The transport mechanism of lignin precursors has been investigated previously by biochemical analyses. In rosette leaves of Arabidopsis thaliana (hereafter, Arabidopsis), ABC-like transporters mediate the translocation of mono- lignol and monolignol glucosides . An ABC transporter of Arabidopsis involved in the transport of p-coumaryl alcohol has been characterized . However, rosette leaves of Arabidopsis contain only small amounts of lignin and lignifying tissue, and H lignin derived from p-coumaryl alcohol is a minor lignin component. More recently, Tsuyama et al.  demonstrated that transport of coniferyl alcohol glucoside (coniferin) is a major transport mechanism in differentiating xylem undergoing vigorous lignification. The process is dependent on a proton gradient created by V-ATPase, and the mechanism occurs in both angiosperm and gymnosperm. Localization of V-ATPase is Part of this article has been presented at the 13th Cell Wall Meeting,
Abstract Lignin is an integral component of the cell wall of vascular plants. The mechanism of supply of lignin precursors from the cytosol into the cell wall of differen- tiating xylem has not yet been elucidated. The present study showed that a certain amount of coniferyl alcohol glucoside (coniferin) occurred in the differentiating xylem of Japanese cypress (Chamaecyparis obtusa), as previously reported in gymnosperms. Coniferin content peaked in the early stages of secondary wall formation and decreased during lignification. In contrast to gymnosperms, coniferin content was limited in the differentiating xylem of poplar (Populus sieboldii 9 Populus grandidentata). Moreover, coniferyl alcohol was not detected in all specimens. In the differentiating xylem of poplar, a higher amount of sinapyl alcohol occurred than glucoside (syringin). However, the phloem contained syringin and not sinapyl alcohol. The sinapyl alcohol content in the xylem peaked in the cells with ceasing cell wall formation, and decreased gradually towards the boundary of the annual ring, where the lignin content kept increasing. Sinapyl alcohol in the differenti- ating xylem of poplar may be used for the lignification of the xylem.
Rabbit anti-PME immunoglobulin G (IgG) antibody was designed based on the amino acid sequence of the PME protein (ASEGSNGNEN); the peptide of this amino acid sequence was used as the antigen to raise the rabbit IgG antibody. To absorb the anti-PME antibody into the antigen, we mixed the anti-PME anti- body and antigen (peptide of ASEGSNGNEN). Following, we incubated the mixture at 4˚C for 2 days. The proteins extracted from the differentiating xylem were separated by sodium dodecyl sulfate-PAGE (SDS-PAGE) (10%) and transferred to a polyvinylidine difluoride membrane (Fluorotrans W-F; Port Washington, NY, Pall Corporation). After washing the membrane in 20 mM Tris-HCl (TBS), pH 7.6, containing 0.1% (v/v) Tween 20 (TBS-T) buffer, we immersed it in TBS-T containing 2% (w/v) ECL Blocking reagent for 1 h at room temperature to block nonspecific antibody binding and washed in TBS-T. The membrane was immersed overnight in anti-PME antibody or absorbed an- ti-PME antibody diluted 100-fold with Can Get Signal Solution 1 (TOYOBO, Osaka, Japan) at 4˚C, and then washed thrice for 20 min in TBS-T. Subsequent- ly, we immersed the membrane for 4 h in anti-rabbit IgG, HRP-linked whole Ab goat (MBL, Aichi, Japan) diluted 20,000-fold with Can Get Signal Solution 2 at room temperature and washed thrice for 20 min in TBS-T containing 0.5 M NaCl and 0.02% SDS. ECL Prime Western Blotting Detection Reagent (GE Healthcare) was used to generate chemiluminescence signals, which were de- tected with an LAS-1000 plus luminescent image analyzer (FUJIFILM, Tokyo, Japan).
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the trees were 20 months old, differentiating xylem, including widely because of the unique properties of specific hy- cells in the stages of cambial cell division, cell expansion, brid genotypes. Crosses between E. grandis and E. globu- and secondary-wall formation, was collected from the first two meters of the entire stem circumference of one ramet of each lus have resulted in progeny populations with wide ge-
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Considering the weight of each section (Fig. 1), lignification should be complete at about section No. 10 in both com- pression and normal woods, and therefore, section No. 13 was analyzed as the mature xylem. Figure 2 shows the yields of thioacidolysis monomers derived from normal and com- pression woods. Yields were expressed as micromoles of product per section (per each volume) to estimate clearly the degree of lignin deposition per single cell. Expressing the yield as the product amount per gram would provide the estimation about the greater number of cells at the early stage of differentiating xylem where the weights of cells are lower than at the late stage. Thus, in this report, the yields cannot be compared precisely between normal and com- pression woods because the yields are not expressed as per gram of the section.
After cell wall components are synthesized in the cytoplasm, they are transported out of the plasma membrane and deposited; hence, cell walls are basically formed from the outer side, inward. Therefore, when making a longitudinal section of differentiating xylem and observing the innermost surface of the cell wall by microscopy, the compo- nent that has been synthesized and deposited most recently should be visible. Observa- tions of the innermost surfaces of cell walls have been observed with electron micro- scopy in Populusnigra , Cryptomeria japonica , and others  . For instance, when the innermost surfaces of tracheids during the thickening stage of the middle layer of the secondary cell walls (S2 layer) were observed in C. japonica grown in the field or in a growth chamber, cellulose microfibrils were evident during the day or the light pe- riod, and a matrix covering the microfibrils was evident at night or during the dark pe- riod. These observations led to the conclusion that the deposition of cell wall compo- nents has diurnal periodicity  .
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volved in dehydrogenative polymerization of monolignols. Peroxidases have the signal sequence at their N-terminus and this suggests that they are transported to extracellular spaces or developing cell walls. In this study, we focused on an anionic peroxidase isozyme encoded by prxA3a, which seems to be related to lignification. To investigate the local- ization of peroxidase in differentiating xylem cells of poplar (Populus sieboldii ⫻ Populus grandidentata), anti-PRX3 antibody was raised against the anionic peroxidase. West- ern blotting and peroxidase activity inhibition assay showed specificity of the antibody. Labeling by anti-PRX3 antibody was localized in vessels and fibers during the secondary wall formation and was observed along the plasma membrane beside the microtubules. The labeling was not seen in the cell wall, where localization of peroxidases was expected during lignification. The peroxidase isozyme, which is sug- gested to be involved in monolignol polymerization, is lo- calized on the plasma membrane and its localization might be regulated by microtubules.
Changes in the morphology and functions of vacuoles provide useful information about the mechanism of cell death. In the present study, we monitored the morphology and contents of vacuoles during the death of ray parenchyma cells in the conifer Cryptomeria japonica. In differentiating xylem, ray parenchyma cells had large central vacuoles. In sapwood, vacuoles in ray parenchyma cells contained proteins, an indication that one of the main functions of these vacuoles might be protein storage. A dramatic decrease in the protein content of some vacuoles was detected in the intermediate wood before the initiation of vacuole rupture. Although vacuole rupture was detected from the intermediate wood to the outermost heartwood, some vacuoles were obviously enlarged in the inner intermediate wood. Condensed nuclei were first observed after the rupture of these large vacuoles in ray parenchyma cells. It seems plausible that the autolysis of the contents of ray parenchyma cells might be caused by the rupture of the enlarged vacuoles in the inner intermediate wood.
Abnormal forms of xylem in the vicinity of the enclosed bark were also visible around the wider growth zone adja- cent to the canker. In this area, disoriented axial cells were more obvious in tangential views (Fig. 4a), while the cells shape was warped toward the canker zone (Fig. 4b). An irregular orientation of xylem elements was commonly observed in transverse views (Fig. 4c). These evidences show that the cambium in the immediate vicinity of the canker actively grew to generate cells for closing the canker. Such disoriented axial cells have frequently been investi- gated in xylem formed after wounding when canker disease is present, for instance, canker formation by inoculation in Fraxinus mandshurica. 11
Acoustic emissions arising from xylem cavitation events were detected with low mass miniature epoxy faced piezoelectric transducers having an operating frequency range of 100 - 1000 kHz (Model S9220, Physical Acoustics Corp., MISTRAS Group, Inc., Princeton Junction, NJ) acoustically coupled to the plant with a small amount of high vacuum silicone stopcock grease placed on the face of the transducer. The transducers were fixed to the upper surface of the petiole or the base of the main stem with black PVC electrical tape. Transducer output was conditioned with a 20 - 1200 kHz active bandpass pre-amplifier set to 60 db gain (Model 2/4/6, Physical Acoustics) and analyzed with a microDiSP™ acoustic digital signal processing and detection system (Physical Acoustics Corp.) driven by a notebook computer running AE Win for DiSP™ (Physical Acoustics Corp.) software configured to detect and log signals > 35 dB (4.75 μV threshold) within a 100 - 200 kHz range. The instrumentation was housed in a large marine grade insulated cooler actively aspirated with muffin fans to minimize heat buildup from solar radiation and electronic equipment.
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fewer ginsenosides than other tissues. For the branch roots of Pq1-4, the cork, xylem and cortex occupied higher contents of ginsenosides than phloem did. Thus, the distribution pattern of ginsenosides in American gin- seng was quite distinct from Asian ginseng. Distinctly, the cork, primary xylem or cortex had more ginsenosides than phloem, secondary xylem and cambium in Ameri- can ginseng. Based on all the above, it was reasonable to deduce that the ratio of total areas of cork, primary xylem and the cortex to the area of whole transection could help to evaluate the quality of American ginsengs.
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Figure 4. Anatomy of Piper betle. (a-d, f: LM; e: fluorescence microscopy, UV Ex = 330 - 380 nm; a, b: stained in safranin/fast green; d, e: stained in toluidine blue; c, f: stained in phloroglucinol/HCl). a: TS of leaf through the midrib; b: TS of leaf through lamina; c: TS of petiole; d: TS of stem showing a ring of mucilage canals between cortical and medullary whorls of vascular bundles and a central mucilage canal; e: a portion of TS of stem showing a secretory cell in the cortex, cortical fibers and endodermis with casparian thickening; f: TS of root. (Cb: cortical bundle, Cf: cortical fibers, Ck: cork, Co: collenchyma, Ct: cuticle, Cx: cortex, En: endodermis, Gt: glandular trichome, Lc: lignified cells, Lh: abaxial hypodermis, Mb: medullary bundle, Mc: mucilage canal, Mr: medullary ray, Nt: nonglandular trichome, Ph: phloem, Sa: sclerenchyma, Sc: secretory cell, Sd: sclereid, Uh: adaxial hypodermis, Xy: xylem, Ph: phloem). Scale bars: a, b, e = 100 µm; c, d, f = 500 µm.
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developed xylem adjacent to the vascular cambium (Figs. 3a,b). The tension wood of the 1-day bending treatment samples did not display any differences when compared with the opposite wood (Figs. 3c,d) or with the control (Figs. 3a,b), in which the staining intensity might have been very low in the differentiating region. After 7 days of bending treatment, however, lignin staining was signifi - cantly reduced in the newly developed xylem tissues adja- cent to the vascular cambium of bent stems (Fig. 3f) compared to that of the opposite wood (Fig. 3e) and upright control (Fig. 3a,b). The differences in lignin deposition between the tension wood and opposite wood were more clearly detected in the 14-day bending treatment samples (Figs. 3g,h). This indicated that the bending treatment of yellow poplar in a 45° confi guration induced an overall reduction of lignin deposition in the tension wood, which is one of the most characteristic chemical features found in tension wood.
Abstract: Graft compatibility is a complex mechanism with a wide range of different physiological, biochemical, and anatomical interactions. In the present paper, features of the graft union on the combination of the European chestnut (Castanea sativa Mill.) on an oak (Quercus vulcanica Boiss.) rootstock based on whip grafting was investigated. The trial was conducted in the greenhouse at the Faculty of Agriculture, Adnan Menderes University, in western Turkey during 2006-7. The grafts were made with scion from chestnut genotype N-3-4 on an oak rootstock. Cross and longitudinal sections of the graft union for examination were taken 30, 60, 90, 120, 150 and 210 days after grafting, and fixed in 70% ethanol. Microscopic observation of the anatomical and histo-cytological structure of the graft union area revealed that new cambium, xylem, and phloem tissues were formed in the samples 120 days after grafting. The results showed that, graft compatibility exists between chestnut and oaks. Callus formation and differentiation of vascular tissues started 150 days after grafting.
The present work has demonstrated the antifungal activity of fisetin and fresh xylem sap from H. courbaril , species largely found in Brazil, against yeasts of C. neo- formans species complex and filamentous fungi as der- matophytes. Numerous assay systems and organisms have been used to screening plant extracts and constitu- ents of active plants for antimicrobial activity. The broth microdilution method used in this work has several ad- vantages. This method is quantitative, allows the use of small quantities of compounds or plant extracts as well as culture media . It was observed MIC values below 256 μg/mL for fisetin and for fresh xylem sap against these fungi. There is no consensus in the literature on the MIC values of a plant extract which qualifies it as promising for fractionation. According to Kuete , the antimicrobial activity of extracts can be classified as follows: significant if MIC values are below100 μg/ml, moderate when 100 < MIC < 625 μg/ml and weak if MIC > 625 μg/ml. Therefore, the overall antifungal activ- ity exhibited in this study varied from moderate to signifi- cant. Previous studies have shown that flavonoids-rich extracts possesses antimicrobial activity [41,42]. Although the identification of mechanism action of flavonoids has been discussed in the literature and there are indications
TS of root composed of an outer piliferous layer, it includes thin superficial periderm which consists of broken epidermal layer. The cortex is composed of parenchyma cells with intercellular spaces; these cells are oval in shape and filled with myrosin. The vascular bundles lie at the center. In matured roots, secondary phloem is thin, continuous layer of sieve elements, parenchyma cells and phloem rays (Fig. 5-a). The phloem elements are small, squarish in shape and are arranged in vertical rows. Secondary xylem cylinder is circular in outline. It includes vessels, sclereids and fibres. The vessels of the secondary xylem are wide, circular and thick walled. The vessels are solitary (Fig. 5-b), the diameter of the vessels increases from centre towards periphery. It includes several straight, thin and one cell thick xylem rays (Fig. 5-c). The pith is well developed, thick walled and filled with myrosin (Fig. 5-d). Calcium oxalate crystals are distributed throughout the root. The crystals are solitary in each parenchyma cells (Fig. 5-d).
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Root pressure is also known as osmosis effect in plant researches. As water moves in the direction of a decreasing water potential, this effect generates a force along the decreasing water potential from pure water, soil, root, xylem, leaf to atmosphere. Mineral ions in soil can be absorbed by the root cells, which would cause the accumulation of ions on the other side of root cell membranes in the cells, with higher concentration comparing with soil environment outside. This would drive the water to flow into the root cells as well. While due to its limitation, it only accounts for the major water migration when transpiration effect is low, such as dur- ing the night. Root pressure is a main reason for gutta- tion, as well as the absorption of minerals by root cells  . It would also contribute to the water refill in xylem, as some plants’ xylem conduits are empty during winter, even though not always the case [31,32] . However, it is now considered as a positive but relatively unimportant force in water migration in xylem, instead of a major one. Therefore, it is not discussed and analysed in detail in this paper.
Measurements of amino acids in the guttation fluid and in the xylem exudates of cut leaves from intact plants provide evidence of the remarkable efficiency with which these nitrogenous compounds are reabsorbed from the xylem sap. This could be achieved by mechanisms involving intercellular transport and/or metabolism. Developmental changes in transcripts and protein showed that transcripts for phosphoenolpyruvate carboxykinase (PEPCK) increased from the base to the leaf tip, and were markedly increased by supplying asparagine. Supplying amino acids also increased the amounts of protein of PEPCK and, to a lesser extent, of pyruvate, Pi dikinase. PEPCK is present in the hydathodes, stomata and vascular parenchyma of rice leaves. Evidence for the role of PEPCK was obtained by using 3-mercap- topicolinic acid (MPA), a specific inhibitor of PEPCK, and by using an activation-tagged rice line that had an increase in PEPCK activity, to show that activation of PEPCK resulted in a decrease in N in the guttation fluid and that treatment by MPA resulted in an increase in amino acids in the guttation fluid and xylem sap towards the leaf tip. Furthermore, increasing PEPCK activity decreased the amount of guttation fluid, whereas decreasing PEPCK activity increased the amount of xylem sap or guttation fluid towards the leaf tip. The findings suggest the following hypotheses: (i) both metabolism and transport are involved in xylem recycling and (ii) excess N is the signal involved in modulating xylem hydraulics, perhaps via nutrient regulation of water-transporting aquaporins. Water relations and vascular metabo- lism and transport are thus intimately linked.
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The loss of leaf conductivity in the field, most likely due to leaf or stem xylem embolism (Domec et al. 2006; Woodruff et al. 2007; Johnson et al. 2009; Blackman et al. 2017), can be accurately predicted from field measure- ments of leaf water potentials and laboratory-generated vulnerability curves (Brodribb and Holbrook 2004; Johnson et al. 2009). The 13 species in this study varied substantially in Loss in the field, from 1.39% in the ever- green I. chinensis to 90.07% in the deciduous S. sebiferum in the field. The large variation in Loss in the karst area in China argued against the hypothesis that plants control xylem tension above threshold for cavitation. Interestingly, Loss in the current study did not significantly depress the efficiency of PSII and/or the photosynthetic rate, suggest- ing that photosynthetic capacity was not affected by xylem cavitation, even in the extreme case of S. sebiferum, which lost 90% hydraulic conductivity at noon.
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plant is one of the most important criteria that affect the robustness of the protoplast system. Healthy plants are necessary for producing high yields of protoplasts with high transfection efficiency. Appropriate temperatures, routine watering and fertilization are necessary for maintaining plants with healthy and vigorous growth in a greenhouse. Healthy greenhouse- grown P. trichocarpa plants have gleaming developing leaves and fully expanded leaves that are flat and bright dark-green colored upper leaf surface. These P. trichocarpa plants usually also develop xylem secretion at the 2 nd to 5 th young stem nodes where auxiliary or lateral buds usually emerge (Fig. 3), indicating active water and nutrient transport and, thus, their vigorous growth status. The stem bark of such plants can be easily peeled off (Fig. 1b,g) and the surface (differentiating xylem cells, Fig. 1d) of the debarked stem is succulent. These plants always give high SDX protoplast yields and transfection efficiency. Therefore xylem secretion at young nodes has always been the best guide for us to select the P. trichocarpa plant for SDX protoplast production. In general, healthy leaves are usually a good indicator of healthy trees for SDX protoplast generation. Trees with stem bark difficult to be peeled off are usually unhealthy or inactive in growth, and should not be used because they give little or no SDX protoplasts.
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