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HD-zip III homeobox genes that include a novel member, ZeHB-13 (Zinnia)/ATHB-15 (Arabidopsis), are involved in procambium and xylem cell differentiation.

HD-Zip III homeobox genes are known to be essential transcriptional factors for vascular development. To further understand the relation of HD-Zip III genes in vascular differentiation, we isolated a new member of the HD-Zip III genes, ZeHB-13, as a Zinnia homolog of ATHB-15, and then characterized the expression profile using a Zinnia xylogenic cell culture and Zinnia plants. We compared the accumulation pattern of transcripts for ZeHB-13 and other HD-Zip III genes and suggested that the expression of ZeHB-13 was restricted to the procambium and was not severely suppressed by brassinazole, an inhibitor of brassinosteroid biosynthesis, unlike other HD-Zip III genes. We also characterized its Arabidopsis counterpart, ATHB-15. A histochemical promoter analysis using ATHB-15::GUS transgenic Arabidopsis plants indicated that ATHB-15 was active specifically in the procambium. These results strongly suggest that ZeHB-13/ATHB-15 is a pivotal transcriptional regulator responsible for early vascular development. Based on these results, we will discuss the regulation of xylem development in light of the functions of HD-Zip III members and brassinosteroids.

Amino Acid Sequence↗

A mathematical and statistical analysis of the curves illustrating vulnerability of xylem to cavitation.

When vulnerability curves are used to assess the susceptibility of plants to drought, the water potential corresponding to 50% loss of conductivity is frequently used as a measure of susceptibility. However, this value does not distinguish between different patterns of conductivity loss, such as a rapid loss over a narrow water potential range versus a gradual decline in conductivity. We have applied an exponential sigmoidal equation to vulnerability curves obtained from four closely related Eucalyptus clones differing in drought tolerance. The coefficients of the equation were evaluated and statictically compared among the clones. If the air-seedling hypothesis of cavitation is accepted, these coefficients have biological relevance. One of the coefficients describes the position of the curve on the water potential axis and is equivalent to the water potential corresponding to 50% loss of conductivity. This coefficient could reflect the size of the largest pit pore per xylem vessel. Another coefficient is related to the slope of the conductivity loss, and could reflect the range in maximum pit pore size per vessel.

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Nitrogen and potassium effects on xylem cavitation and water-use efficiency in poplars.

Effects of N and K nutrition on drought and cavitation resistance were examined in six greenhouse-grown poplar clones: Populus trichocarpa (Torr. & Gray) and its hybrids with P. deltoides Bartr. and P. euramericana (Dole) Guinier, before and after preconditioning to water stress. Both tendency to cavitate and water-use efficiency (WUE) increased when N supply was increased, whereas K supply had little impact on cavitation. Mean xylem vessel diameters increased from 36.6 &mgr;m at low-N supply to 45.2 &mgr;m at high-N supply. Drought-hardy clones, which were relatively resistant to cavitation, had the smallest mean vessel diameters. Vulnerability to cavitation had a weakly positive relationship with vessel diameter, and a negative correlation with transpiration. Drought hardening offered no protection against cavitation in a subsequent drought. Under drought conditions, increasing N supply increased leaf loss and decreased water potentials, whereas increasing K supply decreased leaf loss. Drought-resistant clones exhibited similar WUE to drought-susceptible clones, but had smaller, more numerous stomata and greater leaf retention under drought conditions.

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Spatial variations in xylem sap flux density in the trunk of orchard-grown, mature mango trees under changing soil water conditions.

Circumferential and radial variations in xylem sap flux density in trunks of 13-year-old mango (Mangifera indica L.) trees were investigated with Granier sap flow sensor probes under limiting and non-limiting soil water conditions. Under non-limiting soil water conditions, circumferential variation was substantial, but there was no apparent relationship between sap flux density and aspect (i.e., the radial position of the sensor probes on the trunk relative to the compass). Hourly sap flux densities over 24 hours at different aspects were highly pair-wise correlated. The relationships between different aspects were constant during well-watered periods but highly variable under changing soil water conditions. Sap flux density showed marked radial variation within the trunk and a substantial flux was observed at the center of the trunk. For each selected aspect on each tree, changes in sap flux densities over time at different depths were closely correlated, so flux at a particular depth could be extrapolated as a multiple of flux from 0 to 2 cm beneath the cambium. However, depth profiles of sap flux density differed between trees and even between aspects within a tree, and also varied in an unpredictable manner as soil water conditions changed. Nevertheless, over a period of non-limiting soil water conditions, depth profiles remained relatively constant. Based on the depth profiles obtained during these periods, a method is described for calculating total sap flow in a mango tree from sap flux density at 0-2 cm beneath the cambium. Total daily sap flows obtained were consistent with water use estimated from soil water balance.

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Seasonal changes in levels of cytokinin-like compounds from Douglas-fir xylem extrudate.

High-performance liquid chromatography, immunochromatography, and radioimmunoassay were used to identify cytokinin-like bases and glycosides in xylem sap of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco). Isopentenyladenosine-type (isopentenyladenine and isopentenyladenosine) and zeatin-riboside type (zeatin, zeatin riboside, and dihydrozeatin riboside) cytokinins were detected during springtime. A glucosyl conjugate of zeatin riboside was also present in small amounts. Levels of cytokinin-like compounds varied throughout the spring but were generally highest in late April to early May.

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Xylem dysfunction in Quercus: vessel sizes, tyloses, cavitation and seasonal changes in embolism.

The seasonal progression of xylem dysfunction from tyloses and embolism induced both by cavitation and frost was studied in Quercus rubra L. and Quercus alba L. branches. Vessel lengths and diameters were measured in current-year rings of branches of various ages. Vessels in current-year shoots are about the same size as those in many diffuse porous trees, but vessels in older branches are two to six times larger in diameter and typically more than 10 times longer. Large Quercus vessels were more vulnerable to cavitation than small vessels. The small vessels in current-year shoots were more vulnerable to cavitation than vessels of comparable size in diffuse porous species. Earlywood vessels are completely blocked by tyloses within a year of their formation. Tylose growth starts in winter, but the vessels are not fully blocked until the next summer. Many latewood vessels, by contrast, remain free of complete blockage for several years. In Q. rubra, loss of hydraulic conductivity in current-year shoots due to cavitation reaches 20% by August and > 90% after the first hard frost. Both laboratory and field observations confirm that the role of frost in causing loss of hydraulic conduction by embolism is much more dramatic in Quercus than in conifers and diffuse porous hardwoods.

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Phylogenetic analyses in cornus substantiate ancestry of xylem supercooling freezing behavior and reveal lineage of desiccation related proteins.

The response of woody plant tissues to freezing temperature has evolved into two distinct behaviors: an avoidance strategy, in which intracellular water supercools, and a freeze-tolerance strategy, where cells tolerate the loss of water to extracellular ice. Although both strategies involve extracellular ice formation, supercooling cells are thought to resist freeze-induced dehydration. Dehydrin proteins, which accumulate during cold acclimation in numerous herbaceous and woody plants, have been speculated to provide, among other things, protection from desiccative extracellular ice formation. Here we use Cornus as a model system to provide the first phylogenetic characterization of xylem freezing behavior and dehydrin-like proteins. Our data suggest that both freezing behavior and the accumulation of dehydrin-like proteins in Cornus are lineage related; supercooling and nonaccumulation of dehydrin-like proteins are ancestral within the genus. The nonsupercooling strategy evolved within the blue- or white-fruited subgroup where representative species exhibit high levels of freeze tolerance. Within the blue- or white-fruited lineage, a single origin of dehydrin-like proteins was documented and displayed a trend for size increase in molecular mass. Phylogenetic analyses revealed that an early divergent group of red-fruited supercooling dogwoods lack a similar protein. Dehydrin-like proteins were limited to neither nonsupercooling species nor to those that possess extreme freeze tolerance.

Cornus↗

Can Ca2+ fluxes to the root xylem be sustained by Ca2+-ATPases in exodermal and endodermal plasma membranes?

The pathway of Ca2+ movement from the soil solution into the root stele has been a subject of controversy. If transport through the endodermis is assumed to be through the cytoplasm, the limiting factor is believed to be the active pumping of Ca2+ from the cytoplasm into the stele apoplast through the plasma membrane lying on the stele side of the Casparian band. By analogy, for similar transport through the exodermis, the limiting step would be the active pumping into the apoplast on the central cortical side of the layer. Such effluxes are mediated by Ca2+-ATPases. To assess whether or not known Ca2+ fluxes to the stele in onion (Allium cepa) roots could be supported by Ca2+-ATPases, the percentages of total membrane protein particles required to effect the transport were calculated using measured values of membrane surface areas, an animal literature value for Ca2+-ATPase V(max), plant literature values for Ca2+-ATPase K(m), and protein densities of relevant membranes. Effects of a putative symplastic movement of Ca2+ from the exo- or endodermis into the next cell layer, which would increase the surface areas available for pumping, were also considered. Depending on the assumptions applied, densities of Ca2+ pumps, calculated as a percentage of total membrane protein particles, varied tremendously between three and 1,600 for the endodermis, and between 0.94 and 1,900 for the exodermis. On the basis of the data, the possibility of Ca2+ transport through the cytoplasm and membranes of the exodermis and endodermis cannot be discounted. Thus, it is premature to assign an entirely apoplastic pathway for Ca2+ movement from the soil solution to the tracheary elements of the xylem. To verify any conclusion with certainty, more detailed data are required for the characteristics of exo- and endodermal Ca2+-ATPases.

Arabidopsis↗

Profiling of oligolignols reveals monolignol coupling conditions in lignifying poplar xylem.

Lignin is an aromatic heteropolymer, abundantly present in the walls of secondary thickened cells. Although much research has been devoted to the structure and composition of the polymer to obtain insight into lignin polymerization, the low-molecular weight oligolignol fraction has escaped a detailed characterization. This fraction, in contrast to the rather inaccessible polymer, is a simple and accessible model that reveals details about the coupling of monolignols, an issue that has raised considerable controversy over the past years. We have profiled the methanol-soluble oligolignol fraction of poplar (Populus spp.) xylem, a tissue with extensive lignification. Using liquid chromatography-mass spectrometry, chemical synthesis, and nuclear magnetic resonance, we have elucidated the structures of 38 compounds, most of which were dimers, trimers, and tetramers derived from coniferyl alcohol, sinapyl alcohol, their aldehyde analogs, or vanillin. All structures support the recently challenged random chemical coupling hypothesis for lignin polymerization. Importantly, the structures of two oligomers, each containing a gamma-p-hydroxybenzoylated syringyl unit, strongly suggest that sinapyl p-hydroxybenzoate is an authentic precursor for lignin polymerization in poplar.

Lignin↗

Rice cationic peroxidase accumulates in xylem vessels during incompatible interactions with Xanthomonas oryzae pv oryzae.

A cationic peroxidase, PO-C1 (molecular mass 42 kD, isoelectric point 8.6), which is induced in incompatible interactions between the vascular pathogen Xanthomonas oryzae pv oryzae and rice (Oryza sativa L.), was purified. Amino acid sequences from chemically cleaved fragments of PO-C1 exhibited a high percentage of identity with deduced sequences of peroxidases from rice, barley, and wheat. Polyclonal antibodies were raised to an 11-amino acid oligopeptide (POC1a) that was derived from a domain where the sequence of the cationic peroxidase diverged from other known peroxidases. The anti-POC1a antibodies reacted only with a protein of the same mobility as PO-C1 in extracellular and guttation fluids from plants undergoing incompatible responses collected at 24 h after infection. In the compatible responses, the antibodies did not detect PO-C1 until 48 h after infection. Immunoelectron microscopy was used to demonstrate that PO-C1 accumulated within the apoplast of mesophyll cells and within the cell walls and vessel lumen of xylem elements of plants undergoing incompatible interactions.

Amino Acid Sequence↗

Red Xylem and Higher Lignin Extractability by Down-Regulating a Cinnamyl Alcohol Dehydrogenase in Poplar.

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the last step in the biosynthesis of the lignin precursors, the monolignols. We have down-regulated CAD in transgenic poplar (Populus tremula X Populus alba) by both antisense and co-suppression strategies. Several antisense and sense CAD transgenic poplars had an approximately 70% reduced CAD activity that was associated with a red coloration of the xylem tissue. Neither the lignin amount nor the lignin monomeric composition (syringyl/guaiacyl) were significantly modified. However, phloroglucinol-HCl staining was different in the down-regulated CAD plants, suggesting changes in the number of aldehyde units in the lignin. Furthermore, the reactivity of the cell wall toward alkali treatment was altered: a lower amount of lignin was found in the insoluble, saponified residue and more lignin could be precipitated from the soluble alkali fraction. Moreover, large amounts of phenolic compounds, vanillin and especially syringaldehyde, were detected in the soluble alkali fraction of the CAD down-regulated poplars. Alkaline pulping experiments on 3-month-old trees showed a reduction of the kappa number without affecting the degree of cellulose degradation. These results indicate that reducing the CAD activity in trees might be a valuable strategy to optimize certain processes of the wood industry, especially those of the pulp and paper industry.

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Chemical Composition of Hypodermal and Endodermal Cell Walls and Xylem Vessels Isolated from Clivia miniata (Identification of the Biopolymers Lignin and Suberin).

The occurrence of the biopolymers lignin and suberin was investigated with hypodermal (HCW) and endodermal cell walls (ECW) and xylem vessels (XV) isolated from Clivia miniata Reg. roots. Both biopolymers were detected in HCW and ECW, whereas in XV, typical aliphatic suberin monomers were missing and only representative lignin monomers such as guaiacyl (G) and syringyl (S) units could be detected. The absolute amounts of lignin were about one order of magnitude higher compared with suberin in both HCW and ECW. The ratios of the two aromatic lignin units (G/S) decreased from 39 in XV and 10 in HCW to about 1 in ECW, indicating significant differences in lignin structure and function between the three investigated samples. Additionally, compared with the detectable lignin-derived aromatic units G and S, significantly higher amounts of esterified p-coumaric acid-derived aromatic monomers were obtained with HCW, but not with ECW. This is interpreted as a functional adaption of HCW toward pathogen defense at the root/soil interface. The final aim of this study was to provide a thorough chemical characterization of the composition of HCW, ECW, and XV, which in turn will form the basis for a better understanding of the relevant barriers toward the passive, radial, and apoplastic diffusion of solutes from the soil across the root cortex into the root cylinder.

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A proline-, threonine-, and glycine-rich protein down-regulated by drought is localized in the cell wall of xylem elements.

A cDNA clone encoding a proline-, threonine-, and glycine-rich protein (PTGRP) was isolated from a wild tomato species (Lycopersicon chilense) (L.X. Yu, H. Chamberland, J.G. Lafontain, Z. Tabaeizadeh [1996] Genome 39: 1185-1193). Northern-blot analysis and in situ hybridization studies revealed that PTGRP is down-regulated by drought stress. The level of the mRNA in leaves and stems of 8-d drought-stressed plants decreased 5- to 10-fold compared with that in regularly watered plants. The mRNA re-accumulated when drought-stressed plants were rewatered. Antibodies raised against a glutathione S-transferase/PTGRP fusion protein were used to elucidate the subcellular localization of the protein by immunogold labeling. In regularly watered L. chilense plants, PTGRP protein was found to be localized in xylem pit membranes and disintegrated primary walls. Examination of sections from drought-stressed plants revealed a significant decrease in the levels of labeling. In these samples, only a few scattered gold particles were detected in the same areas. In the leaf tissues of plants that had been rewatered for 3 d following an 8-d drought stress, the labeling pattern was similar to that of the regularly watered plants. To our knowledge, PTGRP is the first drought-regulated protein that has been precisely localized in the cell wall.

Amino Acid Sequence↗

Xylem sap flow as affected by metabolic inhibitors and girdling.

The flow of sap in Populus tremuloides was studied by the thermoelectric method immediately prior to and at several periods of time after radical girdling. Similarly, the flow of sap in Betula papyrifera was studied before and after girdling or injection with metabolic inhibitors or a filtrate of a culture of Ceratocystis ulmi.None of the treatments caused any significant change in the speed at which the sap flowed. However, the rate at which the temperature rose at the upper (downstream) thermocouple decreased significantly in girdled trees and in most trees treated with metabolic inhibitors. That decrease was first noticed 4 days after poisoning with 0.1 m mercuric chloride, and 1 day after poisoning with either 0.01 m picric acid or a culture filtrate of Ceratocystis ulmi. A 0.05 m trichloroacetic acid solution caused a decrease in the rate of temperature rise about 2 hours after the tree was injected. The rate of temperature rise returned to normal the next day after the injection with trichloroacetic but again showed a decrease after 42 days.Increases in the rate of heat rise were noted the day after the birch trees were girdled, and all girdled trees showed a significant decrease in the rate of heat rise sometime after they were girdled. Many of the girdled trees showed no sap flow or even an apparent downward flow of xylem sap sometime after girdling.Laboratory experiments indicated that the blocking of the vessels by air emboli would result in a significant change in the rate of temperature rise, not accompanied by a significant change in the speed at which the sap flowed. Since air emboli caused similar results to those actually experienced in the field, the data are interpreted as the result of blocked or nonfunctional vessels.All of the data agree with that of earlier workers. Cessation of upward flow occurs only in girdled trees after rather long periods of time. It is suggested that cessation could be caused by tyloses and air emboli. All of the data are also in agreement with the passive theories of sap ascent. However, the trichloroacetic acid experiments suggest the existence of a repair mechanism or a second means of sap movement.

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Effects of external cations and respiratory inhibitors on electrical potential of the xylem exudate of excised corn roots.

Glass capillary microelectrodes were used to study the electrical potential difference (PD) between the xylem exudate of excised corn roots, Zea mays L. Golden Bantam hybrid, and the external solution. A survey of the effects of various ions on the PD was made. With 1 mm single salt solutions, the PD was between 25 and 50 mv, exudate negative. The PD responded to concentration differences in single salt solutions of K(+), Na(+), and Ca(2+) in a manner suggestive of cation selectivity and cation diffusion potentials. With Ca(2+) present, the PD was insensitive to concentration changes of other cations. Substitution of NO(3) (-) for Cl(-) in K(+) solutions increased the PD by 2 to 5 mv, although in general the PD showed little response to anion concentration changes. The PD was partially abolished by cyanide. The remaining fraction of the PD was sensitive to concentration changes in external K(+), and we postulate that the PD is the result of both a diffusion potential and an electrogenic pump.

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Lateral transport of ions into the xylem of corn roots: I. Kinetics and energetics.

A technique is described for study of the kinetics of lateral transport of ions across single roots of corn, Zea mays, in short term experiments under steady state conditions. The kinetics of chloride transfer to the vessels reflected the kinetics of absorption of chloride by the root cells. Efflux from the root vacuoles contributed to only a small extent to transport of chloride into the exudate. Lateral transport of chloride was inhibited by bromide at chloride concentrations in the ranges of both mechanisms 1 and 2 in a manner implicating competition. The uncoupler carbonylcyanide m-chlorophenylhydrazone used at 1 mum caused transfer of chloride to cease almost immediately at both low and high concentrations of chloride. Oligomycin depressed transport of chloride to the vessels within 10 to 15 minutes after application at 2 micrograms per milliliter. Inhibition by oligomycin was 75% at 0.5 mm chloride and 55% at 5 mm.It is concluded that lateral transport of chloride across corn roots is mediated by the dual mechanisms of ion absorption which reside in the plasmalemma. Transfer of chloride is inhibited by bromide and depends upon ATP as energy source. Chloride moves from the plasmalemma, the site of carriermediated absorption, to the xylem vessels by way of the symplasm. There is no evidence in these experiments that lateral transport of chloride in corn roots is governed by diffusion at any concentrations of chloride used in these experiments.

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Translocation of nickel in xylem exudate of plants.

Topped plants of tomato (Lycopersicon esculentum), cucumber (Cucumis sativus), corn (Zea mays), carrot (Daucus carota), and peanut (Arachis hypogaea) were treated with 0.5 to 50 micromolar Ni (containing (63)Ni) in nutrient solutions. Xylem exudate was collected for 10 hours or, in the case of corn, for 20 hours at 5-hour intervals. Electrophoresis of nutrient solution distributed all Ni cathodically as inorganic Ni(2+). Low concentrations of Ni in tomato exudate migrated anodically, presumably bound to organic anion (carrier). However, this carrier became saturated at about 2 micromolar Ni in exudate, and excess Ni ran cathodically. Most of the Ni in cucumber, corn, carrot, and peanut exudate ran anodically, and its migration rate was identical for all exudates. Peanut root sap contained 14 to 735 micromolar Ni. The anodic Ni carriers in root sap and exudate appear identical. The carrier in root sap became saturated near 100 micromolar Ni, as shown by cathodic streaking of Ni exceeding that concentration. It appears that all five species translocate low concentrations of Ni in the same anionic form.

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Seasonal variation in the hormone content of willow: I. Changes in abscisic Acid content and cytokinin activity in the xylem sap.

Changes in levels of abscisic acid (ABA) and cytokinin activity in the xylem sap of willow (Salix viminalis, L.) were followed throughout two growth cycles.Growth in spring was preceded by decreasing levels of ABA and an increase in cytokinin activity. The onset of dormancy was associated with low levels of cytokinins and high contents of ABA. A second peak of ABA was found in July which was not related to the dry weight of the sap. The main cytokinin activity in the sap was due to a zeatin riboside-like compound.

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