Identifying the pathways for foliar water uptake in beech (Fagus sylvatica L.): a major role for trichomes.

Foliar water uptake (FWU), the direct uptake of water into leaves, is a global phenomenon, having been observed in an increasing number of plant species. Despite the growing recognition of its functional relevance, our understanding of how FWU occurs and which foliar surface structures are implicated, is limited. In the present study, fluorescent and ionic tracers, as well as microcomputed tomography, were used to assess potential pathways for water entry in leaves of beech, a widely distributed tree species from European temperate regions. Although none of the tracers entered the leaf through the stomatal pores, small amounts of silver precipitation were observed in some epidermal cells, indicating moderate cuticular uptake. Trichomes, however, were shown to absorb and redistribute considerable amounts of ionic and fluorescent tracers. Moreover, microcomputed tomography indicated that 72% of empty trichomes refilled during leaf surface wetting and microscopic investigations revealed that trichomes do not have a cuticle but are covered with a pectin-rich cell wall layer. Taken together, our findings demonstrate that foliar trichomes, which exhibit strong hygroscopic properties as a result of their structural and chemical design, constitute a major FWU pathway in beech.

Intra-annual dynamics of phloem formation and ultrastructural changes in sieve tubes in Fagus sylvatica.

Despite increased interest in the timing and dynamics of phloem formation, seasonal changes in the structure of phloem sieve elements remain largely unexplored. To understand better the dynamics of phloem formation and the functioning of sieve tubes in the youngest phloem in Fagus sylvatica L., we investigated repeatedly taken phloem samples during the growing season of 2017 by means of light microscopy, and transmission and scanning electron microscopy. Phloem formation started with the expansion of the overwintered early phloem sieve tubes adjacent to the cambium and concurrent cambial cell production. The highest phloem growth rate was observed in general 1 week after the onset of cambial cell production, whereas the transition from early to late phloem occurred at the end of May. Cambial cell production ceased at the end of July. The final width of the phloem increment was 184 ± 10 µm, with an early phloem proportion of 59%. Collapse of older phloem tissue is a progressive process, which continuously occurred during the sampling period. Collapse of early phloem sieve tubes started shortly after the cessation of cambial cell production. Prior to the onset of radial growth, late phloem from the previous year represented 80% of the total non-collapsed part; during the growth period, this percentage decreased to 20%. Differences were observed in both sieve tube ultrastructure and sieve plate geometry between the youngest and older phloem. However, sieve plates were never completely occluded by callose, suggesting that processes affecting the functionality of sieve tubes may differ in the case of regular collapse or injury. The youngest parts of the phloem increment from the previous year (i.e., previous late phloem) continue functioning for some time in the current growing season, but the two-step development of overwintered phloem cells also ensures a sufficient translocation pathway for photosynthates to the actively growing tissues.

Microscopic characterization of tension wood cell walls of Japanese beech (Fagus crenata) treated with ionic liquids.

Tension wood that is an abnormal part formed in angiosperms has been barely used for wood industry. In this study, to utilize the tension wood effectively by means of liquefaction using ionic liquid, we performed morphological and topochemical determination of the changes in tension wood of Japanese beech (Fagus crenata) during ionic liquid treatment at the cellular level using light microscopy, scanning electron microscopy and confocal Raman microscopy. Ionic liquid treatment induced cell wall swelling in tension wood. Changes in the tissue morphology treated with ionic liquids were different between normal wood and tension wood, moreover the types of ionic liquids. The ionic liquid 1-ethyl-3-methylimidazolium chloride liquefied gelatinous layers rapidly, whereas 1-ethylpyridinium bromide liquefied slowly but delignified selectively. These novel insights into the deconstruction behavior of tension wood cell walls during ionic liquid treatment provide better understanding of the liquefaction mechanism. The obtained knowledge will contribute to development of an effective chemical processing of tension wood using ionic liquids and lead to efficient use of wood resources.

How does the VPD response of isohydric and anisohydric plants depend on leaf surface particles?

Atmospheric vapour pressure deficit (VPD) is the driving force for plant transpiration. Plants have different strategies to respond to this 'atmospheric drought'. Deposited aerosols on leaf surfaces can interact with plant water relations and may influence VPD response. We studied transpiration and water use efficiency of pine, beech and sunflower by measuring sap flow, gas exchange and carbon isotopes, thereby addressing different time scales of plant/atmosphere interaction. Plants were grown (i) outdoors under rainfall exclusion (OD) and in ventilated greenhouses with (ii) ambient air (AA) or (iii) filtered air (FA), the latter containing <1% ambient aerosol concentrations. In addition, some AA plants were sprayed once with 25 mM salt solution of (NH4 )2 SO4 or NaNO3 . Carbon isotope values (δ(13) C) became more negative in the presence of more particles; more negative for AA compared to FA sunflower and more negative for OD Scots pine compared to other growth environments. FA beech had less negative δ(13) C than AA, OD and NaNO3 -treated beech. Anisohydric beech showed linearly increasing sap flow with increasing VPD. The slopes doubled for (NH4 )2 SO4 - and tripled for NaNO3 -sprayed beech compared to control seedlings, indicating decreased ability to resist atmospheric demand. In contrast, isohydric pine showed constant transpiration rates with increasing VPD, independent of growth environment and spray, likely caused by decreasing gs with increasing VPD. Generally, NaNO3 spray had stronger effects on water relations than (NH4 )2 SO4 spray. The results strongly support the role of leaf surface particles as an environmental factor affecting plant water use. Hygroscopic and chaotropic properties of leaf surface particles determine their ability to form wicks across stomata. Such wicks enhance unproductive water loss of anisohydric plant species and decrease CO2 uptake of isohydric plants. They become more relevant with increasing number of fine particles and increasing VPD and are thus related to air pollution and climate change. Wicks cause a deviation from the analogy between CO2 and water pathways through stomata, bringing some principal assumptions of gas exchange theory into question.

Roles of cell walls and intracellular contents in supercooling capability of xylem parenchyma cells of boreal trees.

The supercooling capability of xylem parenchyma cells (XPCs) in boreal hardwood species differs depending not only on species, but also season. In this study, the roles of cell walls and intracellular contents in supercooling capability of XPCs were examined in three boreal hardwood species, Japanese beech, katsura tree and mulberry, whose supercooling capability differs largely depending on species and season. XPCs in these species harvested in winter and summer were treated by rapid freezing and thawing (RFT samples) or by RFT with further washing (RFTW samples) to remove intracellular contents from XPCs in order to examine the roles of cell walls in supercooling. RFT samples were also treated with glucose solution (RFTG samples) to examine roles of intracellular contents in supercooling. The supercooling capabilities of these samples were examined by differential thermal analysis after ultrastructural observation of XPCs by a cryo-scanning electron microscope to confirm effects of the above treatments. XPCs in RFTW samples showed a large reduction in supercooling capability to similar temperatures regardless of species or season. On the other hand, XPCs in RFTG samples showed a large increase in supercooling capability to similar temperatures regardless of species or season. These results indicate that although cell walls have an important role in maintenance of supercooling, change in supercooling capability of XPCs is induced by change in intracellular contents, but not by change in cell wall properties.

Early events in Populus hybrid and Fagus sylvatica leaves exposed to ozone.

This paper aims to investigate early responses to ozone in leaves of Fagus sylvatica (beech) and Populus maximowiczii x Populus berolinensis (poplar). The experimental setup consisted of four open-air (OA) plots, four charcoal-filtered (CF) open-top chambers (OTCs), and four nonfiltered (NF) OTCs. Qualitative and quantitative analyses were carried out on nonsymptomatic (CF) and symptomatic (NF and OA) leaves of both species. Qualitative analyses were performed applying microscopic techniques: Evans blue staining for detection of cell viability, CeCl3 staining of transmission electron microscope (TEM) samples to detect the accumulation of H2O2, and multispectral fluorescence microimaging and microspectrofluorometry to investigate the accumulation of fluorescent phenolic compounds in the walls of the damaged cells. Quantitative analyses consisted of the analysis of the chlorophyll a fluorescence transients (fast kinetics). The early responses to ozone were demonstrated by the Evans blue and CeCl3 staining techniques that provided evidence of plant responses in both species 1 month before foliar symptoms became visible. The fluorescence transients analysis, too, demonstrated the breakdown of the oxygen evolving system and the inactivation of the end receptors of electrons at a very early stage, both in poplar and in beech. The accumulation of phenolic compounds in the cell walls, on the other hand, was a species-specific response detected in poplar, but not in beech. Evans blue and CeCl3 staining, as well as the multispectral fluorescence microimaging and microspectrofluorometry, can be used to support the field diagnosis of ozone injury, whereas the fast kinetics of chlorophyll fluorescence provides evidence of early physiological responses.

Monitoring of ozone risk for forests in the Czech Republic: preliminary results.

Ozone (O3) is supposed to represent a significant risk for the health of forest ecosystems in Central Europe. So far, however, its impact on stands growing under natural conditions has not been clearly proved. A new project of the National Agency for the Research in Agriculture is focused on the O3 effect on selected parameters of forest health. This paper presents the results of the first year of monitoring, 2005. In 2005, high O3 concentrations were measured, mainly in the spring. In the summer, due to wet and cold weather, the O3 load was comparatively low. In the plots investigated, the concentrations of O3 were higher with the altitude. The amount of epicuticular waxes on 1-year-old Norway spruce needles was the only factor showing significant correlation to O3 concentration. Defoliation of the stands depended only on the stand age. The amount of malondialdehyde (MDA), an oxidative stress marker, was related to the altitude, and only for European beech. The results are preliminary, as the summer O3 development was not typical in 2005, and the results may change over the next monitoring periods.

Use of X-ray computed microtomography for non-invasive determination of wood anatomical characteristics.

Quantitative analysis of wood anatomical characteristics is usually performed using classical microtomy yielding optical micrographs of stained thin sections. It is time-consuming to obtain high quality cross-sections from microtomy, and sections can be damaged. This approach, therefore, is often impractical for those who need quick acquisition of quantitative data on vessel characteristics in wood. This paper reports results of a novel approach using X-ray computed microtomography (microCT) for non-invasive determination of wood anatomy. As a case study, stem wood samples of a 2-year-old beech (Fagus sylvatica L.) and a 3-year-old oak (Quercus robur L.) tree were investigated with this technique, beech being a diffuse-porous and oak a ring-porous tree species. MicroCT allowed non-invasive mapping of 2-D transverse cross-sections of both wood samples with micrometer resolution. Self-developed software 'microCTanalysis' was used for image processing of the 2-D cross-sections in order to automatically determine the inner vessel diameters, the transverse cross-sectional surface area of the vessels, the vessel density and the porosity with computer assistance. Performance of this new software was compared with manual analysis of the same micrographs. The automatically obtained results showed no significant statistical differences compared to the manual measurements. Visual inspection of the microCT slices revealed very good correspondence with the optical micrographs. Statistical analysis confirmed this observation in a more quantitative way, and it was, therefore, argued that anatomical analysis of optical micrographs can be readily substituted by automated use of microCT, and this without loss of accuracy. Furthermore, as an additional application of microCT, the 3-D renderings of the internal microstructure of the xylem vessels for both the beech and the oak sample could be reconstructed, clearly showing the complex nature of vessel networks. It can be concluded that the use of microCT in wood science offers an interesting potential for all those who need quantitative data of wood anatomical characteristics in either the 2-D or the 3-D space.

Chemical and morphological characteristics of key tree species of the Carpathian Mountains.

Concentrations of Al, B, Ca, Cu, Fe, K, Mg, Mn, N, Na, P, S and Zn in the foliage of white fir (Abies alba), Norway spruce (Picea abies) and common beech (Fagus sylvatica) from 25 sites of the Carpathian Mts. forests (Czech Republic, Poland, Romania, Slovakia and Ukraine) are discussed in a context of their limit values. S/N ratio was different from optimum in 90% of localities when compared with the European limit values. Likewise we found increase of Fe and Cu concentrations compared with their background levels in 100% of locations. Mn concentrations were increased in 76% of localities. Mn mobilization values indicate the disturbance of physiological balance leading to the change of the ratio with Fe. SEM-investigation of foliage waxes from 25 sites in the Carpathian Mts. showed, that there is a statistically significant difference in mean wax quality. Epistomatal waxes were damaged as indicated by increased development of net and amorphous waxes. The most damaged stomata in spruce needles were from Yablunitsa, Synevir and Brenna; in fir needles from Stoliky, and in beech leaves from Malá Fatra, Morské Oko and Beregomet. Spruce needles in the Carpathian Mts. had more damaged stomata than fir needles and beech leaves. Spruce seems to be the most sensitive tree species to environmental stresses including air pollution in forests of the Carpathian Mountains. Foliage surfaces of three forest tree species contained Al, Si, Ca, Fe, Mg, K, Cl, Mn, Na, Ni and Ti in all studied localities. Presence of nutrition elements (Ca, Fe, Mg, K and Mn) on foliage surface hinders opening and closing stomata and it is not physiologically usable for tree species.

In situ ageing of fine beech roots (Fagus sylvatica) assessed by transmission electron microscopy and electron energy loss spectroscopy: description of microsites and evolution of polyphenolic substances.

Root biomass is quantitatively and qualitatively important in most ecosystems, but its contribution to the pool of organic matter in the soil is not clear. This work was designed to specify root ageing on an ultrastructural scale by transmission electron microscopy combined with microanalysis by electron energy loss spectroscopy. This approach is very suitable for studying the soil/plant interface, and for semi-quantitative analysis of the evolution of polyphenolic substances during root evolution. Three root segments were studied according to a gradient of root senescence: the apical and basal segments of the mycorrhiza and the mycorrhiza-carrier root. Each segment contained a certain proportion of senescent cells, some of which were of fungal origin, and this proportion increased as the root aged. In the three segments, the soil/plant interfaces were differentiated, and the micro-organisms observed in situ were described. Senescent root cells contained many polyphenolic substances and our results showed that these substances were, according to the root segment, differently associated with Ca, N and Si. When all these ultrastructural data are correlated with more global data, they can be usefully applied to root cell physiology, microbiology and pedology. This approach makes it possible to specify the evolution of organic matter in situ in soils whatever its origin.

Xylan deposition on secondary wall of Fagus crenata fiber.

Delignified and/or xylanase-treated secondary walls of Fagus crenata fibers were examined by field emission scanning electron microscopy. Microfibrils with a smooth surface were visible in the innermost surface of the differentiating fiber secondary wall. There was no ultrastructural difference between control and delignified sections, indicating that lignin deposition had not started in the innermost surface of the cell wall. There was no ultrastructural difference between control and xylanase-treated sections. Microfibrils on the outer part of the differentiating secondary wall surface had globular substances in delignified sections. These globular substances disappeared following xylanase treatment, indicating that these globules are xylan. The globular substances were not visible near the inner part of the differentiating secondary wall but gradually increased toward the outer part of the secondary wall, indicating that xylan penetrated into the cell wall and continuously accumulated on the microfibrils. Mature-fiber secondary walls were also examined by field emission scanning electron microscopy. Microfibrils were not apparent in the secondary wall in control specimens. Microfibrils with many globular substances were observed in the delignified specimens. Following xylanase treatment, the microfibrils had a smooth surface without any globules, indicating that the globular substance is xylan. These results suggest that cellulose microfibrils synthesized on the plasma membrane are released into the innermost surface of the secondary wall and coated with a thin layer of xylan. Successive deposition of xylan onto the cell wall increases the microfibril diameter. The large amounts of xylan that accumulated on microfibrils appear globular but are covered with lignin after they are deposited.