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Background and Aims Pit pairs and their filter-like partition, i.e. pit membranes, play important roles as water pathways, barriers and regulators in the water-conducting system of angiosperms. In Fraxinus species, the intervessel and vessel–parenchyma pit membranes in sapwood are normally encrusted during winter. Although these encrustations inevitably influence the performance of pits, their properties and functions remain unclear. This study aimed to reveal the morphological and chemical characteristics of encrustations in F. mandshurica in order to deepen understanding of the seasonal encrustation of pit membranes. Methods Seasonal and positional variations in the presence and morphology of encrustations were examined by field-emission scanning electron microscopy (FE-SEM). Cryo-FE-SEM for freeze-fixed greenwood samples was conducted to clarify whether encrustations were present in living trees. Chemical components were examined by histochemical staining using light and electron microscopy, immunofluorescence labelling and ultraviolet microspectroscopy. Key Results Encrustations began to deposit in autumn before leaf senescence and disappeared in spring before bud flushing. They infiltrated within the pit membranes, which suggested that they severely limit the permeation of pits. The encrustations differed in morphology among positions: they entirely filled the pit chambers in latewood, while they covered the pit membranes in earlywood. The encrustations were similarly observed in the samples that were freeze-fixed immediately after collection, indicating that they are present in living trees. The encrustations contained polysaccharides, including xyloglucan and homogalacturonan, and phenolic compounds, possibly including flavonoids and coumarins. These chemical components were also detected in droplets found in the latewood vessels with the encrustations, suggesting that the materials constituting encrustations were supplied through the vessel lumens. Conclusions Encrustations undoubtedly cover the pit membranes in living F. mandshurica trees in winter and their morphology and chemical composition indicate that they are impermeable, have positional differences in function and are characterized by elaborate deposition/removal processes. [ABSTRACT FROM AUTHOR]

The structure of the intervascular pit membranes of four dicotyledonous species (Salix sachalinensis, Betula platyphylla var. japonica, Acer mono, and Fraxinus mandshurica var. japonica) was examined by field-emission scanning electron microscopy. The intervascular pit membranes of F. mandshurica var. japonica had thin surface layers and a dense middle layer, while no similar middle layer was detectable in the other three species. In F. mandshurica var. japonica, the entire area of each pit membrane was densely covered with microfibrils. In the other three species, by contrast, openings were found in the pit membranes. In some of the intervascular pit membranes of S. sachalinensis, B. platyphylla var. japonica, and A. mono, microfibrils were sparsely interwoven in small areas of the pit membranes and openings of up to several hundred nanometers in diameter were present in such regions. These porous regions tended to be located in peripheral areas of pit membranes. In S. sachalinensis and B. platyphylla var. japonica, ethanol-soluble extracts, whose chemical nature and function remain unknown, were heavily distributed over the intervascular pit membranes. Our observations suggest that the structure of intervascular pit membranes is more complicated than has previously been acknowledged. Key words: Acer mono; Betula platyphylla var. japonica; field-emission scanning electron microscopy; Fraxinus mandshurica var. japonica; intervascular pit; pit membrane; Salix sachalinensis.

Intervessel pit membranes are recognized as key structures for influencing water flow/embolism resistance. The mechanisms remain largely unclear owing to difficulties in examining them intact in nature. This study investigates ethanol-extractable pit membrane incrustations (PMIs), which were previously reported in certain angiosperms and may affect their water conduction. The presence of PMIs was determined for 40 angiosperms by field-emission scanning electron microscopy (FE-SEM). Candidate components of PMIs were determined by chemical analyses of wood extracts, and their distributions in the xylem were examined by time-of-flight secondary ion mass spectrometry (TOF-SIMS). Cryo-TOF-SIMS and cryo-FE-SEM were also performed to clarify the native distribution of PMIs. PMIs were observed in 11 species. Some of them were categorized as fat trees, which are known to store abundant lipids. Tilia japonica sapwood displaying PMIs contained large amounts of lipids, which were distributed in the dried xylem tissue, consistent with the distribution of the PMIs. In the frozen samples of T. japonica , however, the distributions were restricted to the parenchyma. In conclusion, PMIs consist of an artifactual coating of lipids originated from the parenchyma in dried samples at room temperature. Researchers performing surface analyses of plant cell walls should take strong precautions against such self-coating by these intrinsic chemicals. [ABSTRACT FROM AUTHOR]

The aim of the present study was to investigate the orientation and localization of actin filaments and cortical microtubules in wood-forming tissues in conifers to understand wood formation. Small blocks were collected from the main stems of Abies firma , Pinus densiflora , and Taxus cuspidata during active seasons of the cambium. Bundles of actin filaments were oriented axially or longitudinally relative to the cell axis in fusiform and ray cambial cells. In differentiating tracheids, actin filaments were oriented longitudinally relative to the cell axis during primary and secondary wall formation. In contrast, the orientation of well-ordered cortical microtubules in tracheids changed from transverse to longitudinal during secondary wall formation. There was no clear relationship between the orientation of actin filaments and cortical microtubules in cambial cells and cambial derivatives. Aggregates of actin filaments and a circular band of cortical microtubules were localized around bordered pits and cross-field pits in differentiating tracheids. In addition, rope-like bands of actin filaments were observed during the formation of helical thickenings at the final stage of formation of secondary walls in tracheids. Actin filaments might not play a major role in changes in the orientation of cortical microtubules in wood-forming tissues. However, since actin filaments were co-localized with cortical microtubules during the formation of bordered pits, cross-field pits and helical thickenings at the final stage of formation of the secondary wall in tracheids, it seems plausible that actin filaments might be closely related to the localization of cortical microtubules during the development of these modifications of wood structure. [ABSTRACT FROM AUTHOR]

Wood fibers form thick secondary cell wall (SCW) in xylem tissues to give mechanical support to trees. NAC SECONDARY WALL THICKENING PROMOTING FACTOR3/SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN 1 (NST3/SND1) and NST1 were identified as master regulators of SCW formation in xylem fiber cells in the model plant Arabidopsis thaliana. In Populus species, four NST/SND orthologs have been conserved and coordinately control SCW formation in wood fibers and phloem fibers. However, it remains to be elucidated whether SCW formation in other xylem cells, such as ray parenchyma cells and vessel elements, is regulated by NST/SND orthologs in poplar. We knocked out all NST/SND genes in hybrid aspen using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system and investigated the detailed histological appearance of stem tissues in the knockout mutants. Observation by light microscopy and transmission electron microscopy showed that SCW was severely suppressed in wood fibers, phloem fibers and xylem ray parenchyma cells in the knockout mutants. Although almost all wood fibers lacked SCW, some fiber cells formed thick cell walls. The irregularly cell wall-forming fibers retained primary wall and SCW, and were mainly located in the vicinity of vessel elements. Field emission-scanning electron microscope observation showed that there were no apparent differences in the structural features of pits such as the shape and size between irregularly SCW-forming wood fibers in the knockout mutants and normal wood fibers in wild-type. Cell wall components such as cellulose, hemicellulose and lignin were deposited in the cell wall of irregularly SCW-forming wood fibers in quadruple mutants. Our results indicate that four NST/SND orthologs are master switches for SCW formation in wood fibers, xylem ray parenchyma cells and phloem fibers in poplar, while SCW is still formed in limited wood fibers, which are located at the region adjacent to vessel elements in the knockout mutants. [ABSTRACT FROM AUTHOR]

Lignification of ray parenchyma cells (RPCs) in the sapwood (sW), transition zone (TZ), and heartwood (hW) of Phellodendron amurense Rupr. has been investigated by time-of-flight secondary ion mass spectrometry (TOF-SIMS) and thioacidolysis. The results of TOF-SIMS indicate that the relative ion intensity of lignin in RPCs increased from sW to hW, while there was almost no difference in the case of axial wood fibers. The ratio of syringyl (S) to guaiacyl (G) lignin units (S/G value) in RPCs was higher than that in wood fibers. Samples containing more RPCs were prepared by laser microdissection (LMD) and analyzed by thioacidolysis, including also the subsequent Raney nickel desulfurization, and the resulting monomers and dimers were quantitatively analyzed by GC/MS. The monomer analysis shows that lignin content and S/G values in RPCs rich samples increased from sW towards hW. The dimer analysis reveals that the S-S dimers are dominant in all types of samples. The results of TOF-SIMS and thioacidolysis of LMD samples are interpreted that the lignification of RPCs progresses from the sW towards the hW and that the chemical structure of lignin in RPCs is different from that in axial elements. [ABSTRACT FROM AUTHOR]

The distribution of aluminum (Al) and other inorganic elements has been mapped at the cellular level in the mature xylem of maple by means of time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging. This method permits to image inorganic elements along with organic molecular species with high spatial resolution, and it was demonstrated that Na, K, Ca, and Mg are almost uniformly distributed in several contiguous growth rings of the air-dried wood sample. In contrast, Al was accumulated heterogeneously and intensely in the cell walls of the secondary xylem, including several growth rings, despite the long-term air-drying during storage. These results are interpreted that Al is immobilized within the cell wall, or it is closely interacted with the components of the secondary xylem. TOF-SIMS mapping also shows that Al has a tendency to be localized around vessels in the inner rings. [ABSTRACT FROM AUTHOR]

While it is generally accepted that most plant cells are mononucleate, it has been argued with some vehemence that fusiform cambial cells can be multinucleate. The controversy has not been resolved since to date, studies by conventional microscopy and transmission electron microscopy have failed to confirm unambiguously whether cambial cells are mononucleate or multinucleate. In this study, semi‐thin sections of epoxy‐embedded specimens and thick slices of cambial tissues from the hardwood Kalopanax pictus were analysed by confocal laser scanning microscopy. Tangential sections of cambium, regardless of the thickness of the section, are likely to contain portions of cells in several adjacent layers of cells and, at the lower resolution of conventional microscopy, several adjacent cells can appear to be a single cell with more than one nucleus. The higher resolution in the third dimension of confocal microscopy allowed clearly adjacent layers of cells in the cambium to be distinguished and the number of nuclei per cell to be determined. In this tree, the cambial cells were mononucleate in all cases. [ABSTRACT FROM PUBLISHER]

Some descriptions of the lipids in our paper were careless as pointed out in their commentary. We anticipate their works of the role of amphiphilic lipids in regulation of water flow at intervascular pit membranes to advance more and more. We sincerely thank Dr. Jansen and Dr. Schenk for their commentary on our paper. [Extracted from the article]

We applied a method combining field-emission scanning electron microscopy (FE-SEM) with energy dispersive X-ray spectrometry (EDX) to visualize the deposition and localization of the submicron-sized ammonium sulfate (AS) particles. The AS particles emitted from an aerosol generator in the laboratory were spherical in shape and individually deposited without aggregation on the surface of a silicon substrate. We determined the AS particles on the surfaces of the needles of Japanese larch (Larix kaempferi) and Japanese cedar (Cryptomeria japonica), and the leaves of Japanese beech (Fagus crenata) and Japanese chinquapin (Castanopsis sieboldii), using EDX. The particles were deposited on either the adaxial or abaxial side of the leaves and needles. The AS particles deposited on the surfaces of the leaves and needles did not aggregate, and they were deposited on the surfaces of the leaves and needles in the same manner, regardless of leaf structure. These results, using a new method, highlight the early stages of the deposition and localization of submicron-sized AS particles on the surfaces of the leaves and needles of forest trees. [ABSTRACT FROM AUTHOR]

Monolignols are precursors of lignin, and their glucosides are often found in plants. Glucosylation creates water‐soluble and chemically stable monolignols by protecting the phenolic hydroxyl group. To discuss the role of sinapyl alcohol glucoside, syringin, in planta, the cellular distribution of syringin in the transverse and radial surfaces of quick‐frozen stems of Syringa vulgaris L. (lilac) was visualized by cryo‐time‐of‐flight secondary ion mass spectrometry and scanning electron microscopy (cryo‐TOF‐SIMS/SEM) analyses. The amount and rough distribution of syringin were confirmed by high‐performance liquid chromatography measurements using serial tangential sections of freeze‐fixed lilac stems. The syringin distribution was also discussed with reference to the tissue classification from microscopic observations. Syringin was mainly found in the phloem region. In the xylem region, syringin was evenly distributed irrespective of the cell type from the cambial zone to the early differentiating stage region and selectively distributed in vessels in the later differentiating stage region. After the lignification of wood fibers, syringin was found in rays and some vessels in the initial part of the annual rings. Previously, artificially administered isotope‐labeled syringin was shown to be assimilated into lignin in the differentiating xylem region. Based on this, our present data showing syringin storage in the differentiating xylem region and its variation depending on the lignification stage suggest that syringin works as a lignin precursor. Additionally, detection of syringin in vessels and rays indicates intercellular transportation of syringin in lilac stems. [ABSTRACT FROM AUTHOR]

A scanning electron microscope installed cryo-unit (cryo-SEM) allows specimen observation at subzero temperatures and has been used for exploring water distribution in plant tissues in combination with freeze fixation techniques using liquid nitrogen (LN2). For woody species, however, preparations for observing the xylem transverse-cut surface involve some difficulties due to the orientation of wood fibers. Additionally, higher tension in the water column in xylem conduits can occasionally cause artifactual changes in water distribution, especially during sample fixation and collection. In this study, we demonstrate an efficient procedure to observe the water distribution within the xylem of woody plants in situ by using a cryostat and cryo-SEM. At first, during sample collection, measuring the xylem water potential should determine whether high tension is present in the xylem conduits. When the xylem water potential is low (< ca. -0.5 MPa), a tension relaxation procedure is needed to facilitate better preservation of the water status in xylem conduits during sample freeze fixation. Next, a watertight collar is attached around the tree stem and filled with LN2 for freeze fixation of the water status of xylem. After harvesting, care should be taken to ensure that the sample is preserved frozen while completing the procedures of sample preparation for observation. A cryostat is employed to clearly expose the xylem transverse-cut surface. In cryo-SEM observations, time adjustment for freeze-etching is required to remove frost dust and accentuate the edge of the cell walls on the viewing surface. Our results demonstrate the applicability of cryo-SEM techniques for the observation of water distribution within xylem at cellular and subcellular levels. The combination of cryo-SEM with non-destructive in situ observation techniques will profoundly improve the exploration of woody plant water flow dynamics.

The protocol of freeze stabilization and cryopreparation techniques to examine the distribution of water in living tree stems by X-ray imaging and cryo-scanning electron microscopy have been developed and described. The brief procedures are as follows. Firstly, a portion of transpiring stem is frozen in the standing state with liquid nitrogen to stabilize the water that is present in the conducting tissue. After filling with liquid nitrogen, discs are then collected from the frozen portion of the stem and stored in liquid nitrogen. In a low-temperature room, the samples for X-ray imaging are sectioned with a fine handsaw, and trimmed sample blokes for cryo-scanning electron microscopy are cleanly planed using a sliding microtome. Finally, the frozen sections are irradiated in a soft X-ray apparatus, and the small blocks are examined in cryo-scanning electron microscope after freeze-etching and metal coating.

The C-7 position of jasmonate is practical for synthesis of a probe to use for chemical biological studies. To confirm the utility, we synthesized fluorescent-labeled methyl jasmonate. The synthesized compound exhibited Arabidopsis thaliana root growth inhibitory and meandering activity, and potent fluorescence was observed inside the root and root hairs.