Phloem Fibers Research Articles

Caffeoyl-coenzyme A 3-O-methyltransferase enzyme activity, protein and transcript accumulation in flax (Linum usitatissimum) stem during development.

Flax (Linum usitatissimum) is a commercially important fiber crop in Europe. Lignification of its phloem fibers, although weak, causes a decrease in their commercial quality. In flax, fiber lignin mainly consists of guaiacyl (G) units in contrast to the mixed guaiacyl-syringyl (G-S) lignin type occurring in xylem fibers. G lignins are reported as more condensed polymers due to a higher frequency of 5-5 linkages, whereas the deposition of syringyl end groups in lignins increases the proportion of alky-aryl ether linkages as beta-O-4-bonds. The type of linkages within a lignin polymer depends on the methylation of either the 3-hydroxyl groups or both 3-OH and 5-OH groups, which is controlled by two enzymes: caffeate 3-O-methyltransferase (COMT) and caffeoyl-coenzyme A 3-O-methyltransferase (CCoAOMT). First, we measured the in vitro activity of both OMTs in the flax stem tissues during stem development. CCoAOMT activity varied in the same way as COMT, i.e. increased gradually with stem maturity, from the top to the bottom of the stem, was maximum at the flowering stage and was lower, but still scorable, in the outer fiber-bearing tissues than in the xylem cells. In a second step, we focused our studies on the characterization of CCoAOMT in order to understand the implication of this enzyme in the lignification of flax fiber cells. CCoAOMT activity appeared to be associated with the accumulation of an acidic 33-kDa polypeptide identified as a CCoAOMT after immunological cross-reactivity with a poplar CCoAOMT and microsequencing. The differential accumulation of the CCoAOMT protein was confirmed by immunolocalization on tissue prints and correlated with that of the transcripts, suggesting a transcriptional regulation of CCoAOMT in the flax stem.

Development of the vascular system in the inflorescence stem of Arabidopsis

Summary • The development of the vasculature in the inflorescence stem and pedicel of two common Arabidopsis thaliana ecotypes is reported, describing the tissues and events in primary and secondary growth of the stem vasculature. • Light and transmission electron microscopy was used to investigate the vascular system during plant growth; cellulose, lignin and callose were detected by cytochemistry. • The innermost cortical layer differentiated as a starch sheath during primary growth. The outermost parenchymatous layers of the interfascicular region lignified the walls at the onset of fascicular cambium activity. The secondary structure formed during silique production. In the internodes, the interfascicular cambium was mainly produced by starch sheath cells. In the interfascicular arcs of the internode, the secondary xylem mainly consists of xylem parenchyma whereas in the bundle region, it comprises vessels. Secondary phloem and xylary fibres were produced in limited quantities. Medullary sheath and phloem cap cells also became lignified. • The results show that the secondary vasculature develops in the stem and pedicel, and that, simultaneously, some primary tissues lignify, enhancing stem rigidity. Secondary vasculature is compared with the vascular alterations reported for some mutants.

The last step of syringyl monolignol biosynthesis in angiosperms is regulated by a novel gene encoding sinapyl alcohol dehydrogenase.

Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) has been thought to mediate the reduction of both coniferaldehyde and sinapaldehyde into guaiacyl and syringyl monolignols in angiosperms. Here, we report the isolation of a novel aspen gene (PtSAD) encoding sinapyl alcohol dehydrogenase (SAD), which is phylogenetically distinct from aspen CAD (PtCAD). Liquid chromatography-mass spectrometry-based enzyme functional analysis and substrate level-controlled enzyme kinetics consistently demonstrated that PtSAD is sinapaldehyde specific and that PtCAD is coniferaldehyde specific. The enzymatic efficiency of PtSAD for sinapaldehyde was approximately 60 times greater than that of PtCAD. These data suggest that in addition to CAD, discrete SAD function is essential to the biosynthesis of syringyl monolignol in angiosperms. In aspen stem primary tissues, PtCAD was immunolocalized exclusively to xylem elements in which only guaiacyl lignin was deposited, whereas PtSAD was abundant in syringyl lignin-enriched phloem fiber cells. In the developing secondary stem xylem, PtCAD was most conspicuous in guaiacyl lignin-enriched vessels, but PtSAD was nearly absent from these elements and was conspicuous in fiber cells. In the context of additional protein immunolocalization and lignin histochemistry, these results suggest that the distinct CAD and SAD functions are linked spatiotemporally to the differential biosynthesis of guaiacyl and syringyl lignins in different cell types. SAD is required for the biosynthesis of syringyl lignin in angiosperms.

Alfalfa stem tissues: rate and extent of cell-wall thinning during ruminal degradation

Alfalfa stem internodes of advanced maturity were used to examine the variability among tissues for rate and extent of cell-wall degradation by rumen microorganisms. Thin sections (100 μm) were incubated with rumen fluid in vitro for 0, 2, 4, 8, 16, 24, 48, 72, and 96 h. The degradation of tissue cell walls was evaluated for each fermentation time interval against a nondegraded mirror control section by light microscopy. Cell-wall thickness of alfalfa stem tissues was measured using scanning electron microscopy for both control and fermented sections. Rate and extent of cell-wall degradation were calculated from these measurements. Non-lignified epidermis, collenchyma, chlorenchyma, cambium and primary xylem parenchyma were rapidly and completely degraded within the first 8 h of fermentation. Rates of degradation ranged from 0.04 μm h −1 for thin-walled (0.29 μm) primary xylem parenchyma tissue to 0.11 μm h −1 for thick-walled (0.90 μm) collenchyma tissue. The non-lignified secondary wall (1.70 μm) of the primary phloem fibres required 24 h for complete degradation. Cell walls of some lignified tissues (e.g. pith parenchyma and secondary xylem fibres) were only partially degradable (9.1 to 65.5 %) even after 96 h of fermentation. The primary and secondary xylem vessels appeared to be completely nondegradable. The observed rates of cell-wall degradation for nonlignified alfalfa stem tissues were two to five times faster than previously estimated for nonlignified grass mesophyll tissue. However, extent of degradation for the lignified tissues of alfalfa stems was less than reported for lignified grass stem sclerenchyma. These differences in cell-wall degradation characteristics among tissues within alfalfa and compared to grasses are probably related to cell-wall lignification and polysaccharide composition of individual tissues.

The Brassica napus extA promoter: a novel alternative promoter to CaMV 35S for directing transgene expression to young stem tissues and load bearing regions of transgenic apple trees (Malus pumila Mill.)

The Brassica napus extensin A gene is highly expressed in root tissue of oilseed rape. In an attempt to identify an effective root-specific promoter for biotechnological applications, we have examined the ability of the −940 extA promoter to drive expression of the gusA reporter gene in the vegetative tissues of apple (Malus pumila Mill cv. Greensleeves). Transgenic apple lines were produced by Agrobacterium tumefaciens-mediated transformation and GUS activity was analysed both quantitatively and qualitatively. The extA promoter was active in all tissues of young plants in all 15 clones examined. However Southern blot data suggested that only a proportion of the population contained the entire promoter and that others had suffered deletions of unknown length. This may have contributed to the variation seen in the quantitative and qualitative expression of GUS. Specific GUS activity was highest in the stems where it approached, and in some clones, exceeded that using the constitutive CaMV 35S promoter. Histochemical analysis confirmed that GUS was localised to tissues involved in structural support of the stem. Staining was particularly intense at nodal junctions where high tensile stress is exerted on the tissues. Maturing phloem tissues showed localisation of expression to the phloem parenchyma cells and phloem fibres. Transverse sections of the root revealed staining of primary procambial tissues including the young endodermis but no staining was seen in the cortex. Although the −940 extA promoter is clearly not root-specific in apple, it is likely to have useful biotechnological applications in tree species.

Morphological basis for resistance in sugarcane to Pyrilla perpusilla Walker (Homoptera: Lophopidae)

The morphological basis of sugarcane resistance to the sucking pest Pyrilla perpusilla was studied across a wide genetic range of sugarcane cultivars. The observed mean relative growth rate (MRGR) obtained by confining first and third instar nymphs onto leaves was compared with variation in leaf spine density, thickness of phloem fibre tissues and distance to the phloem from the epidermis. These characteristics were found to determine the growth of first instars (84.4% of the variance in growth rate explained) and third instars (32% variance explained) to a variable degree. The most important characteristics for the antibiotic resistance were spine density and thickness of the phloem fibre layer in the minor vascular bundle, which together determined up to 82.8% of the variance of MRGR in the first instar. The antibiotic resistance is described by a regression model.

REACTION TISSUES IN GNETUM GNEMON A PRELIMINARY REPORT

Gnetum gnemon exhibits Rouxʼs model of tree architecture, with clear differentiation of orthotropic from plagiotropic axes. All axes have similar anatomy and react to displacement in the same way. Secondary xylem of displaced stems shows little eccentricity of development and no reaction anatomy. In contrast, there is considerable eccentricity in extra-xylary tissue involving both primary and secondary production of apparent tension-wood fibres (gelatinous fibres) of three main kinds. Narrow primary fibres occur concentrically in all axes in the outer cortex as a normal developmental feature. In displaced axes gelatinous fibres are developed abundantly and eccentrically on the topographically upper side, from pre-existing and previously undetermined primary cortical cells. They are wide with lamellate cell walls. In addition narrow secondary phloem fibres are also differentiated abundantly and eccentrically on the upper side of displaced axes. These gelatinous fibres are narrow and without obviously lamellate cell walls. Eccentric gelatinous fibres thus occupy a position that suggests they have the function of tension wood fibres as found in angiosperms. This may be the first report in a gymnosperm of fibres with tension capability. Gnetum gne-mon thus exhibits reaction tissues of unique types, which are neither gymnospermous nor angiospermous. Reaction tissues seem important in maintaining the distinctive architecture of the tree.

Anatomía comparada del tallo y de la raíz de las especies del género Neoevansia Marshall (Cactaceae)

Se estudiaron los caracteres anatómicos de las especies de Neoevansia con el objetivo de evaluar la contribución de la anatomía en la sistemática del género. Se analizaron caracteres de los sistemas dérmico, fundamental y vascular de tallo y raíz y compararon con los de algunas especies de Peniocereus, Wilcoxia y Echinocereus. Los resultados mostraron que N. striata se caracteriza por presentar hipodermis no colenquimatosa de un solo estrato y punteaduras intervasculares alternas en elementos de vaso del xilema secundario; mientras que N. lazaro-cardenasii y N. zopilotensis se distinguen anatómicamente entre sí por el tamaño de las células epidérmicas y el grosor de la hipodermis. El incremento en volumen en las raíces tuberosas se relaciona principalmente a las divisiones del parénquima radial. Neoevansia lazaro-cardenasii, N. zopilotensis y N. striata comparten con Peniocereus la presencia de madera fibrosa y fibras sobre el floema. Los resultados apoyan las propuestas de varios autores de transferir a las especies de Neoevansia al género Peniocereus.

Phenylcoumaran benzylic ether reductase, a prominent poplar xylem protein, is strongly associated with phenylpropanoid biosynthesis in lignifying cells.

It has previously been shown (D.R. Gang et al., 1999, J Biol Chem 274: 7516-7527) that the most abundant protein in the secondary xylem of poplar (Populus trichocarpa cv. 'Trichobel') is a phenylcoumaran benzylic ether reductase (PCBER), an enzyme involved in lignan synthesis. Here, the distribution and abundance of PCBER in poplar was studied at both the RNA and protein level. The cellular expression pattern was determined by immunolocalization of greenhouse-grown plants as well as of a field-grown poplar. Compared to other poplar tissues, PCBER is preferentially produced in the secondary xylem of stems and roots and is associated with the active growth period. The protein is present in all cells of the young differentiating xylem, corresponding to the zone of active phenylpropanoid metabolism and lignification. In addition, PCBER is located in young differentiating phloem fibers, in xylem ray parenchyma, and in xylem parenchyma cells at the growth-ring border. Essentially the same expression pattern was observed in poplars grown in greenhouses and in the field. The synthesis of PCBER in phenylpropanoid-synthesizing tissues was confirmed in a bending experiment. Induction of PCBER was observed in the pith of mechanically bent poplar stems, where phenylpropanoid metabolism is induced. These results indicate that the products of PCBER activity are synthesized mainly in lignifying tissues, suggesting a role in wood development.

A xylem-specific cellulose synthase gene from aspen (Populus tremuloides) is responsive to mechanical stress.

Angiosperm trees accumulate an elevated amount of highly crystalline cellulose with a concomitant decrease in lignin in the cell walls of tension-stressed tissues. To investigate the molecular basis of this tree stress response, we cloned a full-length cellulose synthase (PtCesA) cDNA from developing xylem of aspen (Populus tremuloides). About 90% sequence similarity was found between the predicted PtCesA and cotton GhCesA proteins. Northern blot and in situ hybridization analyses of PtCesA gene transcripts in various aspen tissues, and PtCesA gene promoter-beta-glucuronidase (GUS) fusion analysis in transgenic tobacco, demonstrated conclusively that PtCesA expression is confined to developing xylem cells during normal plant growth. During mechanical stress induced by stem bending, GUS expression remained in xylem and was induced in developing phloem fibers undergoing tension stress, but was turned off in tissues undergoing compression on the opposite side of the bend. Our results suggest a unique role for PtCesA in cellulose biosynthesis in both tension-stressed and normal tissues in aspen, and that the on/off control of PtCesA expression may be a part of a signaling mechanism triggering a stress-related compensatory deposition of cellulose and lignin that is crucial to growth and development in trees.

Anatomical structure of stem in the above- and underground portions of Rosa rugosa suckers

Comparitive histological studies were made on the underground and the aboveground stem parts of Rosa rugosa taken from one year old suckers. The underground stem parts were characterized with thicker primary cortex, phloem and pith, weaker phloem fibers, wider cambial zone, medullary rays, xylem and phloem rays as compared with the aboveground stem parts. The most marked differences in the underground stem parts were in the wide cambial zone and in the development of some adventitious roots.

SIMS imaging of Ca and Na, and effect of the ionic interaction Ca/Na on wall maturation of phloem fibres and xylem cells of hypocotyls of beech seedlings

Using SIMS microscopy an unexpected ionic interaction between calcium and sodium has been characterised within the walls of the xylem cells and of the phloem fibres of hypocotyls of beech seedlings: whereas a decrease of the Na signal in the cell walls was always paralleled by an increase of the calcium signal at the same site, unexpectedly an increase of the normalised Na signal in the cell walls of the Naenriched seedlings was paralleled by an increase of the Ca signal. We have also shown that this interaction was involved in the differentiation of the secondary walls.

Developmentally regulated patterns of expression directed by poplar PAL promoters in transgenic tobacco and poplar.

Phenylalanine ammonia-lyase (PAL) catalyzes the first step in phenylpropanoid metabolism and plays a central role in the biosynthesis of phenylpropanoid compounds. We have previously cloned two PAL genes, PALI and PAL2, from a Populus trichocarpa x P. deltoides F1 hybrid. Here, we describe the properties of PALI and PAL2 promoters and their expression patterns in transgenic tobacco and poplar. The promoters were 75% identical in the regions sequenced, and each contained two copies of AC-rich putative cis-acting elements that matched a consensus plant myb transcription factor binding site sequence. In transgenic tobacco, PALI-GUS and PAL2-GUS fusions directed similar patterns of expression in developing primary xylem of leaves, stems, and other organs, and in secondary xylem of stems. Contrary to previously documented patterns of PAL1/2 expression in poplar, no expression of either fusion was detected in epidermal or subepidermal cell layers of young tobacco leaves or stems. In poplar, the PAL2-GUS fusion directed the highest levels of expression in roots and young leaves and stems. In young leaves and stems, high GUS activity was detected in epidermal or subepidermal cells as well as in primary xylem and phloem fibers. GUS activity was low in woody stems, and was weak or absent in developing secondary xylem. The patterns of PAL2-GUS expression in poplar are very similar to those of PAL1/2 mRNA accumulation in poplar. However, the distinct patterns of expression directed by the PAL2 promoter in poplar and tobacco show that PAL2-GUS expression in tobacco does not accurately reflect all aspects of PAL2 expression in poplar.

Variability of Bark Structure in Plantation-Grown Eucalyptus Globulus

The bark structure of Eucalyptus globulus Labill. grown in plantations in Central Portugal is described, based on specimens extracted at six height levels from ten 15-year-old trees. No significant variation of qualitative features between trees was observed. The non-collapsed phloem is characterised by multiseriate tangential rows of phloem parenchyma alternating with rows of phloem fibres, interspersed with large sieve tubes and their respective companion cells, and uniseriate rays . With the onset of sieve tube collapse (collapsed phloem ), some parenchyma cells expand and sclerify, the course of rays becomes irregular, and ray dilatation is initiated. The periderm is composed of a phellem of lignified cells with horseshoe thickening (phelloids), followed by a layer of cells with suberised tangential walls, and a phelloderm with a variable number of layers of thin-walled cells. Age-related secondary changes give rise to a specific within-tree pattern of axial variation. Both the intensity of sclerification of phloem parenchyma cells and the degree of ray dilatation increase with tree age.

Anatomy of relictual members of Subfamily Cactoideae, IOS Group 1a (Cactaceae)

The anatomy of IOS Group la (part of Leptocereeae and part of Echinocereeae) was examined to determine if these genera have characters that are relictual in Cactoideae. Echinocereus lacks fibrous wood and seemed distinct from genera with fibrous wood, which constitute the majority of Group la. Excluding Echinocereus and other genera with non-fibrous wood, Group la has many features presumed to be relictual: epidermal cells with ordinary shapes and thin walls, a tendency to have patches of multiseriate epidermis, hypodermis of a few layers with walls of medium thickness, cortical bundles with caps of primary phloem fibers but lacking clusters of terminal tracheids, presence of medullary bundles (and these have xylary fibers), fiber caps next to primary phloem of the stele, presence of fibrous wood and lack of any derived wood types. Derived characters that do occur are not universally present or even widespread in the group. Little evolutionary modification has occurred in Group la, except for Echinocereus.

Multiple forms of extracellular superoxide dismutase in needles, stem tissues and seedlings of Scots pine

which is involved in lignification of cell walls (Dean and Eriksson, 1992), defence reactions against microbial infecExtracellular superoxide dismutase was found in extra- tions (Levine et al., 1994) and cross-linking of structural xylematic bark tissues and seedlings of Pinus syl- proteins of the cell wall (Bradley et al., 1992). Its generavestris L. The isozymes were partially purified by tion in cell walls of diVerentiating xylem and in cell walls infiltration and analysed by polyacrylamide gel electro- of phloem fibres has been demonstrated with histochemphoresis. One of the major isoforms was purified to ical assays (Cassab, 1993; Czaninski et al., 1993). homogeneity and the N-terminal amino acid sequence Superoxide dismutase (SOD; EC1.15.1.1), which catawas determined. The isoelectric point of these extra- lyses the dismutation of superoxide anion radicals (O’ 2 )

Taxonomic significance of extracellular crystals on the phloem fibres of Taxaceae

Summary A preliminary study of extracellular crystals deposited on the outer surface of phloem fibres in 12 species belonging to 5 genera of Taxaceae has shown that the occurrence and characteristics of crystals are of taxonomic significance. Lack of crystals on the outer surface of phloem fibres in Austrotaxus suggests that it is necessary to raise it as a separate tribe under Taxaceae. Based on differences in the nature of the crystals in the other four genera of Taxaceae, three distinct groups were recognized. Therefore, it seems more appropriate to divide the family into four tribes Taxeae, Amentotaxeae and Torreyaxeae as well as Austrotaxeae.

Immunolocalization of cinnamyl alcohol dehydrogenase 2 (CAD 2) indicates a good correlation with cell-specific activity of CAD 2 promoter in transgenic poplar shoots.

Cinnamyl alcohol dehydrogenase 2 (CAD 2) localization and the cell-specific activity of the eucalyptus CAD 2 promoter were investigated by CAD 2 immunogold localization and promoter beta-glucuronidase (GUS) histochemistry in apical and mature parts of stable transformed poplar (Populus tremula x P. alba) stems. Both CAD 2 protein and GUS activity were found to be confined in the same types of cells in the shoot apices, particularly in the determined meristematic cells in leaf axils and shell zones, procambium and developing tracheids. Within mature stems, CAD 2 and GUS were also identified in cambium and in fully or partially lignified cells derived from it (young xylem, developing phloem fibres, chambered parenchyma cells around phloem). Additionally, GUS activity was found in the scale leaves of apical shoot buds and in the roots (namely in the procambium, cambium, phellogen, young xylem, pericycle) of transformed plants. By employing immunogold cytochemistry, CAD 2 was shown to be localized in the cytoplasm within cambial, ray and young xylem cells in stems, the gold particles being randomly attached to endoplasmic reticulum and Golgi-derived vesicles. These results support a crucial role for CAD 2 in lignification and indicate a new role for this enzyme in branching events within the shoot apex and during lateral root formation.

Montigena (Fabaceae), a new genus endemic to New Zealand

Montigena, a new monotypic genus, is distinguished from Swainsona by a strongly rhizomatous habit, terminal innovation shoots, stems with persistent pith, vessel elements with helical thickenings, well developed ray parenchyma, and slender and flexuose phloem fibres. A new combination Montigena novae‐zelandiae replaces Swainsona novae‐zelandiae as the name for this legume that is endemic to the dry eastern mountains of the South Island of New Zealand, where it grows on partially stable screes. A botanical description is presented, followed by detailed notes on growth habit, rhizome anatomy, leaf anatomy, distribution, and habitats, with an assessment of conservation status.

Wood and Bark Anatomy of Caricaceae; Correlations with Systematics and Habit

Wood and bark anatomy are described for four species of three genera of Caricaceae; both root and stem material were available for Jacaratia hassleriana. Wood of all species lacks libriform fibers in secondary xylem, and has axial parenchyma instead. Cylicomorpha parviflora has paratracheal parenchyma cells with thin lignified walls; otherwise, all cell walls of secondary xylem in Caricaceae except those of vessels have only primary walls. Vessels have alternate laterally elongate (pseudoscalariform) pits on vessel-vessel interfaces, but wide, minimally bordered scalariform pits on vessel-parenchyma contacts. Laticifers occur commonly in tangential plates in fascicular secondary xylem, and rarely in xylem rays. Proliferation of axial parenchyma by zones of tangential divisions is newly reported for the family. Bark is diverse in the species, although some features (e.g., druses) are common to all. Wood of Caricaceae is compared to that of two species of Moringaceae, recently designated the sister family of Caricaceae. Although the wood and bark of Moringa oleifera, a treelike species, differ from those of Caricaceae, wood and bark of the stem succulent M. hildebrandtii, the habit of which resembles those in Caricaceae, simulate wood and bark of Caricaceae closely. Counterparts to laticifers in Moringaceae are uncertain, however. Phloem fibers of Caricaceae form an expansible peripheral cylinder of mechanical tissue that correlates with the stem succulence of most species of Caricaceae.