Understanding airspace in leaves: 3D anatomy and directional tortuosity.

The leaf intercellular airspace is a tortuous environment consisting of cells of different shapes, packing densities, and orientation, all of which have an effect on the travelling distance of molecules from the stomata to the mesophyll cell surfaces. Tortuosity, the increase in displacement over the actual distance between two points, is typically defined as encompassing the whole leaf airspace, but heterogeneity in pore dimensions and orientation between the spongy and palisade mesophyll likely result in heterogeneity in tortuosity along different axes and would predict longer traveling distance along the path of least tortuosity, such as vertically within the columnar cell matrix of the palisade layer. Here, we compare a previously established geometric method to a random walk approach, novel for this analysis in plant leaves, in four different Eucalyptus species. The random walk method allowed us to quantify directional tortuosity across the whole leaf profile, and separately for the spongy and palisade mesophyll. For all species tortuosity was higher in the palisade mesophyll than the spongy mesophyll and horizontal (parallel to the epidermis) tortuosity was consistently higher than vertical (from epidermis to epidermis) tortuosity. We demonstrate that a random walk approach improves on previous geometric approaches and is valuable for investigating CO2 and H2 O transport within leaves.

Analysis of Orthologous SECONDARY WALL-ASSOCIATED NAC DOMAIN1 (SND1) Promotor Activity in Herbaceous and Woody Angiosperms.

SECONDARY WALL-ASSOCIATED NAC DOMAIN1 (SND1) is a master regulator of fibre secondary wall deposition in Arabidopsis thaliana (Arabidopsis), with homologs in other angiosperms and gymnosperms. However, it is poorly understood to what extent the fibre-specific regulation of the SND1 promoter, and that of its orthologs, is conserved between diverged herbaceous and woody lineages. We performed a reciprocal reporter gene analysis of orthologous SND1 promoters from Arabidopsis (AthSND1), Eucalyptus grandis (EgrNAC61) and Populus alba × P. grandidentata (PagWND1A) relative to secondary cell wall-specific Cellulose Synthase4 (CesA4) and CesA7 promoters, in both a non-woody (Arabidopsis) and a woody (poplar) system. ß-glucuronidase (GUS) reporter analysis in Arabidopsis showed that the SND1 promoter was active in vascular tissues as previously reported and showed interfascicular and xylary fibre-specific expression in inflorescence stems, while reporter constructs of the woody plant-derived promoters were partial to the (pro)cambium-phloem and protoxylem. In transgenic P. tremula × P. alba plants, all three orthologous SND1 promoters expressed the GUS reporter similarly and preferentially in developing secondary xylem, ray parenchyma and cork cambium. Ours is the first study to reciprocally test orthologous SND1 promoter specificity in herbaceous and woody species, revealing diverged regulatory functions in the herbaceous system.

Micromorphological observation of seed coat of Eucalyptus species (Myrtaceae) using scanning electron microscopy technique.

Seed shape, surface cells shape, arrangement, anticlinal wall pattern, and periclinal wall protuberances were recorded for nine species of Eucalyptus (Myrtaceae) using scanning electron microscopy to determine the importance of seed morphological characters as an additional tool for identification. Most of the seeds were found ovate and some seeds were elliptic and cuboid in shape. Almost reticulate regular seed surface patterns were observed. Four types of surface cells were examined; diamond, elliptic, oblong, and irregular. Majority of the seeds showed raised anticlinal wall level and diversity from wavy to puzzle in pattern. Periclinal wall may be glabrous or having protuberances that were rhombus and bullate in shape. Both macro- and micromorphological characters can provide basis for classification and delimitation of genus Eucalyptus. RESEARCH HIGHLIGHTS: Seeds quantitative characters of nine Eucalyptus species as seed length, width, and weight. Macromorphological characters of seeds including seed color, helium position, and seed shape. Micromorphological characters include seed surface, periclinal wall, and anticlinal wall investigation under scanning electron microscope. Ultra-seed sculpturing features as an additional tool in identification.

Phloem as capacitor: radial transfer of water into xylem of tree stems occurs via symplastic transport in ray parenchyma.

The transfer of water from phloem into xylem is thought to mitigate increasing hydraulic tension in the vascular system of trees during the diel cycle of transpiration. Although a putative plant function, to date there is no direct evidence of such water transfer or the contributing pathways. Here, we trace the radial flow of water from the phloem into the xylem and investigate its diel variation. Introducing a fluorescent dye (0.1% [w/w] fluorescein) into the phloem water of the tree species Eucalyptus saligna allowed localization of the dye in phloem and xylem tissues using confocal laser scanning microscopy. Our results show that the majority of water transferred between the two tissues is facilitated via the symplast of horizontal ray parenchyma cells. The method also permitted assessment of the radial transfer of water during the diel cycle, where changes in water potential gradients between phloem and xylem determine the extent and direction of radial transfer. When injected during the morning, when xylem water potential rapidly declined, fluorescein was translocated, on average, farther into mature xylem (447 ± 188 µm) compared with nighttime, when xylem water potential was close to zero (155 ± 42 µm). These findings provide empirical evidence to support theoretical predictions of the role of phloem-xylem water transfer in the hydraulic functioning of plants. This method enables investigation of the role of phloem tissue as a dynamic capacitor for water storage and transfer and its contribution toward the maintenance of the functional integrity of xylem in trees.

Programmed cell death and necrosis during cryopreparative drying of in vitro Eucalyptus grandis axillary buds.

In preparation for cryopreservation, Eucalyptus grandis in vitro axillary buds were dried over silica gel. Pretreatment of the buds with 5 mg per L ABA resulted in partial resistance to water loss (0.76 to 0.45 g per g fresh mass basis) as compared with untreated buds (0.76 to 0.33 g per g) and was associated with the retention of viability (70 vs. 55%). The loss of viability of the dried buds was protracted over several days. Ultrastructural examination and vital staining demonstrated cellular and tissue responses to drying. The meristem appeared to withstand drying and 72 h of rehydration whilst the leaf primordia were destroyed immediately after drying. High reactive oxygen species (ROS) activity was associated with bud excision and drying. Caspase-3-like protease activity was detected after rehydration, thereby providing evidence that the dried buds, that had ultimately died, had undergone programmed cell death. ROS production is considered to be the trigger for programmed cell death.

Multiphoton fluorescence lifetime imaging shows spatial segregation of secondary metabolites in Eucalyptus secretory cavities.

Multiphoton fluorescence lifetime imaging provides an excellent tool for imaging deep within plant tissues while providing a means to distinguish between fluorophores with high spatial and temporal resolution. Ideal candidates for the application of multiphoton fluorescence lifetime imaging to plants are the embedded secretory cavities found in numerous species because they house complex mixtures of secondary metabolites within extracellular lumina. Previous investigations of this type of structure have been restricted by the use of sectioned material resulting in the loss of lumen contents and often disorganization of the delicate secretory cells; thus it is not known if there is spatial segregation of secondary metabolites within these structures. In this paper, we apply multiphoton fluorescence lifetime imaging to investigate the spatial arrangement of metabolites within intact secretory cavities isolated from Eucalyptus polybractea R.T. Baker leaves. The secretory cavities of this species are abundant (up to 10 000 per leaf), large (up to 6 nL) and importantly house volatile essential oil rich in the monoterpene 1,8-cineole, together with an immiscible, non-volatile component comprised largely of autofluorescent oleuropeic acid glucose esters. We have been able to optically section into the lumina of secretory cavities to a depth of ∼80 µm, revealing a unique spatial organization of cavity metabolites whereby the non-volatile component forms a layer between the secretory cells lining the lumen and the essential oil. This finding could be indicative of a functional role of the non-volatile component in providing a protective region of low diffusivity between the secretory cells and potentially autotoxic essential oil.

Quantitative electron microscopy of cellulose nanofibril structures from Eucalyptus and Pinus radiata kraft pulp fibers.

This work comprises the structural characterization of Eucalyptus and Pinus radiata pulp fibers and their corresponding fibrillated materials, based on quantitative electron microscopy techniques. Compared to hardwood fibers, the softwood fibers have a relatively open structure of the fiber wall outer layers. The fibrillation of the fibers was performed mechanically and chemi-mechanically. In the chemi-mechanical process, the pulp fibers were subjected to a TEMPO-mediated oxidation to facilitate the homogenization. Films were made of the fibrillated materials to evaluate some structural properties. The thicknesses and roughnesses of the films were evaluated with standardized methods and with scanning electron microscopy (SEM), in backscattered electron imaging mode. Field-emission SEM (FE-SEM) and transmission electron microscopy (TEM) were performed to quantify the nanofibril morphology. In this study, we give additional and significant evidences about the suitability of electron microscopy techniques for quantification of nanofibril structures. In addition, we conclude that standard methods are not suitable for estimating the thickness of films having relatively rough surfaces. The results revealed significant differences with respect to the morphology of the fibrillated material. The differences are due to the starting raw material and to the procedure applied for the fibrillation.

Alterations in energy properties of eucalyptus wood and bark subjected to torrefaction: the potential of mass loss as a synthetic indicator.

Torrefaction is a mild pyrolysis process (usually up to 300 degrees C) that changes the chemical and physical properties of biomass. This process is a possible pre-treatment prior to further processes (transport, grinding, combustion, gasification, etc) to generate energy or biofuels. In this study, three eucalyptus wood species and bark were subjected to different torrefaction conditions to determine the alterations in their structural and energy properties. The most severe treatment (280 degrees C, 5h) causes mass losses of more than 35%, with severe damage to anatomical structure, and an increase of about 27% in the specific energy content. Bark is more sensitive to heat than wood. Energy yields are always higher than mass yields, thereby demonstrating the benefits of torrefaction in concentrating biomass energy. The overall mass loss is proposed as a relevant parameter to synthesize the effect of torrefaction conditions (temperature and duration). Accordingly, all results are summarised by analytical expressions able to predict the energy properties as a function of the overall mass loss. These expressions are intended to be used in any optimization procedure, from production in the field to the final use.

Gene expression in Eucalyptus branch wood with marked variation in cellulose microfibril orientation and lacking G-layers.

In response to gravitational stresses, angiosperm trees form tension wood in the upper sides of branches and leaning stems in which cellulose content is higher, microfibrils are typically aligned closely with the fibre axis and the fibres often have a thick inner gelatinous cell wall layer (G-layer). Gene expression was studied in Eucalyptus nitens branches oriented at 45 degrees using microarrays containing 4900 xylem cDNAs, and wood fibre characteristics revealed by X-ray diffraction, chemical and histochemical methods. Xylem fibres in tension wood (upper branch) had a low microfibril angle, contained few fibres with G-layers and had higher cellulose and decreased Klason lignin compared with lower branch wood. Expression of two closely related fasciclin-like arabinogalactan proteins and a beta-tubulin was inversely correlated with microfibril angle in upper and lower xylem from branches. Structural and chemical modifications throughout the secondary cell walls of fibres sufficient to resist tension forces in branches can occur in the absence of G-layer enriched fibres and some important genes involved in responses to gravitational stress in eucalypt xylem are identified.

Freeze/thaw-induced embolism depends on nadir temperature: the heterogeneous hydration hypothesis.

Freeze/thaw-induced embolism was studied in leaves of field-grown snow gum (Eucalyptus pauciflora) subject to frequent morning frosts. Juvenile trees were grown in buried pots, brought to the laboratory at different stages of acclimation and subjected to simulated frost-freezes (at 2 degrees C h(-1)) to nadir temperatures of -3 or -6 degrees C, which snow gums commonly experience. Frost-frozen and subsequently thawed leaves were cryo-fixed to preserve the distribution of water and were then examined by cryo-scanning electron microscopy. No embolisms were found in leaves frozen to -3 degrees C and thawed. In contrast, 34% of vessels were embolized in thawed leaves that had been frozen to -6 degrees C. This difference was seen also in the extent of extracellular ice blocks in the mid-vein expansion zones in leaves frozen to -3 and -6 degrees C, which occupied 3 and 14% of the mid-vein area, respectively. While the proportion of embolism depended on nadir temperature, it was independent of season (and hence of acclimation state). From the observation that increased embolism at lower nadir temperature was related to the freeze-induced redistribution of water, we hypothesize that the dehydration of cell walls and cells caused by the redistribution exerts sufficient tension on xylem water to induce cavitation on thawing.

Leaf water content and palisade cell size.

The palisade cell sizes in leaves of Eucalyptus pauciflora were estimated in paradermal sections of cryo-fixed leaves imaged in the cryo-scanning electron microscope, as a quantity called the cell area fraction (CAF). Cell sizes were measured in detached leaves as a function of leaf water content, in intact leaves in the field during a day"s transpiration as a function of balance pressure of adjacent leaves, and on leaf disks equilibrated with air of relative humidities from 100 to 58%. Values of CAF ranged from 0.82 at saturation to approx. 0.3 in leaves dried to a relative water content (RWC) of 0.5, and in the field to approx. 0.58 at 15 bar (1.5 MPa) balance pressure. At a CAF of 0.58, the moisture content of the cell walls is in equilibrium with air at 90% relative humidity, which is the estimated relative humidity in the intercellular spaces. It is shown that at this moisture content, the cell walls could be exerting a pressure of approx. 50 bar on the cell contents.

The effect of xylanase on lignocellulosic components during the bleaching of wood pulps.

HPLC, SEM and XRD techniques have been proposed as methods for ascertaining the changes occurring in polysaccharides (cellulose and xylans) and fibres during the xylanase bleaching processes. TCF and ECF bleached pulps with and without enzyme pretreatment were analysed. The ratio of carbohydrates present in the pulp, observation of changes occurring in the surface of the fibres and the crystallinity and accessibility of the bleached fibres were determinated. These characteristics have been related with pulp properties. Xylan content decreased when pulp was bleached. Xylanase treatment substantially reduced the xylose content present in pulp, measured by HPLC after the hydrolysis method of the sample. Morphological changes in the fibres occurred when the enzymatic treatment was applied. Bleaching increased the crystallinity of the pulp and enzyme pretreatment also affected the crystallinity of cellulose fibres

[Research on microstructure of caulis herb].

OBJECTIVE: To provide reference for the microscopic identification of caulis herb. METHOD: Systematic arrangement and comparative studies were carried out on the microstructure of medicinal herb of different groups and shapes. RESULT: The rule and characteristics of the microstructure of caulis herb were discussed, and the sorting search list of the microstructure of common caulis herb was established. CONCLUSION: The microstructure characteristics of caulis herb, as the reference of the microscopic identification, are of research value.

Microanalytical studies of metal localization in biological tissues by environmental SEM.

The presence and distribution of Al and Mn in floral and seed tissues of eucalypts from Al-contaminated soils was analyzed using energy-dispersive X-ray microanalysis (EDS) in an environmental scanning electron microscope (ESEM). EDS by ESEM determined the distribution of elements between tissue types was suitable for intact samples or those with lower available moisture or intact specimens. The analytical technique was not appropriate for highly vascular samples. Other factors influencing the detection of elements by ESEM-EDS were electron scattering and the relative concentration and localization of elements within the tissues. EDS-detectable levels were significantly correlated with tissue concentrations determined by atomic absorption spectrophotometry for Mn but not for Al.

Digestion and metabolism of high-tannin Eucalyptus foliage by the brushtail possum (Trichosurus vulpecula) (Marsupialia: Phalangeridae).

The digestion and metabolism of Eucalyptus melliodora foliage was studied in captive brushtail possums (Trichosurus vulpecula). The foliage was low in nitrogen and silica but high in lignified fibre and phenolics compared with diets consumed by most other herbivores. The high lignin content was suggested as the main cause of the low digestibility of E. melliodora cell walls (24%); microscopic observations of plant fragments in the caecum and faeces revealed few bacteria attached to lignified tissues. The conversion of digestible energy (0.34 MJ X kg-0.75 X d-1) to metabolizable energy (0.26 MJ X kg-0.75 X d-1) was low compared to most other herbivores, probably because of excretion of metabolites of leaf essential oils and phenolics in the urine. When the inhibitory effect of leaf tannins on fibre digestion was blocked by supplementing the animals with polyethylene glycol (PEG), intake of dry matter, metabolizable energy and digestible fibre increased. These effects were attributed to the reversal by PEG of tannin-microbial enzyme complexes. It was concluded that the gut-filling effect of a bulk of indigestible fibre is a major reason why the brushtail possum does not feed exclusively on Eucalyptus foliage in the wild.