliar phenolics were higher in reproductive trees than in saplings, whereas leaf toughness, water and nitrogen concentration did not vary between ontogenetic stages. Survival rates of lepidopteran larvae exposed to natural enemies were equivalent in reproductive trees and saplings. Given the greater herbivore density on saplings, equal survival rates implied a greater foraging effort of predators on reproductive trees. Furthermore, observed foraging of parasitoid wasps was restricted to reproductive trees. I propose that herbivore density, and as a consequence, leaf damage were lower in reproductive trees than in saplings due to both traits influencing food quality, and architectural or unmeasured indirect defensive traits influencing foraging preference of natural enemies of herbivores.

Environmental and auxin regulation of wood formation involves members of the Aux/IAA gene family in hybrid aspen.

Indole acetic acid (IAA/auxin) profoundly affects wood formation but the molecular mechanism of auxin action in this process remains poorly understood. We have cloned cDNAs for eight members of the Aux/IAA gene family from hybrid aspen (Populus tremula L. x Populus tremuloides Michx.) that encode potential mediators of the auxin signal transduction pathway. These genes designated as PttIAA1-PttIAA8 are auxin inducible but differ in their requirement of de novo protein synthesis for auxin induction. The auxin induction of the PttIAA genes is also developmentally controlled as evidenced by the loss of their auxin inducibility during leaf maturation. The PttIAA genes are differentially expressed in the cell types of a developmental gradient comprising the wood-forming tissues. Interestingly, the expression of the PttIAA genes is downregulated during transition of the active cambium into dormancy, a process in which meristematic cells of the cambium lose their sensitivity to auxin. Auxin-regulated developmental reprogramming of wood formation during the induction of tension wood is accompanied by changes in the expression of PttIAA genes. The distinct tissue-specific expression patterns of the auxin inducible PttIAA genes in the cambial region together with the change in expression during dormancy transition and tension wood formation suggest a role for these genes in mediating cambial responses to auxin and xylem development.

Independent activation of cold acclimation by low temperature and short photoperiod in hybrid aspen.

Temperate zone woody plants cold acclimate in response to both short daylength (SD) and low temperature (LT). We were able to show that these two environmental cues induce cold acclimation independently by comparing the wild type (WT) and the transgenic hybrid aspen (Populus tremula x Populus tremuloides Michx.) line 22 overexpressing the oat (Avena sativa) PHYTOCHROME A gene. Line 22 was not able to detect the SD and, consequently, did not stop growing in SD conditions. This resulted in an impaired freezing tolerance development under SD. In contrast, exposure to LT resulted in cold acclimation of line 22 to a degree comparable with the WT. In contrast to the WT, line 22 could not dehydrate the overwintering tissues or induce the production of dehydrins (DHN) under SD conditions. Furthermore, abscisic acid (ABA) content of the buds of line 22 were the same under SD and long daylength, whereas prolonged SD exposure decreased the ABA level in the WT. LT exposure resulted in a rapid accumulation of DHN in both the WT and line 22. Similarly, ABA content increased transiently in both the WT and line 22. Our results indicate that phytochrome A is involved in photoperiodic regulation of ABA and DHN levels, but at LT they are regulated by a different mechanism. Although SD and LT induce cold acclimation independently, ABA and DHN may play important roles in both modes of acclimation.

Seasonal changes of plasma membrane H(+)-ATPase and endogenous ion current during cambial growth in poplar plants.

The plasma membrane H(+)-ATPase (PM H(+)-ATPase), potassium ions, and endogenous ion currents might play a fundamental role in the physiology of cambial growth. Seasonal changes of these parameters were studied in twigs of Populus nigra and Populus trichocarpa. Monoclonal and polyclonal antibodies against the PM H(+)-ATPase, x-ray analysis for K(+) localization and a vibrating electrode for measurement of endogenous ion currents were used as probes. In dormant plants during autumn and winter, only a slight immunoreactivity against the PM H(+)-ATPase was found in cross sections and tissue homogenates, K(+) was distributed evenly, and the density of endogenous current was low. In spring during cambial growth, strong immunoreactivity against a PM H(+)-ATPase was observed in cambial cells and expanding xylem cells using the monoclonal antibody 46 E5 B11 F6 for fluorescence microscopy and transmission electron microscopy. At the same time, K(+) accumulated in cells of the cambial region, and strong endogenous current was measured in the cambial and immature xylem zone. Addition of auxin to dormant twigs induced the formation of this PM H(+)-ATPase in the dormant cambial region within a few days and an increase in density of endogenous current in shoot cuttings within a few hours. The increase in PM H(+)-ATPase abundance and in current density by auxin indicates that auxin mediates a rise in number and activity of an H(+)-ATPase in the plasma membrane of cambial cells and their derivatives. This PM H(+)-ATPase generates the necessary H(+)-gradient (proton-motive force) for the uptake of K(+) and nutrients into cambial and expanding xylem cells.

PtABI3 impinges on the growth and differentiation of embryonic leaves during bud set in poplar.

The Arabidopsis ABSCISIC ACID-INSENSITIVE3 (ABI3) protein plays a crucial role during late seed development and has an additional function at the vegetative meristem, particularly during periods of growth-arresting conditions and quiescence. Here, we show that the ABI3 homolog of poplar (PtABI3) is expressed in buds during natural bud set. Expression occurs clearly after perception of the critical daylength that initiates bud set and dormancy in poplar. In short-day conditions mimicking natural bud set, the expression of a chimeric PtABI3::beta-glucuronidase (GUS) gene occurred in those organs and cells of the apex that grow actively but will undergo arrest: the young embryonic leaves, the subapical meristem, and the procambial strands. If PtABI3 is overexpressed or downregulated, bud development in short-day conditions is altered. Constitutive overexpression of PtABI3 resulted in apical buds with large embryonic leaves and small stipules, whereas in antisense lines, bud scales were large and leaves were small. Thus, PtABI3 influences the size and ratio of embryonic leaves and bud scales/stipules that differentiate from the primordia under short-day conditions. These observations, together with the expression of PtABI3::GUS in embryonic leaves but not in bud scales/stipules, support the idea that wild-type PtABI3 is required for the relative growth rate and differentiation of embryonic leaves inside the bud. These experiments reveal that ABI3 plays a role in the cellular differentiation of vegetative tissues, in addition to its function in seeds.

Light-regulated leaf expansion in two Populus species: dependence on developmentally controlled ion transport.

Leaf growth responses to light have been compared in two species of Populus, P. deltoides and P. trichocarpa. These species differ markedly in morphology, anatomy, and dependence on light during leaf expansion. Light stimulates the growth rate and acidification of cell walls in P. trichocarpa but not in P. deltoides, whereas leaves of P. deltoides maintain growth in the dark. Light-induced growth is promoted in P. deltoides when cells are provided 50-100 mM KCl. In both species, light initially depolarizes, then hyperpolarizes mesophyll plasma membranes. However, in the dark, the resting E(m) of mesophyll cells in P. deltoides, but not in P. trichocarpa, is relatively insensitive to decade changes in external [K+]. Results suggest that light-stimulated leaf growth depends on developmentally regulated cellular mechanisms controlling ion fluxes across the plasma membrane. These developmental differences underlie species-level differences in growth and physiological responses to the photoenvironment.

Structure and function of shisham forests in central Himalaya, India: dry matter dynamics.

The biomass and net primary productivity (NPP) of 5- to 15-year-old Shisham (Dalbergia sissoo Roxb.) forests growing in central Himalaya were estimated. Allometric equations were developed for all above- and below-ground components of trees and shrubs for each stand. Understorey forest floor biomass and litter fall were also estimated in forest stands. The biomass (dry matter), forest floor biomass (standing crop litter), tree litter fall and NPP of trees and shrubs increased with increasing age of the forest stand, whereas the dry matter and herb NPP decreased significantly (P < 0.001) with increasing age of the forest. Total forest biomass and NPP ranged from 58.7 (5-year-old stand) to 136.1 t ha(-1) (15-year-old stand) and 12.6 (5-year-old stand) to 20.3 t ha(-1) year(-1) (15-year-old stand), respectively. Of these values, tree biomass accounted for 85.7 (5-year-old stand) to 90.1% (15-year-old) of total forest biomass, and tree NPP for 72.2 (5-year-old) to 82.3% (15-year-old) of total forest NPP. The biomass accumulation ratio (BAR) of the bole component (bole wood + bole bark) increased with increasing age of the forest stand. The bole BAR was 5.8 (5-year-old stand) to 7.9 (15-year-old stand). However, total BAR of the forest stand ranged from 5.5 (5-year-old) to 7.5 (15-year-old).

Laccase down-regulation causes alterations in phenolic metabolism and cell wall structure in poplar.

Laccases are encoded by multigene families in plants. Previously, we reported the cloning and characterization of five divergent laccase genes from poplar (Populus trichocarpa) xylem. To investigate the role of individual laccase genes in plant development, and more particularly in lignification, three independent populations of antisense poplar plants, lac3AS, lac90AS, and lac110AS with significantly reduced levels of laccase expression were generated. A repression of laccase gene expression had no effect on overall growth and development. Moreover, neither lignin content nor composition was significantly altered as a result of laccase suppression. However, one of the transgenic populations, lac3AS, exhibited a 2- to 3-fold increase in total soluble phenolic content. As indicated by toluidine blue staining, these phenolics preferentially accumulate in xylem ray parenchyma cells. In addition, light and electron microscopic observations of lac3AS stems indicated that lac3 gene suppression led to a dramatic alteration of xylem fiber cell walls. Individual fiber cells were severely deformed, exhibiting modifications in fluorescence emission at the primary wall/middle lamella region and frequent sites of cell wall detachment. Although a direct correlation between laccase gene expression and lignification could not be assigned, we show that the gene product of lac3 is essential for normal cell wall structure and integrity in xylem fibers. lac3AS plants provide a unique opportunity to explore laccase function in plants.

Mycorrhizal colonization of transgenic aspen in a field trial.

Mycorrhizal colonization of genetically modified hybrid aspen (Populus tremula x P. tremuloides Michx.) was investigated over 15 months in a field experiment. The aspen carried the rolC gene from Agrobacterium rhizogenes under control of either the constitutive cauliflower mosaic virus 35S promoter or the light-inducible rbcS promoter. Arbuscular mycorrhizas (AMs) were rare in all root samples, while fully developed ectomycorrhizas (EMs) were found in all samples. No significant differences in the degree of mycorrhizal colonization between aspen lines were seen with either AMs or EMs. The EM community on the release area was dominated by four fungal species that formed more than 90% of all mycorrhizas, while eleven EM types were found occasionally. Mycorrhizal diversity did not differ between transgenic and non-transgenic trees. The structure of mycorrhizal communities was similar for most aspen lines. The sole significant difference was found in the abundance and development of one of the four common EM morphotypes, which was rare and poorly developed on roots from the transgenic aspen line Esch5:35S-rolC-#5 compared with non-transgenic controls. This effect is clone specific as the formation of this EM type was not affected by the transgene expression in the other transgenic line, Esch5:35S-rolC-#1. This is the first demonstration of a clonal effect influencing the ability of a transgenic plant to form a mycorrhizal symbiosis with a potential fungal partner.

Understanding the role of the cytoskeleton in wood formation in angiosperm trees: hybrid aspen (Populus tremula x P. tremuloides) as the model species.

The involvement of microfilaments and microtubules in the development of the radial and axial components of secondary xylem (wood) in hybrid aspen (Populus tremula L. x P. tremuloides Michx.) was studied by indirect immunofluorescent localization techniques. In addition to cambial cells, the differentiated cell types considered were early- and late-wood vessel elements, axial parenchyma, normal-wood fibers and gelatinous fibers, and contact and isolation ray cells. Microfilaments were rare in ray cambial cells, but were abundant and axially arranged in their derivatives once cell elongation had begun, and persisted in that orientation in mature ray cells. Microfilaments were axially arranged in fusiform cambial cells and persisted in that orientation in all xylem derivatives of those cells. Microtubules were randomly oriented in ray and fusiform cells of the cambial zone. Dense arrays of parallel-aligned microtubules were oriented near axially in the developing gelatinous fibers, but at a wide range of angles in normal-wood fibers. Ellipses of microfilaments were associated with pit development in fiber cells and isolation ray cells. Rings of co-localized microtubules and microfilaments were associated with developing inter-vessel bordered pits and vessel-contact ray cell contact pits, and, in the case of bordered pits, these rings decreased in diameter as the over-arching pit border increased in size. Although only microtubules were seen at the periphery of the perforation plate of vessel elements, a prominent meshwork of microfilaments overlaid the perforation plate itself. A consensus view of the roles of the cytoskeleton during wood formation in angiosperm trees is presented.