Ascorbic acid and xylem development in trunks of the Siberian larch trees

The contents of ascorbic acid (AA) and its oxidized form, dehydroascorbic acid (DHA), were assessed as related to the tracheid differentiation in the course of early and late wood development in the Siberian larch (Larix sibirica Ldb.) trees. The samples of the cambium, cell enlargement zone and mature cells were collected at the successive developmental stages by scraping tissues off layer by layer from trunk segments of the 20-year-old trees according to anatomical and histochemical criteria. While cambium initials were rapidly dividing, the AA contents per dry weight and per cell considerably exceeded the corresponding values characteristic of the late xylem development; such difference corresponded to the higher number of early tracheids per annual ring, as compared to the late tracheids. The AA content decreased as cells enlarged. The radial growth of the early wood tracheids, as compared to the late wood tracheids, was accompanied with a threefold increase in the AA and a decline in the DHA contents. The AA/DHA ratio was in line with the early tracheid enlargement. The maximum AA content was observed at the early stage of the secondary cell wall thickening in the tracheids of early and late xylem preceding lignification. During this stage of early wood development, the DHA content exceeded sixfold the corresponding value in the late xylem; as a result, the initial rates of lignification were different in two tissues. The rate of lignification in a newly developing layer of the early xylem increased gradually and was the highest in the completely differentiated tracheids. In the late xylem, the lignification rate was at its highest at the very beginning and then declined in the course of tracheid maturation. The dissimilar patterns of lignification in the early and late xylem were primarily associated with the DHA content, which increased in the early xylem and decreased in the maturing late xylem. Thus, the AA content and its accessibility to oxidation in the growing and mature xylem cells exhibited the diverse developmental patterns in the early and late xylem: two tissues differed in the tracheid number and radial diameter as well as in the rate of lignification.Translated from Fiziologiya Rastenii, Vol. 52, No. 1, 2005, pp. 97–107.Original Russian Text Copyright © 2005 by Antonova, Chaplygina, Varaksina, Stasova.     

Changes in phenolic acids during maturation and lignification of scots pine xylem

The content and fractional composition of alcohol soluble phenolic acids (PhA) in cells with different degree maturation and lignification in the course of early and late wood formation in the pine (Pinus sylvestris L.) stem during vegetation were studied. Phenolic compounds (PhC), extracted by 80% ethanol, were divided into free and bound fractions of PhA. In turn, the esters and ethers were isolated from bound PhA. The contents of all substances were calculated per dry weight and per cell. Considerable differences have been found to exist in both the contents and the composition of the fractions PhA on successive stages of tracheid maturation of early and late xylem. Early wood tracheids at all secondary wall thickening steps contained PhC less and free PhA more than late wood tracheids. Throughout earlywood tracheid maturation, the pool of free PhA per cell declined at the beginning of lignification and then increased gradually while that of bound PhA decreased. The maturation of late wood tracheids were accompanied by the rise of free PhA pool and the diminution of bound PhA pool. In the composition of bound PhA, the ethers were always dominant, and the amount of that in earlywood cells was less than in latewood cells. The cells of early xylem at all steps of maturation contained more of esters. The sum total of free hydroxycinnamic acids, precursors of monolignols, gradually decreased during early xylem lignification as the result of the reduction of the pools of p-coumaric, caffeic, ferulic and synapic acids, while that of their esters rised. In the course of late xylem lignification, the pools of free p-coumaric, ferulic and, especially, synapic acids increased. Simultaneously, the amount of ferulic acid ester and synapic acid ether increased too. According to the data, lignin biosynthesis in early xylem and late xylem occurs with different dynamics and the structure of lignins of two xylem types might be different too.     

Seasonal development of phloem in scots pine stems

The formation of phloem was studied for two years in stems of 50 to 60 year old trees of Scots pine (Pinus sylvestris L.) growing in nature. The development of phloem of the current year begins 10 to 20 days before the xylem formation and is completed with the termination of shoot growth in the end of June. Observations over the seasonal activity of cambium producing sieve cells of phloem and duration of their differentiation as compared to the xylem derivatives of cambium have shown that the maxima of formation of phloem and xylem cells could coincide or not coincide by season, while the activities of their differentiation were always in antiphase. The sieve cells of early phloem were separated from those of late phloem by a layer of tannin-containing cells, which are formed simultaneously with the formation of late xylem cells by the cambium. Seasonal dynamics of accumulation of starch grain in structural elements of the phloem is related to the xylem development. The content of metabolites in differentiating and mature phloem elements, in the cambium zone, and in the xylem cells growing in the radial direction depended on cell specificity, stage of their development, and type of forming wood, early or late, which differ in the cell wall parameters and, hence, requirement of assimilates. Significant differences were described between the content of low molecular weigh carbohydrates, amino acids, organic acids, and phenol compounds using two methods of calculation: per dry weight and per cell.     

Seasonal development of phloem in Scots pine stems

The formation of phloem was studied for two years in stems of 50 to 60 year old trees of Scots pine (Pinus sylvestris L.) growing in nature. The development of phloem of the current year begins 10 to 20 days before the xylem formation and is completed with the termination of shoot growth in the end of June. Observations over the seasonal activity of cambium producing sieve-like cells of phloem and duration of their differentiation as compared to the xylem derivatives of cambium have shown that the maxima of formation of phloem and xylem cells could coincide or not coincide by season, while the activities of their differentiation were always at antiphase. The sieve-like cells of early phloem were separated from those of late phloem by a layer of tannin-containing cells, which are formed simultaneously with the formation of late xylem cells by the cambium. Seasonal dynamics of accumulation of starch grain in structural elements of the phloem is related to the xylem development. The content of metabolites in differentiating and mature phloem elements, in the cambium zone, and in the xylem cells growing in the radial direction depended on cell specificity, stage of their development, and type of forming wood, early or late, which differ in the cell wall parameters and, hence, requirement of assimilates. Significant differences were described between the content of low molecular weigh carbohydrates, amino acids, organic acids, and phenol compounds using two methods of calculation: per dry weight and per cell.     

Seasonal development of phloem in Siberian larch stems

The seasonal development of phloem in the stems of Siberian larch (Larix sibirica Ldb.) was studied over two seasons on 50–60-year-old trees growing in a natural stand in the Siberian forest-steppe zone. Trees at the age of 20–25 years were used to study metabolites in differentiating and mature phloem elements, cambial zone, and radially growing xylem cells in the periods of early and late wood formation. The development of the current-year phloem in the stems of 50–60-year-old trees started, depending on climatic conditions, in the second-third decades of May, 10–20 days before the xylem formation, and ended together with the shoot growth cessation in late July. Monitoring of the seasonal activity of cambium producing phloem sieve cells and the duration of their differentiation compared to the xylem derivatives in the cambium demonstrated that the top production of phloem and xylem cells could coincide or not coincide during the season, while their differentiation activity was always in antiphase. Sieve cells in the early phloem are separated from those in the late phloem by a layer of tannin-containing cells, which are formed in the period when late xylem formation starts. The starch content in the structural elements of phloem depends on the state of annual xylem layer development. The content of low molecular weight carbohydrates, amino acids, organic acids, and phenols in phloem cells, cambial zone, and xylem derivatives of the cambium depends on the cell type and developmental stage as well as on the type of forming wood (early or late) differing by the cell wall parameters and, hence, by the requirement for assimilates. Significant differences in the dynamics of substances per dry weight and cell were observed during cell development.     

Changes in content and composition of phenolic acids during growth of xylem cells of Scots pine

The content and composition of alcohol soluble phenolic acids (PhAs) were studied during cell xylem growth in course of wood annual increment formation in the trunks of Scots pine. Cells of the cambium zone, two stages of expansion growth, and outset of secondary thickening zone (before lignification) within the period of formation of early wood xylem were subsequently isolated from trunk segments of 25-year-old trees with constant anatomical and histochemical control. The amount of free and bound forms of phenolic acids extracted from tissues by 80% ethanol, as well as their ethers and esters, were calculated both per dry weight and per cells. The substantial alteration in content, proportion of fractions and composition of acids has been found between the cambium zone and the outset of secondary thickening of tracheids, and the character of variation depended on the calculation method. The amount of free and bound PhAs and esters and especially ethers calculated per cell had increased at the first stage of extension growth, reduced at the second, and increased in the outset of secondary wall deposition. The pool of bound acids was more than acids by 2–5 times depending on the stage of development of the cells. Sinapic and ferulic acids dominate among free hydroxycinnamic acids. The composition and the content of hydroxycinnamic acids in esters and ethers also depended on the stage of development of the cells. p-Coumaric and sinapic acids were the main aglycons in ethers in the cambium and sinapic and caffeic acids were in the other stages. The esters from cambium included mostly p-coumaric acid and those at other stages of development were sinapic and ferulic acids. The esters included benzoic acid at the first stages of growth. The pool of these esters decreased from the first phase of growth until the outset of cell wall thickening. The level of free benzoic acid increased respectively.     

Alfalfa Stem Tissues: Cell-wall Development and Lignification

Alfalfa stems contain a variety of tissues with different patternsof cell-wall development. Development of alfalfa cell wallswas investigated after histochemical staining and with polarizedlight using light microscopy and scanning electron microscopy.Samples of the seventh internode, from the base of stems grownon cut stems, were harvested at five defined stages of developmentfrom early internode elongation through to late maturity. Internodeseven was elongating up to the third sample harvest and internodediameter increased throughout the entire sampling period. Chlorenchyma,cambium, secondary phloem, primary xylem parenchyma and pithparenchyma stem tissues all had thin primary cell walls. Pithparenchyma underwent a small amount of cell-wall thickeningand lignification during maturation. Collenchyma and primaryphloem tissues developed partially thickened primary walls.In contrast to a recent report, the formation of a ring shaped,lignified portion of the primary wall in a number of cells inthe exterior part of the primary phloem was found to precedethe deposition of a thick, non-lignified secondary wall whichwas degradable by rumen microbes. In numerous xylem fibres fromthe fourth harvest date onwards, an additional highly degradablesecondary wall layer was deposited against a previously depositedlignified and undegradable secondary wall. The pattern of lignificationobserved in alfalfa stem tissues suggests that polymerizationof monolignols by peroxidases at the luminal border of the primarycell wall creates an impermeable zone which restricts lignificationof the middle lamella region of tissues with thick primary walls.Copyright1998 Annals of Botany Company Alfalfa,Medicago sativaL., stem tissue, cell wall, development, lignification, degradation.     

Periodicity of Cambial Activity and Changes of Starch Content in Broussonetia papyrifera

Methods of sampling and sections preparaction were the same as reported previously. Except that sampling was made at monthly intervals between May 20 and July 30, then at 7–14 day-intervals between July 30 and October 14, and then at monthly intervals between October 14 and March 25 in the next year. The stored starch in various tissues was stained with PAS reaction. During active period of cambium in Broussonetia papyrifera after July 30, the cell layers of immature xylem and phloem decreased progressively, and the formation of mature xylem and phloem increased rapidly. The formation of late wood started early in August, formation of xylem ceased after September 5, followed by ceasation of phloem formation about 1.5 months later. Increasing and decreasing of stored starch were closely related to the periodicity of cambial activity during the year. Starch grains decreased progressively after cambial activity was resumed in early spring until they disappeared in all the stem tissues. Then, starch accumulated progressively again after cambial activity slowed down, particularly after the ceasation of xylem formation. However, after the formation of phloem had ceased, the stored starch once again disappeared progressively until the end of December, and accumulated again. Such changes might be related to the transition of cambium activity involving two periods of dormancy.     

Changes of Peroxidase and Estease Isozymes During Periodicity of Cambial Activity in Broussonetia papyrifera (L.) Vent.

Methods of sampling and sections preparaction were the same as reported previously. Except that sampling was made at monthly intervals between May 20 and July 30, then at 7–14 day-intervals between July 30 and October 14, and then at monthly intervals between October 14 and March 25 in the next year. The stored starch in various tissues was stained with PAS reaction. During active period of cambium in Broussonetia papyrifera after July 30, the cell layers of immature xylem and phloem decreased progressively, and the formation of mature xylem and phloem increased rapidly. The formation of late wood started early in August, formation of xylem ceased after September 5, followed by ceasation of phloem formation about 1.5 months later. Increasing and decreasing of stored starch were closely related to the periodicity of cambial activity during the year. Starch grains decreased progressively after cambial activity was resumed in early spring until they disappeared in all the stem tissues. Then, starch accumulated progressively again after cambial activity slowed down, particularly after the ceasation of xylem formation. However, after the formation of phloem had ceased, the stored starch once again disappeared progressively until the end of December, and accumulated again. Such changes might be related to the transition of cambium activity involving two periods of dormancy.     

Differentiation Between Tissues from Carnation (Dianthus caryophyllus) Stems by Pyrolysis-Mass Spectrometry

Small pieces of different tissues from stems of young and oldcarnation plants were analyzed for lignification (lignin/celluloseratios) and lignin composition by means of pyrolysis-(gas chromatography)-massspectrometry. The epidermis and phloem of young and old stemswere essentially non-lignified. Pith parenchyma was only lignifiedin mature and senescing tissues. The type of lignin in sclerenchymadiffered from that in xylem and pith. Lignification in the xylemof very young tissues was a mainly guaiacyl-type lignin, whichgradually changed into a mixed guaiacyl-syringyl lignin in oldertissues. In mature tissues, the sclerenchyma was more highlylignified than the xylem. All tissues yielded comparatively large amounts of dihydroferulicacid, a compound which may be specific for carnation. Carnation, Dianthus caryophyllus, epidermis, cortex, sclerenchyma, phloem, xylem, pith, lignification, aging, dihydroferulic acid, pyrolysis-(gas chromatography)-mass spectrometry     

构树形成层的活动周期及其淀粉贮量的变化

在构树（Ｂｒｏｕｓｓｏｎｅｔｉａ ｐａｐｙｒｉｆｅｒａ （Ｌ．） Ｖｅｎｔ．）形成层活动周期中,每年７月末以后,未成熟的木质部和韧皮部逐渐减少,成熟的木质部和韧皮部急剧增多。８月初开始分化晚材。进入９月后木质部的形成逐渐停止,而一个半月以后才停止形成韧皮部。淀粉贮量的消长与形成层的活动周期有很强的相关关系。早春形成层恢复活动后,淀粉贮量逐渐减少直至消失。尔后,形成层活动减慢,特别是木质部分化停止后,淀粉又开始积累。当韧皮部分化也停止后,淀粉又消失,直至翌年１月才重新积累,这似乎与两个休眠期的转化有关     

Changes of Polysaccharide Grain Content and Amylase Isozyme in Relation to Periodicity of Cambial Activity in Pinus bungeana Shoot

The cambium of Pinus bungeana Zucc. resumed its activities in early April with cell proliferation and increase in immature xylem and phloem cells. Some mature xylem cells occurred dunng the last ten days of April. The xylem and phloem were rapidly formed after May. The late- wood was firstly formed in the beginning of June. It ceased to produce new xylem in early August, mid phloem cells in mid-September. The seasonal changes of polysaccharide grain content in the tissues of P. bungeana evidenced significant correlation with the annual cycle of cambial activity. Polysaccharide grains continued to increase before and after cambial reactivity and then decreased gradaally from June onwards after the late-wood had been firstly formed, until almost disappeared by next January, and again were gradually accumulated after March. Isoenzymic study revealed only one band of amylase after cambium reactivity, three peculiar bands after ceasing to produce xylem, and another two peculiar bands that occurred in early December. These 5 bands all disappeared after reactivity of cambium.     

Fractionation and polypeptide analysis of phloem tissue of Pinus sabiniana Dougl.

The polypeptide complement of phloem tissue and xylem tissue in trunks of Pinus sabiniana collected in spring was compared by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Two polypeptides at 24 and 25 kdalton represented over 20% of the total polypeptide complement of the phloem but were absent in xylem tissue. In an attempt to relate the 24- and 25-kdalton phloem polypeptides (PPP) to cellular constitutents, phloem tissue was fractionated by sequential differential and density-gradient centrifugation utilizing the PPP as biochemical markers. In borate buffer, the fractions containing PPP pelleted at less than 12,000 g and were subsequently enriched in sucrose gradients at densities greater than 1.22. However, the cytological entities containing the PPP were almost completely dissociated when phloem tissue was processed with a 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris) buffer containing mercaptoethanol, and the PPP were then found in the supernatant of material spun at 45,000 g for 3 h. In electron micrographs of PPP-enriched fractions processed with borate buffer an assortment of structures that are associated with mature sieve cells of pine were found, including filaments, cup-shaped arrays, polyhedral crystals, and paracrystalline bodies. Similar structures were not found in identically processed xylem tissue or in phloem tissue processed with the Tris buffer. It is suggested that the PPP represent a proteinaceous component common to these phloem structures.     

An anatomical study of the haustoria of Rhinanthus minor attached to roots of different hosts

Roots of a range of potential hosts responded differently when Rhinanthus minor attempted to form haustoria. Roots of Fabaceae show the weakest reaction as apart from slight lignification, no reaction was observed at the interface between the endophyte and the cortical tissue of the host root. Grass roots react with strong lignification of all cells within the stele with the exception of a small number of phloem cells whilst the endodermis fully enters the tertiary stage. In the case of Phleum bertolonii the cortical cells also become lignified. The lignification is even observed in the host root tissue in a distance of about 1 mm from the haustorium (both apically and basipetally). In the case of Leucanthemum vulgare, strong suberisation can be observed in the cell walls of the interface between endophyte (tip of the sucker) and host. Plantago lanceolata exhibits the strongest reactions against the haustorial tissues. Cells of the interface between the endophyte and the host cortex are completely destroyed, as well as a few cell layers outside the central xylem cylinder, even in some distance from the haustorium. Thus, host xylem is completely isolated from the haustorium in this case. Extraction of sap from xylem vessels is likely to be drastically impaired in such a situation.     

Distribution of an Indoleacetic Acid-oxidase-inhibitor in the Storage Root of Daucus carota

Indoleacetic acid (IAA)-oxidase from both secondary phloem and xylem was dependent on 2,4-dichlorophenol for activity, and was enhanced by addition of Mn²⁺. The pH optimum was 6.0 from both tissues. IAA-oxidase and its inhibitors were distributed differently in the secondary phloem and secondary xylem of carrot root. In the phloem a high IAA-oxidase activity was distributed uniformly along the radius but in the xylem a somewhat lower concentration decreased from the cambium. IAA-oxidase inhibitor in the phloem increased exponentially from a very low concentration near the cambium, whereas in the xylem an appreciable concentration was present near the cambium, decreasing linearly with distance from the cambium. Longitudinal gradients in the xylem parallel studies by other workers with the greatest IAA-destroying capacity present in older tissues. In the xylem inhibitor decreased and IAA-oxidase increased from the root apex. In the phloem IAA-oxidase was uniform, whereas the inhibitor increased in older tissue.

The IAA-oxidase inhibitors in phloem and xylem may be different. In the xylem the IAA-oxidase inhibitor may be a lignin precursor present in young cells which disappears as lignification proceeds. In the phloem IAA-oxidase reacting with endogenous IAA appears to form a physiologically active product.

     

Isolation and characterization of a Pinus radiata lignin biosynthesis-related O-methyltransferase promoter

A putative promoter fragment of a Pinus radiata gene encoding a multi-functional O-methyltransferase (AEOMT) was isolated from genomic DNA. Sequence analysis revealed a number of putative cis elements, including AC-rich motifs common in promoters of genes related to the phenylpropanoid pathway. The isolated promoter was fused to the GUS reporter gene and its expression profile analyzed in transgenic tobacco and in transient transformation experiments with P. radiata embryogenic and xylogenic tissue. The promoter conferred weak expression in embryogenic tissue but caused strong GUS activity in both ray parenchyma cells and developing tracheary elements of xylem strips. Histochemical analysis in transgenic tobacco plants revealed that the AEOMT promoter induced GUS expression in cell types associated with lignification, such as developing vessels, phloem and wood fibers and xylem parenchyma as well as in non-lignifying phloem parenchyma. The isolated promoter was activated by challenge of the tissue with a fungal pathogen. Our results also indicate that the control of lignin-related gene expression is conserved and can be compared in evolutionarily distant species such as tobacco and pine.     

Localization of cell wall polysaccharides in normal and compression wood of radiata pine: relationships with lignification and microfibril orientation

The distribution of noncellulosic polysaccharides in cell walls of tracheids and xylem parenchyma cells in normal and compression wood of Pinus radiata, was examined to determine the relationships with lignification and cellulose microfibril orientation. Using fluorescence microscopy combined with immunocytochemistry, monoclonal antibodies were used to detect xyloglucan (LM15), β(1,4)-galactan (LM5), heteroxylan (LM10 and LM11), and galactoglucomannan (LM21 and LM22). Lignin and crystalline cellulose were localized on the same sections used for immunocytochemistry by autofluorescence and polarized light microscopy, respectively. Changes in the distribution of noncellulosic polysaccharides between normal and compression wood were associated with changes in lignin distribution. Increased lignification of compression wood secondary walls was associated with novel deposition of β(1,4)-galactan and with reduced amounts of xylan and mannan in the outer S2 (S2L) region of tracheids. Xylan and mannan were detected in all lignified xylem cell types (tracheids, ray tracheids, and thick-walled ray parenchyma) but were not detected in unlignified cell types (thin-walled ray parenchyma and resin canal parenchyma). Mannan was absent from the highly lignified compound middle lamella, but xylan occurred throughout the cell walls of tracheids. Using colocalization measurements, we confirmed that polysaccharides containing galactose, mannose, and xylose have consistent correlations with lignification. Low or unsubstituted xylans were localized in cell wall layers characterized by transverse cellulose microfibril orientation in both normal and compression wood tracheids. Our results support the theory that the assembly of wood cell walls, including lignification and microfibril orientation, may be mediated by changes in the amount and distribution of noncellulosic polysaccharides.     

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.     

Gradients and dynamics of inner bark and needle osmotic potentials in Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies L. Karst)

Preconditions of phloem transport in conifers are relatively unknown. We studied the variation of needle and inner bark axial osmotic gradients and xylem water potential in Scots pine and Norway spruce by measuring needle and inner bark osmolality in saplings and mature trees over several periods within a growing season. The needle and inner bark osmolality was strongly related to xylem water potential in all studied trees. Sugar concentrations were measured in Scots pine, and they had similar dynamics to inner bark osmolality. The sucrose quantity remained fairly constant over time and position, whereas the other sugars exhibited a larger change with time and position. A small osmotic gradient existed from branch to stem base under pre‐dawn conditions, and the osmotic gradient between upper stem and stem base was close to zero. The turgor in branches was significantly driven by xylem water potential, and the turgor loss point in branches was relatively close to daily minimum needle water potentials typically reported for Scots pine. Our results imply that xylem water potential considerably impacts the turgor pressure gradient driving phloem transport and that gravitation has a relatively large role in phloem transport in the stems of mature Scots pine trees.