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The Role of Auxin and Gibberellin in Controlling Lignin Formation in Primary Phloem Fibers and in Xylem of Coleus blumei Stems.

The hypothesis that auxin (IAA) and gibberellic acid (GA(3)) control the formation of lignin is confirmed for the primary phloem fibers and for the secondary xylem in the stem of Coleus blumel Benth. Indoleacetic acid alone, or a combination of high IAA/low GA(3) (w/w), induced short phloem fibers with thick secondary walls, that contained lignin rich in syringyl units (high ratio of syringyl/guaiacyl). On the other hand, a combination of high GA(3)/low IAA (w/w), which promoted the differentiation of long phloem fibers with thin walls, decreased the relative content of the syringyl units (low syringyl/guaiacyl ratio). In the secondary xylem, these hormonal treatments yielded only slight changes in the noncondensed monomeric guaiacyl units, confirming the relative stability of the guaiacyl lignification pattern in this tissue. In the xylem, indoleacetic acid alone, or a combination of high IAA/low GA(3) induced lignin poor in syringyl units (low syringyl/guaiacyl ratio). A combination of high GA(3)/low IAA promoted a relatively slight increase in syringyl yield, indicating greater responsiveness of the syringyl lignification pattern to growth regulators. The possible functional and technological significance of our results is discussed.

Journal Article↗

Ethylene Biosynthesis-Inducing Endoxylanase Is Translocated through the Xylem of Nicotiana tabacum cv Xanthi Plants.

Ethylene biosynthesis-inducing xylanase (EIX) from the fungus Trichoderma viride elicits enhanced ethylene production and tissue necrosis in whole tobacco (Nicotiana tabacum cv Xanthi) plants at sites far removed from the point of EIX application when applied through a cut petiole. Symptoms develop in a specific pattern, which appears to be determined by the interconnections of the tobacco xylem. Based on results of tissue printing experiments, EIX enters the xylem of the stem from the point of application and rapidly moves up and down the stem, resulting in localized foliar symptoms on the treated side of the plant above and below the point of EIX application. The observation that a fungal protein that elicits plant defense responses can be translocated through the xylem suggests that plants respond to pathogen-derived extracellular proteins in tissues distant from the invading pathogen.

Journal Article↗

Adaptation to high salinity in poplar involves changes in xylem anatomy and auxin physiology.

To investigate the physiological basis of salt adaptation in poplar, we compared the effect of salt stress on wood anatomy and auxin physiology of the salt-resistant Populus euphratica and salt-sensitive Populus x canescens. Both poplar species showed decreases in vessel lumina associated with increases in wall strength in response to salt, however, in P. euphratica at three-fold higher salt concentrations than in P. x canescens. The predicted hydraulic conductivity of the wood formed under salt stress decreased in P. x canescens, while in P. euphratica, no significant effects of salt on conductivity and transpiration were observed. The concentration of free indole-3-acetic acid (IAA) decreased under salt stress in the xylem of both poplar species, but to a larger extent in P. x canescens than in P. euphratica. Only salt-treated P. euphratica exhibited an increase in IAA-conjugates in the xylem. Genes homologous to the auxin-amidohydrolase ILL3 were isolated from the xylems of P. euphratica and P. x canescens. For functional analysis, the auxin-amidohydrolase from P. x canescens was overexpressed in Arabidopsis. Transgenic Arabidopsis plants were more resistant to salt stress than the wild-type plants. Increased sensitivity of the transgenic Arabidopsis to IAA-Leu showed that the encoded hydrolase used IAA-Leu as a substrate. These results suggest that poplar can use IAA-amidoconjugates in the stem as a source of auxin to balance the effects of salt stress on auxin physiology.

Adaptation, Physiological↗

ATPase in mature and differentiating phloem and xylem.

A cytochemical study using a lead precipitation technique has been made of the distribution of adenosine triphosphatase (ATPase) in mature and differentiating phloem and xylem cells of Nicotiana tabacum and Pisum sativum. The sites of ATPase localization in tobacco phloem were the plasma membrane, endoplasmic reticulum, mitochondria, dictyosomes, plasmodesmata, and the dispersed P proteins of mature sieve elements. In pea phloem sieve elements ATPase was localized in the endoplasmic reticulum, but was not associated with the P proteins or plasma membranes at any stage of their differentiation. In pea transfer cells ATPase activity was associated with the endoplasmic reticulum at all stages of their differentiation and with the plasma membrane of transfer cells that had formed wall ingrowths. In xylem cells of both tobacco and pea the patterns of ATPase activity was similar. At early stages of differentiation ATPase activity was associated with the plasma membrane and the endoplasmic reticulum. At intermediate stages of differentiation ATPase activity continued to be associated with the endoplasmic reticulum, but was no longer associated with the plasma membrane. At later stages of xylem element differentiation ATPase activity was associated with disintegrating organelles and with the hydrolyzing cell walls.

Adenosine Triphosphatases↗

The relationship between xylem conduit diameter and cavitation caused by freezing.

The centrifuge method for measuring the resistance of xylem to cavitation by water stress was modified to also account for any additional cavitation that might occur from a freeze-thaw cycle. A strong correlation was found between cavitation by freezing and mean conduit diameter. On the one extreme, a tracheid-bearing conifer and diffuse-porous angiosperms with small-diameter vessels (mean diameter <30 μm) showed no freezing-induced cavitation under modest water stress (xylem pressure = -0.5 MPa), whereas species with larger diameter vessels (mean >40 μm) were nearly completely cavitated under the same conditions. Species with intermediate mean diameters (30-40 μm) showed partial cavitation by freezing. These results are consistent with a critical diameter of 44 μm at or above which cavitation would occur by a freeze-thaw cycle at -0.5 MPa. As expected, vulnerability to cavitation by freezing was correlated with the hydraulic conductivity per stem transverse area. The results confirm and extend previous reports that small-diameter conduits are relatively resistant to cavitation by freezing. It appears that the centrifuge method, modified to include freeze-thaw cycles, may be useful in separating the interactive effects of xylem pressure and freezing on cavitation.

Journal Article↗

The graviosmotic hypothesis of xylem transport of water in plants.

This paper presents a full version of the graviosmotic hypothesis postulated earlier in outline (Kargol 1978), which concerns xylem rise of water in plants. According to this hypothesis water is transported by xylem vessels (at respective development stages of these vessels) using the graviosmotic mechanisms. A detailed description of hypothesis is introduced by a discussion of the development stages of xylem vessels and a presentation of graviosmotic mechanisms postulated to be involved. These mechanisms include: convective graviosmosis and related effects, gravidiffusional graviosmosis, and osmotic transport aided by gravitational force in multi-membrane systems. The presented hypothesis does not contradict the theory of transpiration-cohesion or that of root pressure; rather, it is complementary to them.

Biological Transport, Active↗

Cytoskeletal organization during xylem cell differentiation.

The water and mineral conductive tube, the xylem vessel and tracheid, is a highly conspicuous tissue due to its elaborately patterned secondary-wall deposition. One constituent of the xylem vessel and tracheid, the tracheary element, is an empty dead cell that develops secondary walls in the elaborate patterns. The wall pattern is appropriately regulated according to the developmental stage of the plant. The cytoskeleton is an essential component of this regulation. In fact, the cortical microtubule is well known to participate in patterned secondary cell wall formation. The dynamic rearrangement of the microtubules and actin filaments have also been recognized in the cultured cells differentiating into tracheary elements in vitro. There has recently been considerable progress in our understanding of the dynamics and regulation of cortical microtubules, and several plant microtubule associated proteins have been identified and characterized. The microtubules have been observed during tracheary element differentiation in living Arabidopsis thaliana cells. Based on this recently acquired information on the plant cytoskeleton and tracheary element differentiation, this review discusses the role of the cytoskeleton in secondary cell wall formation.

Cell Differentiation↗

Mobilisation of nutrients and transport via the xylem sap in a shrub (Ligustrum ovalifolium) during spring growth: N and C compounds and interactions.

In open-field soilless culture there can be great deal of leaching, particularly in rainy springs. Ligneous plants have the capacity to store large quantities of nutrients in perennial organs. Knowledge of the plant's internal nutrient mobilisation during spring to supply growing organs could lead to reduction of fertiliser application. To quantify the fraction of storage mobilisation available for growth of new organs during spring, Ligustrum ovalifolium shrubs were grown for 2 years with or without fertilisation in the second spring. Nitrogen (N) absorption and N and carbon (C) mobilisation from storage were followed during spring growth via the sap quality. A mathematical combination of the sap composition with flow velocity provided the transported quantities of N and C. Nitrogen and C mobilisation towards new shoots took place during all the spring growth from bud break onwards. In unfertilised plants, C was mobilised primarily as sugars (stachyose, mannose and sucrose) and starch. In fertilised plants, the same sugars were transported in the xylem sap, but at lower concentrations. Stachyose concentration was lower in fertilised than in unfertilised plants and decreased during spring growth. Nitrogen was transported in the xylem sap mainly as amino acids in both fertilisation treatments. Glutamine was the predominant form at bud break and during shoot elongation. In fertilised plants, arginine became predominant after shoot elongation, and was related to low C availability. The interactions of N with C are discussed; specifically, insufficient availability of N limits the use of C, more of which is directed to aerial organs by sap flow.

Biological Transport↗

Long-term acclimatization of hydraulic properties, xylem conduit size, wall strength and cavitation resistance in Phaseolus vulgaris in response to different environmental effects.

Phaseolus vulgaris grown under various environmental conditions was used to assess long-term acclimatization of xylem structural characteristics and hydraulic properties. Conduit diameter tended to be reduced and 'wood' density (of 'woody' stems) increased under low moisture ('dry'), increased soil porosity ('porous soil') and low phosphorus ('low P') treatments. Dry and low P had the largest percentage of small vessels. Dry, low light ('shade') and porous soil treatments decreased P50 (50% loss in conductivity) by 0.15-0.25 MPa (greater cavitation resistance) compared with 'controls'. By contrast, low P increased P50 by 0.30 MPa (less cavitation resistance) compared with porous soil (the control for low P). Changes in cavitation resistance were independent of conduit diameter. By contrast, changes in cavitation resistance were correlated with wood density for the control, dry and porous soil treatments, but did not appear to be a function of wood density for the shade and low P treatments. In a separate experiment comparing control and porous soil plants, stem hydraulic conductivity (kh), specific conductivity (ks), leaf specific conductivity (LSC), total pot water loss, plant biomass and leaf area were all greater for control plants compared to porous soil plants. Porous soil plants, however, demonstrated higher midday stomatal conductance to water vapour (gs), apparently because they experienced proportionally less midday xylem cavitation.

Light↗

Protein extraction from xylem and phloem sap.

It is well known that phloem and xylem vessels transport small nutrient molecules over long distances in higher plants. The finding that proteins also occur in both transport fluids was unexpected, and the function of most of these proteins is not yet well understood. This chapter outlines how proteins can be obtained and purified from xylem and phloem saps to perform subsequent proteomic analyses.

Chemical Precipitation↗

Activation of carboxylesterases in root cortical parenchymal cells of Pisum sativum during xylem induction in vitro.

A quantitative cytochemical study of naphthol AS-D esterase activity in explants from roots of Pisum sativum grown on basal medium for 3 or 6 days showed similar levels of activity to those seen in sections of cortex and stele from intact roots of similar ages. Explants grown in the presence of auxins or cytokinin alone showed a threefold or twofold increase in cortical parenchymal activity, respectively. On adding both hormones to initiate xylem element formation, there was also a threefold increase in activity in the cortex. In all three cases, the stimulated activity was totally inhibited by either 10(-4) M diisopropylfluorophosphate (DFP) or 10(-4) M diethyl p-nitrophenylphosphate (E600), indicating carboxylesterase activity. The low level of activity normally present in cortical cells was inhibitor resistant, so indicating acetylesterase activity. Thus, auxins and cytokinins appear to activate mainly similar carboxylesterases during the initiation of xylem elements from cortical parenchyma cells.

2,4-Dichlorophenoxyacetic Acid↗

Expression of endo-1,4-beta-glucanase (cel1) in Arabidopsis thaliana is associated with plant growth, xylem development and cell wall thickening.

Arabidopsis thaliana CEL1 protein was detected in young expanding tissues. Immunostaining revealed that CEL1 accumulated mostly in xylem cells. The primary, as well as the secondary xylem showed considerable CEL1 staining. CEL1 was also observed in young epidermal cells, in which the thicker lateral and tangential walls stained more intensely than the inner walls. In newly formed cell walls, the lateral tangential walls were labeled more intensively than the inner walls. Cellulase activity was found to be significantly higher in growing tissue compared to mature parts of the plant. Cel1 expression concurrently with cellulase activity could be restored in detached matured leaves by sucrose treatment after 48 h in the culture medium.

Antibodies↗

Chromosomes associate premeiotically and in xylem vessel cells via their telomeres and centromeres in diploid rice ( Oryza sativa).

Studies of the meiosis of diploid plants such as Arabidopsis, maize and diploid progenitors of wheat have revealed no premeiotic association of chromosomes. Premeiotic and somatic association of chromosomes has only been previously observed in the anther tissues and xylem vessel cells of developing roots in polyploid plants such as hexaploid and tetraploid wheat, polyploid relatives of wheat and artificial polyploids made from the progenitor diploids of wheat. This suggested that this association was confined specifically to polyploids or was induced by polyploidy. However, we developed procedures for in situ hybridization on structurally well-preserved tissue sections of rice, and analysed two diploid rice species ( Oryza sativa and O. punctata). Contrary to expectation, this has revealed that centromeres and telomeres also associate both in the xylem vessel cells of developing root and in undifferentiated anther cells in these diploids. However, in contrast to wheat and related polyploids, where the initial association in undifferentiated anthers is between either non-homologous or related chromosomes, and not homologous chromosomes, the initial association of rice chromosomes seems to be between homologues. Thus, in contrast to the diploid dicot model Arabidopsis, meiotic studies on the diploid model cereal, rice, will now need to take into account the effects of premeiotic chromosome association.

Centromere↗

Localization of dibenzodioxocin substructures in lignifying Norway spruce xylem by transmission electron microscopy-immunogold labeling.

The lignification process in mature Norway spruce [ Picea abies (L.) H. Karsten] xylem cell walls was studied using transmission electron microscopy (TEM)-immunogold detection with a polyclonal antibody raised against a specific lignin substructure, dibenzodioxocin. The study reveals for the first time the exact location of this abundant eight-ring structure in the cell wall layers of wood. Spruce wood samples were collected in Southern Finland at the time of active growth and lignification of the xylem cell walls. In very young tracheids where secondary cell wall layers were not yet formed, the presence of the dibenzodioxocin structure could not be shown at all. During secondary cell wall thickening, the dibenzodioxocin structure was more abundant in the secondary cell wall layers than in the middle lamella. The highest number of gold particles revealing dibenzodioxocin was in the S2+S3 layer. Statistically significant differences were found in the frequency of gold particles present in various cell wall layers. For comparison, wood sections were also cut with a cryomicrotome for light and fluorescence microscopy.

Cell Wall↗

The dibenzodioxocin lignin substructure is abundant in the inner part of the secondary wall in Norway spruce and silver birch xylem.

A specific condensed lignin substructure, dibenzodioxocin, was immunolocalized in differentiating cell walls of Norway spruce ( Picea abies (L.) H. Karsten) and silver birch ( Betula pendula Roth) xylem. A fluorescent probe, Alexa 488 was used as a marker on the dibenzodioxocin-specific secondary antibody. For the detection of this lignin substructure, 25-microm cross-sections of xylem were viewed with a confocal laser-scanning microscope with fluorescein isothiocyanate fluorescence filters. In mature cells, fluorescence was detected in the S3 layer of the secondary wall in both tree species, but it was more intense in Norway spruce than in silver birch. In silver birch most of the signal was detected in vessel walls and less in fiber cell walls. In very young tracheids of Norway spruce and vessels and fibers of silver birch, where secondary cell wall layers were not yet formed, the presence of the dibenzodioxocin structure could not be shown.

Betula↗

Xylem-specific and tension stress-responsive coexpression of KORRIGAN endoglucanase and three secondary wall-associated cellulose synthase genes in aspen trees.

In nature, angiosperm trees develop tension wood on the upper side of their leaning trunks and drooping branches. Development of tension wood is one of the straightening mechanisms by which trees counteract leaning or bending of stem and resume upward growth. Tension wood is characterized by the development of a highly crystalline cellulose-enriched gelatinous layer next to the lumen of the tension wood fibers. Thus experimental induction of tension wood provides a system to understand the process of cellulose biosynthesis in trees. Since KORRIGAN endoglucanases (KOR) appear to play an important role in cellulose biosynthesis in Arabidopsis, we cloned PtrKOR, a full-length KOR cDNA from aspen xylem. Using RT-PCR, in situ hybridization, and tissue-print assays, we show that PtrKOR gene expression is significantly elevated on the upper side of the bent aspen stem in response to tension stress while KOR expression is significantly suppressed on the opposite side experiencing compression stress. Moreover, three previously reported aspen cellulose synthase genes, namely, PtrCesA1, PtrCesA2, and PtrCesA3 that are closely associated with secondary cell wall development in the xylem cells exhibited similar tension stress-responsive behavior. Our results suggest that coexpression of these four proteins is important for the biosynthesis of highly crystalline cellulose typically present in tension wood fibers. Their simultaneous genetic manipulation may lead to industrially relevant improvement of cellulose in transgenic crops and trees.

Arabidopsis Proteins↗

Water resource partitioning, stem xylem hydraulic properties, and plant water use strategies in a seasonally dry riparian tropical rainforest.

This study investigated seasonal variation in the origin of water used by plants in a riparian tropical rainforest community and explored linkages between plant water source, plant xylem hydraulic conductivity and response to the onset of dry conditions. The study focused on five co-dominant canopy species, comprising three tree species ( Doryphora aromatica, Argyrodendron trifoliolatum, Castanospora alphandii) and two climbing palms ( Calamus australis and Calamus caryotoides). Stable isotope ratios of oxygen in water (delta(18)O) from soil, groundwater, stream water and plant xylem measured in the wet season and the subsequent dry season revealed water resource partitioning between species in the dry season. Measurement of stem-area-specific hydraulic conductivity ( K(S)) in the wet season and subsequent dry season showed a significant dry-season loss of K(S) in three of the five species ( Castanospora alphandii, Calamus australis and C. caryotoides) and a decrease in mean K(S) for all species. This loss of hydraulic conductivity was positively correlated with the difference between wet-season and dry-season midday leaf water potentials and with leaf carbon isotope discrimination, indicating that plants that were less susceptible to loss of conductivity had greater control over transpiration rate and were more water-use efficient.

Adaptation, Physiological↗

Metabolite profiling reveals a role for atypical cinnamyl alcohol dehydrogenase CAD1 in the synthesis of coniferyl alcohol in tobacco xylem.

In angiosperms, lignin is built from two main monomers, coniferyl and sinapyl alcohol, which are incorporated respectively as G and S units in the polymer. The last step of their synthesis has so far been considered to be performed by a family of dimeric cinnamyl alcohol dehydrogenases (CAD2). However, previous studies on Eucalyptus gunnii xylem showed the presence of an additional, structurally unrelated, monomeric CAD form named CAD1. This form reduces coniferaldehyde to coniferyl alcohol, but is inactive on sinapaldehyde. In this paper, we report the functional characterization of CAD1 in tobacco (Nicotiana tabacum L.). Transgenic tobacco plants with reduced CAD1 expression were obtained through an RNAi strategy. These plants displayed normal growth and development, and detailed biochemical studies were needed to reveal a role for CAD1. Lignin analyses showed that CAD1 down-regulation does not affect Klason lignin content, and has a moderate impact on G unit content of the non-condensed lignin fraction. However, comparative metabolic profiling of the methanol-soluble phenolic fraction from basal xylem revealed significant differences between CAD1 down-regulated and wild-type plants. Eight compounds were less abundant in CAD1 down-regulated lines, five of which were identified as dimers or trimers of monolignols, each containing at least one moiety derived from coniferyl alcohol. In addition, 3-trans-caffeoyl quinic acid accumulated in the transgenic plants. Together, our results support a significant contribution of CAD1 to the synthesis of coniferyl alcohol in planta, along with the previously characterized CAD2 enzymes.

Alcohol Oxidoreductases↗