EgPHI-1, a PHOSPHATE-INDUCED-1 gene from Eucalyptus globulus, is involved in shoot growth, xylem fiber length and secondary cell wall properties.

MAIN CONCLUSION: EgPHI-1 is a member of PHI-1/EXO/EXL protein family. Its overexpression in tobacco resulted in changes in biomass partitioning, xylem fiber length, secondary cell wall thickening and composition, and lignification. Here, we report the functional characterization of a PHOSPHATE-INDUCED PROTEIN 1 homologue showing differential expression in xylem cells from Eucalyptus species of contrasting phenotypes for wood quality and growth traits. Our results indicated that this gene is a member of the PHI-1/EXO/EXL family. Analysis of the promoter cis-acting regulatory elements and expression responses to different treatments revealed that the Eucalyptus globulus PHI-1 (EgPHI-1) is transcriptionally regulated by auxin, cytokinin, wounding and drought. EgPHI-1 overexpression in transgenic tobacco changed the partitioning of biomass, favoring its allocation to shoots in detriment of roots. The stem of the transgenic plants showed longer xylem fibers and reduced cellulose content, while the leaf xylem had enhanced secondary cell wall thickness. UV microspectrophotometry of individual cell wall layers of fibers and vessels has shown that the transgenic plants exhibit differences in the lignification of S2 layer in both cell types. Taken together, the results suggest that EgPHI-1 mediates the elongation of secondary xylem fibers, secondary cell wall thickening and composition, and lignification, making it an attractive target for biotechnological applications in forestry and biofuel crops.

The plastid and mitochondrial genomes of Eucalyptus grandis.

BACKGROUND: Land plant organellar genomes have significant impact on metabolism and adaptation, and as such, accurate assembly and annotation of plant organellar genomes is an important tool in understanding the evolutionary history and interactions between these genomes. Intracellular DNA transfer is ongoing between the nuclear and organellar genomes, and can lead to significant genomic variation between, and within, species that impacts downstream analysis of genomes and transcriptomes. RESULTS: In order to facilitate further studies of cytonuclear interactions in Eucalyptus, we report an updated annotation of the E. grandis plastid genome, and the second sequenced and annotated mitochondrial genome of the Myrtales, that of E. grandis. The 478,813 bp mitochondrial genome shows the conserved protein coding regions and gene order rearrangements typical of land plants. There have been widespread insertions of organellar DNA into the E. grandis nuclear genome, which span 141 annotated nuclear genes. Further, we identify predicted editing sites to allow for the discrimination of RNA-sequencing reads between nuclear and organellar gene copies, finding that nuclear copies of organellar genes are not expressed in E. grandis. CONCLUSIONS: The implications of organellar DNA transfer to the nucleus are often ignored, despite the insight they can give into the ongoing evolution of plant genomes, and the problems they can cause in many applications of genomics. Future comparisons of the transcription and regulation of organellar genes between Eucalyptus genotypes may provide insight to the cytonuclear interactions that impact economically important traits in this widely grown lignocellulosic crop species.

First insights into the functional role of vasicentric tracheids and parenchyma in eucalyptus species with solitary vessels: do they contribute to xylem efficiency or safety?

The relationship between hydraulic specific conductivity (ks) and vulnerability to cavitation (VC) with size and number of vessels has been studied in many angiosperms. However, few of the studies link other cell types (vasicentric tracheids (VT), fibre-tracheids, parenchyma) with these hydraulic functions. Eucalyptus is one of the most important genera in forestry worldwide. It exhibits a complex wood anatomy, with solitary vessels surrounded by VT and parenchyma, which could serve as a good model to investigate the functional role of the different cell types in xylem functioning. Wood anatomy (several traits of vessels, VT, fibres and parenchyma) in conjunction with maximum ks and VC was studied in adult trees of commercial species with medium-to-high wood density (Eucalyptus globulus Labill., Eucalyptus viminalis Labill. and Eucalyptus camaldulensis Dehnh.). Traits of cells accompanying vessels presented correlations with functional variables suggesting that they contribute to both increasing connectivity between adjacent vessels-and, therefore, to xylem conduction efficiency-and decreasing the probability of embolism propagation into the tissue, i.e., xylem safety. All three species presented moderate-to-high resistance to cavitation (mean P50 values = -2.4 to -4.2 MPa) with no general trade-off between efficiency and safety at the interspecific level. The results in these species do not support some well-established hypotheses of the functional meaning of wood anatomy.

Glycosylation of Chrysin by Cultured Cells of Eucalyptus perriniana.

The biotransformation of chrysin was investigated using cultured plant cells of Eucalyptus perriniana as biocatalysts. Chrysin was glucosylated to chrysin 7- 0-ß-D-glucoside and chrysin 7-ß-p-gentiobioside.

Assessment of quality of air in Palermo by chemical (ICP-OES) and cytological analyses on leaves of Eucalyptus camaldulensis.

In this work, we studied the influence of air pollution on the morpho-structural, biochemical and chemical composition of Eucalyptus camaldulensis leaves. Analyses were carried out on 22 samples collected in Palermo (Italy) area. Considering the mean concentrations (in unwashed leaves) of investigated metals, nutrient elements as Fe (214 mg kg(-1) dry weight (d.w.)), Mn (160 mg kg(-1) d.w.) and Zn (39 mg kg(-1) d.w.) were the most abundant, whereas Pb (5.6 mg kg(-1) d.w.) and Cd (0.072 mg kg(-1) d.w.) showed the lowest concentrations. The values of metal pollution index (MPI) ranged from 6.0 (station no. 15) to 25 (station no. 8) and from 4.0 (station no. 16) to 17 (stations no. 7 and no. 15) for unwashed and washed leaves, respectively. The station no. 8, located in an area interested by traffic mostly caused by the activities of university, showed the highest value of MPI. The station no. 15 (Industrial area) showed the lowest MPI value, which is similar to those determined in the reference stations. Considering that the station considered is located in a large and open area interested only by commercial activities and there are no production activities, this data is not surprising. In this study, the washing of the leaves with distilled water has caused a little reduction of metal concentrations. Microphotography reveals a correlation between zones of necrosis, modified cuticles and accumulations of acid phosphatases in the leaves collected in polluted areas. The diaphanized leaves from the more polluted areas show irregular areolas, several idioblasts both on the ribs and scattered in the mesophyll. With polarized light, we observe many crystal deposits near the ribs.

A model system to study the lignification process in Eucalyptus globulus.

Recalcitrance of plant biomass is closely related to the presence of the phenolic heteropolymer lignin in secondary cell walls, which has a negative effect on forage digestibility, biomass-to-biofuels conversion and chemical pulping. The genus Eucalyptus is the main source of wood for pulp and paper industry. However, when compared to model plants such as Arabidopsis thaliana and poplar, relatively little is known about lignin biosynthesis in Eucalyptus and only a few genes were functionally characterized. An efficient, fast and inexpensive in vitro system was developed to study lignification in Eucalyptus globulus and to evaluate the potential role of candidate genes in this biological process. Seedlings were grown in four different conditions, in the presence or absence of light and with or without sucrose in the growth medium, and several aspects of lignin metabolism were evaluated. Our results showed that light and, to a lesser extent, sucrose induced lignin biosynthesis, which was followed by changes in S/G ratio, lignin oligomers accumulation and gene expression. In addition, higher total peroxidase activity and differential isoperoxidase profile were observed when seedlings were grown in the presence of light and sucrose. Peptide sequencing allowed the identification of differentially expressed peroxidases, which can be considered potential candidate class III peroxidases involved in lignin polymerization in E. globulus.

The tonoplast intrinsic aquaporin (TIP) subfamily of Eucalyptus grandis: Characterization of EgTIP2, a root-specific and osmotic stress-responsive gene.

Aquaporins have important roles in various physiological processes in plants, including growth, development and adaptation to stress. In this study, a gene encoding a root-specific tonoplast intrinsic aquaporin (TIP) from Eucalyptus grandis (named EgTIP2) was investigated. The root-specific expression of EgTIP2 was validated over a panel of five eucalyptus organ/tissues. In eucalyptus roots, EgTIP2 expression was significantly induced by osmotic stress imposed by PEG treatment. Histochemical analysis of transgenic tobacco lines (Nicotiana tabacum SR1) harboring an EgTIP2 promoter:GUS reporter cassette revealed major GUS staining in the vasculature and in root tips. Consistent with its osmotic-stress inducible expression in eucalyptus, EgTIP2 promoter activity was up-regulated by mannitol treatment, but was down-regulated by abscisic acid. Taken together, these results suggest that EgTIP2 might be involved in eucalyptus response to drought. Additional searches in the eucalyptus genome revealed the presence of four additional putative TIP coding genes, which could be individually assigned to the classical TIP1-5 groups.

Regioselective hydroxylation and glucosylation of alpha- and beta-pinenes with cultured cells of Eucalyptus perriniana.

Cultured plant cells of Eucalyptus perriniana regioselectively hydroxylated (+)- and (-)-alpha-pinenes to the corresponding (+)- and (-)-verbenols. In addition, (+)- and (-)-verbenols were converted into mono-beta-D-glucosides. On the other hand, (+)- and (-)-beta-pinenes were transformed into (+)- and (-)-pinocarveol 3-O-beta-D-glucosides via (+)- and (-)-pinocarveols.

Glucosylation of taxifolin with cultured plant cells.

Cultured plant cells of Eucalyptus perriniana glucosylated taxifolin to its 3'- and 7-O-beta-D-glucosides and 3',7-O-beta-D-diglucoside. On the other hand, taxifolin was converted into 3'- and 7-O-beta-D-glucosides by cultured cells of Nicotiana tabacum and Catharanthus roseus.

High spatial resolution infrared micro-spectroscopy reveals the mechanism of leaf lignin decomposition by aquatic fungi.

Organic carbon is a critical component of aquatic systems, providing energy storage and transfer between organisms. Fungi are a major decomposer group in the aquatic carbon cycle, and are one of few groups thought to be capable of breaking down woody (lignified) tissue. In this work we have used high spatial resolution (synchrotron light source) infrared micro-spectroscopy to study the interaction between aquatic fungi and lignified leaf vein material (xylem) from River Redgum trees (E. camaldulensis) endemic to the lowland rivers of South-Eastern Australia. The work provides spatially explicit evidence that fungal colonisation of leaf litter involves the oxidative breakdown of lignin immediately adjacent to the fungal tissue and depletion of the lignin-bound cellulose. Cellulose depletion occurs over relatively short length scales (5-15 µm) and highlights the likely importance of mechanical breakdown in accessing the carbohydrate content of this resource. Low bioavailability compounds (oxidized lignin and polyphenols of plant origin) remain in colonised leaves, even after fungal activity diminishes, and suggests a possible pathway for the sequestration of carbon in wetlands. The work shows that fungi likely have a critical role in the partitioning of lignified material into a biodegradable fraction that can re-enter the aquatic carbon cycle, and a recalcitrant fraction that enters long-term storage in sediments or contribute to the formation of dissolved organic carbon in the water column.

High-resolution temperature responses of leaf respiration in snow gum (Eucalyptus pauciflora) reveal high-temperature limits to respiratory function.

We tested whether snow gum (Eucalyptus pauciflora) trees growing in thermally contrasting environments exhibit generalizable temperature (T) response functions of leaf respiration (R) and fluorescence (Fo). Measurements were made on pot-grown saplings and field-grown trees (growing between 1380 and 2110 m a.s.l.). Using a continuous, high-resolution protocol, we quantified T response curves of R and Fo--these data were used to identify an algorithm for modelling R-T curves at subcritical T's and establish variations in heat tolerance. For the latter, we quantified Tmax [T where R is maximal] and Tcrit [T where Fo rises rapidly]. Tmax ranged from 51 to 57 °C, varying with season (e.g. winter summer). Tcrit ranged from 41 to 49 °C in summer and from 58 to 63 °C in winter. Thus, surprisingly, leaf energy metabolism was more heat-tolerant in trees experiencing ice-encasement in winter than warmer conditions in summer. A polynomial model fitted to log-transformed R data provided the best description of the T-sensitivity of R (between 10 and 45 °C); using these model fits, we found that the negative slope of the Q10 -T relationship was greater in winter than in summer. Collectively, our results (1) highlight high-T limits of energy metabolism in E. pauciflora and (2) provide a framework for improving representation of T-responses of leaf R in predictive models.

Regioselective formation of silybin-23-beta-D-glucoside by glucosylation of silybin with cultured plant cells of Eucalyptus perriniana.

Time course experiment revealed that cultured plant cells of Eucalyptus perriniana regioselectively glucosylated silybin to silybin-23-beta-D-glucoside in up to 70% yield.

Glycosylation of vanillin and 8-nordihydrocapsaicin by cultured Eucalyptus perriniana cells.

Glycosylation of vanilloids such as vanillin and 8-nordihydrocapsaicin by cultured plant cells of Eucalyptus perriniana was studied. Vanillin was converted into vanillin 4-O-ß-D-glucopyranoside, vanillyl alcohol, and 4-O-ß-D-glucopyranosylvanillyl alcohol by E. perriniana cells. Incubation of cultured E. perriniana cells with 8-nordihydrocapsaicin gave 8-nordihydrocapsaicin 4-O-ß-D-glucopyranoside and 8-nordihydrocapsaicin 4-O-ß-D-gentiobioside.

Structural and chemical characterization of hardwood from tree species with applications as bioenergy feedstocks.

Eucalypt species are a group of flowering trees widely used in pulp production for paper manufacture. For several decades, the wood pulp industry has focused research and development efforts on improving yields, growth rates and pulp quality through breeding and the genetic improvement of key tree species. Recently, this focus has shifted from the production of high quality pulps to the investigation of the use of eucalypts as feedstocks for biofuel production. Here the structure and chemical composition of the heartwood and sapwood of Eucalyptus dunnii, E. globulus, E. pillularis, E. urophylla, an E. urophylla-E. grandis cross, Corymbia citriodora ssp. variegata, and Acacia mangium were compared using nuclear magnetic resonance spectroscopy (NMR), X-ray diffraction (XRD) and biochemical composition analysis. Some trends relating to these compositions were also identified by Fourier transform near infrared (FT-NIR) spectroscopy. These results will serve as a foundation for a more comprehensive database of wood properties that will help develop criteria for the selection of tree species for use as biorefinery feedstocks.

Increase in enzyme accessibility by generation of nanospace in cell wall supramolecular structure.

An energy efficient nanofibrillation method that combines disk milling and mild hot-compressed water (HCW) treatment was developed to improve enzymatic accessibility of Eucalyptus wood. In this method, the residual product of HCW treatment was fibrillated by disk milling under wet conditions. Relatively moderate HCW treatment conditions (temperature below 180 degrees C and reaction time of 30 min) were adopted, and the amount of water used was only five times that of wood. These conditions were sufficient for the partial removal of hemicellulose and lignin from cell wall supramolecular structure to create nanospace between cellulose microfibrils. This morphological characteristic effectively improved nanofibrillation by disk milling. The fibrillated products with a size of less than 20 nm can be obtained after very short milling time, and this process drastically improved the enzymatic saccharification yield. The energy consumption was much lower than that of other mechanical methods for size reduction to give the same monosaccharide-recovery yield.

CAMBIUM, a process-based model of daily xylem development in Eucalyptus.

In hardwoods such as Eucalyptus spp., xylem (wood) is a heterogeneous tissue consisting of multiple cell types. As such, xylem development involves multiple complex interactions. To describe and understand xylem development, and ultimately predict the resultant wood properties, a process-based approach to modelling wood property variation is potentially very useful. In this paper, a new model (CAMBIUM), which incorporates concepts of these processes, is described. CAMBIUM predicts how wood density and fibre and vessel anatomical properties vary from pith-to-bark at a daily time step as a function of changing environmental conditions and a set of simulated physiological processes. Simulations from an existing process-based model of stand development (CABALA) are used as inputs. A key feature of CAMBIUM is a model of the interaction between different xylem cell types. Some weaknesses were identified in the ability of the model to simulate vessel spatial patterns and frequencies, emphasizing the complexities inherent in this aspect of angiosperm wood formation. The model was, however, able to provide realistic estimates of short-term variation and temporal ranges in eucalypt fibre diameter and secondary wall development and wood density.

Biotransformation of naringin and naringenin by cultured Eucalyptus perriniana cells.

The biotransformation of naringin and naringenin was investigated using cultured cells of Eucalyptus perriniana. Naringin (1) was converted into naringenin 7-O-beta-D-glucopyranoside (2, 15%), naringenin (3, 1%), naringenin 5,7-O-beta-D-diglucopyranoside (4, 15%), naringenin 4',7-O-beta-D-diglucopyranoside (5, 26%), naringenin 7-O-[6-O-(beta-D-glucopyranosyl)]-beta-d-glucopyranoside (6, beta-gentiobioside, 5%), naringenin 7-O-[6-O-(alpha-l-rhamnopyranosyl)]-beta-D-glucopyranoside (7, beta-rutinoside, 3%), and 7-O-beta-D-gentiobiosyl-4'-O-beta-d-glucopyranosylnaringenin (8, 1%) by cultured cells of E. perriniana. On the other hand, 2 (14%), 4 (7%), 5 (13%), 6 (2%), 7 (1%), naringenin 4'-O-beta-D-glucopyranoside (9, 4%), naringenin 5-O-beta-D-glucopyranoside (10, 2%), and naringenin 4',5-O-beta-D-diglucopyranoside (11, 5%) were isolated from cultured E. perriniana cells, that had been treated with naringenin (3). Products, 7-O-beta-D-gentiobiosyl-4'-O-beta-D-glucopyranosylnaringenin (8) and naringenin 4',5-O-beta-D-diglucopyranoside (11), were hitherto unknown.

Leaf anatomy and morphometry in three eucalypt clones treated with glyphosate.

This work aimed to evaluate the effects of simulated drift of glyphosate on the morphoanatomy of three eucalypt clones and to correlate the intoxication symptoms on a microscopic scale with those observed in this visual analysis. The effects of glyphosate drift were proportional to the five doses tested, with Eucalyptus urophylla being more tolerant to the herbicide than E. grandis and urograndis hybrid. The symptoms of intoxication which were similar for the different clones at 7 and 15 days after application were characterized by leaf wilting, chlorosis and curling and, at the highest rates, by necrosis, leaf senescence and death. Anatomically glyphosate doses higher than 86.4 g.ha-1 caused cellular plasmolysis, hypertrophy and hyperplasia, formation of the cicatrization tissue and dead cells on the adaxial epidermis. The spongy parenchyma had a decrease, and the palisade parenchyma and leaf blade thickness had an increase. The increased thickness in leaf blade and palisade parenchyma may be related to the plant response to glyphosate action, as a form of recovering the photosynthetically active area reduced by necroses and leaf senescence caused by the herbicide.

Live biospeckle laser imaging of root tissues.

Live imaging is now a central component for the study of plant developmental processes. Currently, most techniques are extremely constraining: they rely on the marking of specific cellular structures which generally apply to model species because they require genetic transformations. The biospeckle laser (BSL) system was evaluated as an instrument to measure biological activity in plant tissues. The system allows collecting biospeckle patterns from roots which are grown in gels. Laser illumination has been optimized to obtain the images without undesirable specular reflections from the glass tube. Data on two different plant species were obtained and the ability of three different methods to analyze the biospeckle patterns are presented. The results showed that the biospeckle could provide quantitative indicators of the molecular activity from roots which are grown in gel substrate in tissue culture. We also presented a particular experimental configuration and the optimal approach to analyze the images. This may serve as a basis to further works on live BSL in order to study root development.

Leaf anatomy and morphometry in three eucalypt clones treated with glyphosate / Anatomia e morfometria foliar em clones de eucalipto tratados com glyphosate

This work aimed to evaluate the effects of simulated drift of glyphosate on the morphoanatomy of three eucalypt clones and to correlate the intoxication symptoms on a microscopic scale with those observed in this visual analysis. The effects of glyphosate drift were proportional to the five doses tested, with Eucalyptus urophylla being more tolerant to the herbicide than E. grandis and urograndis hybrid. The symptoms of intoxication which were similar for the different clones at 7 and 15 days after application were characterized by leaf wilting, chlorosis and curling and, at the highest rates, by necrosis, leaf senescence and death. Anatomically glyphosate doses higher than 86.4 g.ha-1 caused cellular plasmolysis, hypertrophy and hyperplasia, formation of the cicatrization tissue and dead cells on the adaxial epidermis. The spongy parenchyma had a decrease, and the palisade parenchyma and leaf blade thickness had an increase. The increased thickness in leaf blade and palisade parenchyma may be related to the plant response to glyphosate action, as a form of recovering the photosynthetically active area reduced by necroses and leaf senescence caused by the herbicide.

Este trabalho teve como objetivo avaliar os efeitos da deriva simulada de glyphosate na morfoanatomia de três clones de eucalipto e correlacionar os sintomas de intoxicação em escala microscópica com aqueles observados à vista desarmada. Os efeitos da deriva do glyphosate foram proporcionais às doses testadas, sendo Eucalyptus urophylla mais tolerante ao herbicida que E. grandis e o híbrido urograndis. Os sintomas de intoxicação foram semelhantes para os diferentes clones testados, tanto aos 7 quanto aos 15 dias após a aplicação, sendo caracterizados, morfologicamente, por murcha, clorose e enrolamento foliar e, no caso das maiores doses, por necrose, senescência foliar e morte das plantas de eucalipto. Anatomicamente, doses de glyphosate superiores a 86,4 g.ha-1 provocaram plasmólise, hipertrofia e hiperplasia celular, formação de tecido de cicatrização e morte das células da face adaxial da epiderme. Observou-se diminuição na espessura do parênquima lacunoso e aumento na espessura do parênquima paliçádico e da lâmina foliar. O aumento na espessura da folha e do parênquima paliçádico podem estar relacionados à resposta das plantas ao glyphosate, como forma de compensar a área fotossinteticamente reduzida pelas necroses e senescência causadas pelo herbicida.