Role of auxin and gibberellin in differentiation of primary Phloem fibers

The hypothesis that auxin and gibberellic acid (GA₃) control the differentiation of primary phloem fibers is confirmed for the stem of Coleus blumei Benth. Indoleacetic acid (IAA) alone sufficed to cause the differentiation of a few primary phloem fibers. In long term experiments auxin induced a considerable number of fibers in mature internodes. GA₃ by itself did not exert any effect on fiber differentiation. Combinatiosn of IAA with GA₃ completely replaced the role of the leaves in primary phloem fiber differentiation qualitatively and quantitatively. Although the combined effect of the two growth hormones diminished considerably with increasing distance from the source of induction, auxin with GA₃ or IAA alone induced fibers in a few internodes below the application site. When various combinations of both hormones were applied, high concentrations of IAA stimulated rapid differentiation of fibers with thick secondary walls, while high levels of GA₃ resulted in long fibers with thin walls. The size of the primary phloem fibers correlated with the dimensions of the differentiating internode, thereby providing evidence that both growth regulators figure in the control of stem extension. High IAA/low GA₃ concentrations have an inhibitory effect on internode elongation, whereas low IAA/high GA₃ concentrations promote maximal stem elongation.     

The regulation from guaiacyl to syringyl lignin in the differentiating xylem of Robinia pseudoacacia

13C- and deuterium (D)-labeled ferulic acid and sinapic acid ([8-(13)C, 3-OCD3]-ferulic acid and [8-(13)C, 3,5-OCD3]-sinapic acid) were administered to robinia (Robinia pseudoacacia L.) shoots. To estimate the distribution of the label from administrated ferulic or sinapic acid, continuous 50-microm-thick tangential sections cut from the cambium of robinia were subjected to lignin chemical analysis by the DFRC method. Labeled ferulic acid was incorporated into guaiacyl and syringyl lignin. The incorporation of labeled ferulic acid into syringyl units was observed only in the later stage of lignification. Labeled sinapic acid was incorporated into syringyl lignin in the early stage and the later stage of lignification. In general, syringyl lignin was deposited in the later stage of cell wall lignification. Thus, the incorporation of sinapic acid to syringyl lignin in the early stage of lignification was abnormal. Taken together, the aromatic ring-modifying reactions (the conversion from guaiacyl to syringyl moiety, including the hydroxylation and methylation) were more important for the regulation of the sinapyl alcohol biosynthesis than the reducing reactions (the reduction of acids to alcohols) in the differentiating xylem.     

Lignification in the flax stem: evidence for an unusual lignin in bast fibers

In the context of our research on cell wall formation and maturation in flax (Linum usitatissimum L) bast fibers, we (1) confirmed the presence of lignin in bast fibers and (2) quantified and characterized the chemical nature of this lignin at two developmental stages. Histochemical methods (Weisner and Maüle reagents and KMnO₄-staining) indicating the presence of lignin in bast fibers at the light and electron microscope levels were confirmed by chemical analyses (acetyl bromide). In general, the lignin content in flax bast fibers varied between 1.5% and 4.2% of the dry cell wall residues (CWRs) as compared to values varying between 23.7% and 31.4% in flax xylem tissues. Immunological and chemical analyses (thioacidolysis and nitrobenzene oxidation) indicated that both flax xylem- and bast fiber-lignins were rich in guaiacyl (G) units with S/G values inferior to 0.5. In bast fibers, the highly sensitive immunological probes allowed the detection of condensed guaiacyl-type (G) lignins in the middle lamella, cell wall junctions, and in the S1 layer of the secondary wall. In addition, lower quantities of mixed guaiacyl–syringyl (GS) lignins could be detected throughout the secondary cell wall. Chemical analyses suggested that flax bast-fiber lignin is more condensed than the corresponding xylem lignin. In addition, H units represented up to 25% of the monomers released from bast-fiber lignin as opposed to a value of 1% for the corresponding xylem tissue. Such an observation indicates that the structure of flax bast-fiber lignin is significantly different from that of the more typical woody plant lignin, thereby suggesting that flax bast fibers represent an interesting system for studying an unusual lignification process.     

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.

     

Relations polyphenols-croissance: Lignification et limitation de croissance chez Lycopersicum esculentum

Abstract Polyphenols and growth – Lignification and limitation of growth in Lycopersicum esculentum. When fed with quinic acid, young tomato seedlings (Lycopersicum esculentum Mill. cv. St. Pierre) exhibited reduced growth and marked changes in cell wall composition: cellulose concentrations decreased whereas those of lignin increased. Exogenously supplied growth substances (IAA, GA₃) affected both the size of the plants and the lignification process: IAA treatments resembled quinic acid induced modifications with regard to both size and lignification: GA₃ applied to quinate treated plants counteracted the effects of this compound by reducing the lignin content and improving the growth. The possible effect of abnormal lignification as a limiting factor in cell growth is supported by the characterization of lignins in tissues different from xylem.     

Photoregulation of the incorporation of guaiacyl units into lignins

When fed with [¹⁴C] phenylalanine in the light, xylem tissues isolated from poplar stems were able to incorporate part of the radioactivity into both the guaiacyl and the syringyl residues of lignins. In the dark, only syringyl units were integrated into the polymer whereas the guaiacyl residues remained unlabeled.When a membrane perturber (isopropanol) was added to the incubation mixture, the label was incorporated into the guaiacyl units either in the light or in the dark. Conversely, a membrane stabilizer (CaCl₂) prevented the labeling of the guaiacyl units even when the tissues were illuminated. These results suggest that light acts through the modification of membrane permeabilities, altering specifically the synthesis and the transport or the polymerization of guaiacyl-type units during the process of lignification.Abbreviations S syringyl residue - G guaiacyl residue This is paper No. 4 in a series. For paper No. 3 see Grand and Ranjeva, in press     

Effect of laterally applied gibberellin A4/7 on cambial growth and the level of indole-3-acetic acid in Pinus sylvestris shoots

Gibberellin A_4/7 (GA_4/7) was applied in lanolin or ethanol around the circumference at the midpoint of the previous-year terminal of dormant Pinus sylvestris seedlings. After cultivating the seedlings under environmental conditions favorable for growth for up to 10 weeks, cambial growth was measured as the radial widths of xylem and phloem, and the level of indole-3-acetic acid (IAA) was determined by combined gas chromatography-mass spectrometry using [¹³₆](IAA) as the internal standard. In intact seedlings, both 1 mg GA_4/7 g^?1 lanolin and 50 mg GA_4/7 I^?1 ethanol increased phloem production and the cambial region IAA level in the current-year terminal, without significantly altering its longitudinal growth. In the previous-year terminal, 1 mg GA_4/7 g^?1 lanolin promoted phloem production at the application point and increased the cambial region IAA level above this point, whereas 50 mg GA_4/7 I^?1 ethanol stimulated the production of both xylem and phloem at the treatment site and elevated the cambial region IAA level beneath it. Laterally applied GA_4/7 at 50 mg I^?1 ethanol stimulated xylem and phloem production in debudded previous-year terminals treated at the apical cut surface with 1 mg IAA g^?1 lanolin, but not in those treated with plain lanolin. However, the promotion of cambial growth in debudded terminals treated apically with 1 mg IAA g^?1 lanolin and laterally with 50 mg GA_4/7 I^?1 ethanol was not associated with an elevated IAA content in the cambial region. The results indicate that exogenous GA_4/7 can promote xylem and phloem production provided an IAA source is present, and that it or a metabolic product acts directly, rather than indirectly by stimulating longitudinal growth and/or raising the cambial region IAA level.     

Cambial activity,annual rhythm of xylem production in relation to phenology and climatic factors and lignification pattern during xylogenesis in drum-stick tree (Moringa oleifera)

The interrelationship among seasonality of cambium, wood formation, cell size variation, lignification, tree phenology and climatic factors has been examined in Moringa oleifera, a tropical evergreen tree. The vascular cambium in Moringa is a storied with a distinct seasonal variation in its structure due to dimensional changes in rays. Though cambium remains active throughout the year it is sensitive to water availability. Peak cambial cell division and rate of xylem differentiation are influenced by average rainfall during the monsoon period. Cambial cell division reaches higher up in the tree trunk when it is supporting a high number of branches and leaves. Statistical analysis of cell size variation and climate factors revealed that xylem cell development is greatly influenced by rainfall and rarely by temperature. Lengths of fusiform initials and vessel elements are positively correlated. The pattern of lignification during xylogenesis shows that the vessels are the first element to develop lignified walls and ray cells are the last elements to become lignified. Fiber cell walls show more syringyl lignin, while the cell walls of other xylem elements are characterized by relatively more guaiacyl lignin units.     

Lignin composition in cambial tissues of poplar

The cambial tissues of a Populus balsamifera, Balsam poplar clone were studied during a growth season. The Klason and acid-soluble lignin contents were determined as well as the carbohydrate monomer distribution and the protein content. Both the phloem and the xylem sides of the cambial region were examined. The samples were analyzed by thioacidolysis and structures of dimeric products were determined by mass spectrometry after desulphuration. Chemical analysis of samples during the growth season was combined with microscopy of embedded specimens that showed the state of cell differentiation at the time of sampling. In spring and early summer, growth is very rapid and the intention was to collect tissue in which exclusively the middle lamella/primary cell wall had begun to lignify. The Klason lignin, protein content and carbohydrate monomer distribution showed that all the specimens from the cambial tissues sampled during a growth season contained predominantly middle lamella and primary walls; except for the developing xylem sampled in August where the carbohydrate composition showed that secondary walls were present. Thioacidolysis showed that the lignin from the cambial tissues had more condensed structures than the lignin from the reference balsam poplar clone wood. More guaiacyl than syringyl units were detected and mass spectrometry showed that the cambial tissues contained more lignin structures with end-groups than the reference sample. These results suggest that lignification in the cambial layer and early developing xylem may take place predominantly in a bulk fashion during the summer.     

Lignification and lignin heterogeneity for various age classes of bamboo (Phyllostachys pubescens) stems

The lignification process and lignin heterogeneity of fibre, vessel and parenchyma cell walls for various age classes of bamboo stems of Phyllostachys pubescens Mazel were investigated. It was shown that protoxylem vessels lignified in the early stage of vascular bundle differentiation, metaxylem vessel and fibre walls initiated lignification from the middle lamella and cell corners after the completion of vascular bundle differentiation. Most of the parenchyma cell walls lignified after the stem reached its full height, while a few parenchyma cells remained non-lignified even in the mature culm. The cell walls of fibres and most parenchyma cells thickened further during the stem growth to form polylamellate structure and the lignification process of these cells may last even up to 7 years. The fibre walls were rich in guaiacyl lignin in the early stage of lignification, and lignin rich in syringyl units were deposited in the later stage. Vessel walls mainly contained guaiacyl lignin, while both guaiacyl and syringyl lignin were present in the fibre and parenchyma cell walls.     

Regulation of Syringyl to Guaiacyl Ratio in Lignin Biosynthesis

p -hydroxyphenyl (H)-, guaiacyl (G)- and syringyl (S) propane, in situ is described. New pathways that regulate the ratio of S to G moieties operating at the stages of cinnamoyl CoA, cinnamyl aldehyde and cinnamyl alcohol are introduced. The roles of monolignol glucoside in the lignification of tree xylem are discussed. The results of gene manupulations that alter the lignin structures are also introduced. Received 15 September 2001/ Accepted in revised form 16 October 2001     

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.     

Guaiacyl Lignin Associated with Vessels in Aspen Callus Cultures

Lignin from aspen (Populus tremuloides Michx.) tissue cultures containing only mature vessels and undifferentiated parenchymatous cells is exclusively of the guaiacyl type normally associated with gymnosperms. This supports the theory that the guaiacyl and syringyl lignin in angiosperm wood is compartmentalized, with guaiacyl lignin in vessels and syringyl lignin in fibers and ray cells.     

Effects of prohexadione on cambial and longitudinal growth and the levels of endogenous gibberellins A1, A3, A4, and A9 and indole-3-acetic acid in Pinus sylvestris shoots

Prohexadione, a gibberellin (GA) biosynthesis inhibitor, was applied in ethanol around the circumference at the midpoint of the previous year terminal shoot of dormant Pinus sylvestris seedlings. After cultivating the seedlings under environmental conditions favorable for growth for 10 weeks, longitudinal and cambial growth were measured, and the endogenous levels of GA₁, GA₃, GA₄, GA₉, and indole-3-acetic acid (IAA) were determined by combined gas chromatography-mass spectrometry, using deuterated GAs and [¹³C₆]IAA as internal standards. Prohexadione application inhibited elongation and xylem and phloem production in the current year terminal shoot and xylem production in the previous year terminal shoots. Concomitantly, in both ages of shoots the cambial region contents of GA₁; GA₃, and GA₄ were decreased, whereas the level of GA₉ was increased. However, the IAA content was not altered in the terminal bud on the current year terminal shoot or in the cambial region of the current year or previous year terminal shoots. The results provide additional evidence that: (1) GAs are involved in the regulation of cambial growth, as well as longitudinal growth, in Pinus sylvestris shoots; (2) they act directly, rather than indirectly, by altering the IAA level; and (3) the GA₉ GA₄ GA₁ pathway is a major route of GA biosynthesis in conifer species.Abbreviations GA gibberellin - IAA indole-3-acetic acid - HPLC high performance liquid chromatography - GC gas chromatography - SIM selected ion monitoring - MS mass spectrometry     

Distribution and Chemical Composition of Lignin in Secondary Xylem of Cactaceae

Cactaceae family has heterogeneity in the accumulation of lignocellulose due to the diversity of shapes and anatomy of the wood. Most studies focus on fibrous and dimorphic species; but the non-fibrous species are poorly studied. The aims of this work were to analyze the syringyl/guaiacyl ratio of lignin and its distribution in secondary xylem, especially in non-fibrous species. The syringyl/guaiacyl (S/G) ratio was quantified from 34 species of cacti by nitrobenzene oxidation of free-extractive wood. The distribution of lignocellulose in wood sections stained with safranin O/fast green was determined with epifluorescence microscopy. The S/G ratio was heterogeneous; most of the non-fibrous species had a higher percentage of syringyl, while the fibrous ones accumulate guaiacyl. Fluorescence emission showed that vessel elements and wide-band tracheids had similar tonalities. It is hypothesized that the presence of a higher percentage of syringyl in most cacti is part of the defense mechanism against pathogens, which together with the succulence of the stem represent adaptations that contribute to survival in their hostile environments.     

PRIMARY PHLOEM REGENERATION WITHOUT CONCOMITANT XYLEM REGENERATION: ITS HORMONE CONTROL IN COLEUS

Phloem regeneration in the absence of xylem regeneration was evoked in number 5 internodes of Coleus blumei Benth. by severing xylemless phloem bundles. Its quantitative extent was estimated. To determine whether phloem regeneration is directly affected by auxin, or whether it is a secondary consequence of the auxin-dependent xylem regeneration which usually accompanies it, phloem regeneration was measured in decapitated plants from which auxin-producing leaves and buds had been removed (i.e., in “plant stumps”). In these stumps, 1% IAA in lanolin completely restored phloem regeneration to the intact plant level. In such stumps from which roots had been excised, and in excised internodes, IAA failed to restore it to this level. However, zeatin or zeatin riboside in aqueous solution applied to the bases of excised internodes receiving IAA at their apical ends restored phloem regeneration to the level of that in whole plants. When similarly tested, other cytokinins (kinetin, kinetin riboside, 2iP, and 2iPA), gibberellic acid (GA₃), glutamine, proline, sucrose, and a mixture of mineral salts failed to promote phloem regeneration. Glutamic acid, tested only once, was slightly promotive of it.