Immunocytochemical characterization of early-developing flax fiber cell walls

Summary The deposition and formation of a thick secondary wall is a major event in the differentiation of flax (Linum usitatissimum) fibers. This wall is cellulose-rich; but it also contains significant amounts of other matrix polymers which are noncellulosic such as pectins. We have used immunocytochemical techniques with antibodies specific for various epitopes associated with either pectins or arabinogalactan proteins (AGPs) to investigate the distribution of these polymers within the walls of differentiating young fibers of 1- and 2-week-old plants. Our results show that different epitopes exhibit distinct distribution patterns within fiber walls. Unesterified pectins recognized by polygalacturonic acid-rhamnogalacturonan I (PGA/RG-I) antibodies and rhamnogalacturonan II recognized by anti-RG-II-borate complex antibodies are localized all over the secondary wall of fibers. PGA/RG-I epitopes, but not RG-II epitopes, are also present in the middle lamellae and cell junctions. In marked contrast, -(14) galactans recognized by the LM5 monoclonal antibody and AGP epitopes recognized by anti--(16) galactan and LM2 antibodies are primarily located in the half of the secondary wall nearest the plasma membrane. LM2 epitopes, present in 1-week-old fibers, are undetectable later in development, suggesting a regulation of the expression of certain AGP epitopes. In addition, localization of cellulose with the cellobiohydrolase I-gold probe reveals distinct subdomains within the secondary walls of young fibers. These findings indicate that, in addition to cellulose, early-developing flax fibers synthesize and secrete different pectin and AGP molecules.     

Distribution of pectin and arabinogalactan protein epitopes during organogenesis from androgenic callus of wheat

The distribution of several arabinogalactan protein and pectic epitopes were studied during organogenesis in androgenic callus of wheat. In cell wall of mature and degenerating parenchyma cells, the arabinogalactan epitopes JIM4, JIM14, JIM16 or LM2 were expressed differently according to the cells location. LM2 was observed also in meristematic cells of regenerated shoot buds and leaves. Anti-pectin JIM7 labelled the wall of meristematic cells but fluorescence was strongest in outer walls of surface cells of callus and shoot buds coated by extracellular matrix surface network (ECMSN). During leaves growth the ECMSN disappeared, and JIM7 fluorescence decreased. JIM5 epitope was abundant in the cell walls lining the intercellular spaces of callus parenchyma and in tricellular junctions within regenerated buds and leaves.     

Deposition of glucuronoxylans on the secondary cell wall of Japanese beech as observed by immuno-scanning electron microscopy

Summary Glucuronoxylans (GXs), the main hemicellulosic component of hardwoods, are localized exclusively in the secondary wall of Japanese beech and gradually increase during the course of fiber differentiation. To reveal where GXs deposit within secondary wall and how they affect cell wall ultrastructure, immuno-scanning electron microscopy using anti-GXs antiserum was applied in this study. In fibers forming the outer layer of the secondary wall (S₁), cellulose fibrils were small in diameter and deposited sparsely on the inner surface of the cell wall. Fine fibrils with approximately 5 nm width aggregated and formed thick fibrils with 12 nm width. Some of these thick fibrils further aggregated to form bundles which labelled positively for GXs. In fibers forming the middle layer of the secondary wall (S₂), fibrils were thicker than those found in S₁ forming fibers and were densely deposited. The S₂ layer labelled intensely for GXs with no preferential distribution recognized. Compared with newly formed secondary walls, previously formed secondary walls were composed of thick and highly packed microfibrils. Labels against GXs were much more prevalent on mature secondary walls than on newly deposited secondary walls. This result implies that the deposition of GXs into the cell wall may occur continuously after cellulose microfibril deposition and may be responsible for the increase in diameter of the microfibrils.Abbreviations GXs glucuronoxylans - PBS phosphate-buffered saline - RFDE rapid-freeze and deep-etching technique - FE-SEM field emission scanning electron microscope - TEM transmission electron microscope     

Immunohistochemical Localization of Hemicelluloses and Pectins Varies During Tissue Development in the Bamboo Culm

The distribution of hemicelluloses and pectins in bamboo internodes was studied immunocytochemistrically at various stages of development. The ultra-structures of bamboo cell walls have been reported previously at various stages. The internodes were identically classified into three developmental phases: primary wall stage (phase I), unlignified secondary wall stage (phase II) and lignified wall stage (phase III), using the same bamboo culm. (1→,1→4)-β-Glucans were distributed in nearly all tissues in an actively elongating stage. Limited amounts of β-glucans were deposited in primary walls and the middle lamellae, but were limited to the phloem in secondary walls. This suggests that the function of β-glucans might be different in phloem vis-à-vis other tissues. Highly-substituted xylans were located in nearly all tissues of early phase I, but had disappeared in all tissues immediately prior to lignification. In contrast, low-branched xylan epitopes were present only in the protoxylem in phase I, but were present in all tissues immediately prior to lignification in phase II. In phase III, the epitopes were densely localized in lignified walls, suggesting that the substitution of xylans is closely related to maturation. Methyl-esterified (but not unesterified) pectins were present in all tissues of early phase I. Just before and after lignification, both types of pectins were concentrated in the phloem and protoxylem. Xyloglucans were largely distributed in the phloem and in lignified tissues, suggesting that they might be closely correlated with maturation. This represents the first account of the distribution of hemicelluloses and pectins at the tissue and ultrastructural level in bamboo internodes at various stages of development.     

Immunohistochemical localization of hemicelluloses and pectins varies during tissue development in the bamboo culm

The distribution of hemicelluloses and pectins in bamboo internodes was studied immunocytochemistrically at various stages of development. The ultra-structures of bamboo cell walls have been reported previously at various stages. The internodes were identically classified into three developmental phases: primary wall stage (phase I), unlignified secondary wall stage (phase II) and lignified wall stage (phase III), using the same bamboo culm. (1-->3, 1-->4)-Beta-glucans were distributed in nearly all tissues in an actively elongating stage. Limited amounts of beta-glucans were deposited in primary walls and the middle lamellae, but were limited to the phloem in secondary walls. This suggests that the function of beta-glucans might be different in phloem vis-à-vis other tissues. Highly-substituted xylans were located in nearly all tissues of early phase I, but had disappeared in all tissues immediately prior to lignification. In contrast, low-branched xylan epitopes were present only in the protoxylem in phase I, but were present in all tissues immediately prior to lignification in phase II. In phase III, the epitopes were densely localized in lignified walls, suggesting that the substitution of xylans is closely related to maturation. Methyl-esterified (but not unesterified) pectins were present in all tissues of early phase I. Just before and after lignification, both types of pectins were concentrated in the phloem and protoxylem. Xyloglucans were largely distributed in the phloem and in lignified tissues, suggesting that they might be closely correlated with maturation. This represents the first account of the distribution of hemicelluloses and pectins at the tissue and ultrastructural level in bamboo internodes at various stages of development.     

Pectin esterification is spatially regulated both within cell walls and between developing tissues of root apices

Monoclonal antibodies recognizing un-esterified (JIM5) and methyl-esterified (JIM7) epitopes of pectin have been used to locate these epitopes by indirect immunofluorescence and immunogold electron microscopy in the root apex of carrot (Daucus carota L.). Both antibodies labelled the walls of cells in all tissues of the developing root apex. Immunogold labelling observed at the level of the electron microscope indicated differential location of the pectin epitopes within the cell walls. The un-esterified epitope was located to the inner surface of the primary cell walls adjacent to the plasma membrane, in the middle lamella and abundantly to the outer surface at intercellular spaces. In contrast, the epitope containing methyl-esterified pectin was located evenly throughout the cell wall. In root apices of certain other species the JIM5 and JIM7 epitopes were found to be restricted to distinct tissues of the developing roots. In the root apex of oat (Avena sativa L.), JIM5 was most abundantly reactive with cell walls at the region of intercellular spaces of the cortical cells. JIM7 was reactive with cells of the cortex and the stele. Neither epitope occurred in walls of the epidermal or root-cap cells. These pattern of expression were observed to derive from the very earliest stages of the development of these tissues in the oat root meristem and were maintained in the mature root. In the coleoptile and leaf tissues of oat seedlings, JIM5 labelled all cells abundantly whereas JIM7 was unreactive. Other members of the Gramineae and also the Chenopodiaceae are shown to express similar restricted spatial patterns of distribution of these pectin epitopes in root apices.Abbreviations CDTA 1,2-diaminocyclohexane tetraacetic acid - RG rhamnogalacturonan J.P.K. was supported by the Agricultural and Food Research Council Cell Signalling and Recognition Programme. We thank J. Cooke and N. Stacey for technical assistance, H.A. Schols, Drs. P. Albersheim and A. Darvill for pectic polysaccharides, and Dr. R.R. Selvendran and M. McCann for useful discussions.     

Gibberellin signaling

A study of stem anatomy and the sclerenchyma fibre cells associated with the phloem tissues of hemp (Cannabis sativa L.) plants is of interest for both understanding the formation of secondary cell walls and for the enhancement of fibre utility as industrial fibres and textiles. Using a range of molecular probes for cell wall polysaccharides we have surveyed the presence of cell wall components in stems of hemp in conjunction with an anatomical survey of stem and phloem fibre development. The only polysaccharide detected to occur abundantly throughout the secondary cell walls of phloem fibres was cellulose. Pectic homogalacturonan epitopes were detected in the primary cell walls/intercellular matrices between the phloem fibres although these epitopes were present at a lower level than in the surrounding parenchyma cell walls. Arabinogalactan-protein glycan epitopes displayed a diversity of occurrence in relation to fibre development and the JIM14 epitope was specific to fibre cells, binding to the inner surface of secondary cell walls, throughout development. Xylan epitopes were found to be present in the fibre cells (and xylem secondary cell walls) and absent from adjacent parenchyma cell walls. Analysis of xylan occurrence in the phloem fibre cells of hemp and flax indicated that xylan epitopes were restricted to the primary cell walls of fibre cells and were not present in the secondary cell walls of these cells.     

Cell wall proteins of flax phloem fibers

A cell wall has been isolated from single-type cells, phloem fibers of flax (Linum usitatissimum L.) being at the stage of the active formation of a thick secondary cell wall. Weakly bound proteins of the cell wall of phloem fibers were extracted and separated, and their mass spectra were recorded. The identification of the proteins and their assignment to a particular cell compartment were performed using a variety of bioinformatics methods. In all, 93 proteins were identified of which many proteins were defined as predicted, putative, and hypothetical. Twenty one proteins were identified as cell-wall proteins. The absence of the marker proteins of primary cell walls such as xyloglucan endotransglycosylase and expansins indirectly confirms the predominance of the secondary cell wall in a sample for protein extraction.     

Developmental and Tissue-Specific Structural Alterations of the Cell-Wall Polysaccharides of Arabidopsis thaliana Roots

The plant cell wall is a dynamic structure that plays important roles in growth and development and in the interactions of plants with their environment and other organisms. We have used monoclonal antibodies that recognize different carbohydrate epitopes present in plant cell-wall polysaccharides to locate these epitopes in roots of developing Arabidopsis thaliana seedlings. An epitope in the pectic polysaccharide rhamnogalacturonan I is observed in the walls of epidermal and cortical cells in mature parts of the root. This epitope is inserted into the walls in a developmentally regulated manner. Initially, the epitope is observed in atrichoblasts and later appears in trichoblasts and simultaneously in cortical cells. A terminal [alpha]-fucosyl-containing epitope is present in almost all of the cell walls in the root. An arabinosylated (1->6)-[beta]-galactan epitope is also found in all of the cell walls of the root with the exception of lateral root-cap cell walls. It is striking that these three polysaccharide epitopes are not uniformly distributed (or accessible) within the walls of a given cell, nor are these epitopes distributed equally across the two walls laid down by adjacent cells. Our results further suggest that the biosynthesis and differentiation of primary cell walls in plants are precisely regulated in a temporal, spatial, and developmental manner.     

Xyloglucan endotransglycosylases have a function during the formation of secondary cell walls of vascular tissues

Xyloglucan transglycosylases (XETs) have been implicated in many aspects of cell wall biosynthesis, but their function in vascular tissues, in general, and in the formation of secondary walls, in particular, is less well understood. Using an in situ XET activity assay in poplar stems, we have demonstrated XET activity in xylem and phloem fibers at the stage of secondary wall formation. Immunolocalization of fucosylated xylogucan with CCRC-M1 antibodies showed that levels of this species increased at the border between the primary and secondary wall layers at the time of secondary wall deposition. Furthermore, one of the most abundant XET isoforms in secondary vascular tissues (PttXET16A) was cloned and immunolocalized to fibers at the stage of secondary wall formation. Together, these data strongly suggest that XET has a previously unreported role in restructuring primary walls at the time when secondary wall layers are deposited, probably creating and reinforcing the connections between the primary and secondary wall layers. We also observed that xylogucan is incorporated at a high level in the inner layer of nacreous walls of mature sieve tube elements.     

Reciprocal influence of ethylene and gibberellins on response-gene expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A cortical band of fiber cells originate de novo in tendrils of redvine [Brunnichia ovata (Walt.) Shiners] when these convert from straight, supple young filaments to stiffened coiled structures in response to touch stimulation. We have analyzed the cell walls of these fibers by in situ localization techniques to determine their composition and possible role(s) in the coiling process. The fiber cell wall consists of a primary cell wall and two lignified secondary wall layers (S₁ and S₂) and a less lignified gelatinous (G) layer proximal to the plasmalemma. Compositionally, the fibers are sharply distinct from surrounding parenchyma as determined by antibody and affinity probes. The fiber cell walls are highly enriched in cellulose, callose and xylan but contain no homogalacturonan, either esterified or de-esterified. Rhamnogalacturonan-I (RG-I) epitopes are not detected in the S layers, although they are in both the gelatinous layer and primary wall, indicating a further restriction of RG-I in the fiber cells. Lignin is concentrated in the secondary wall layers of the fiber and the compound middle lamellae/primary cell wall but is absent from the gelatinous layer. Our observations indicate that these fibers play a central role in tendril function, not only in stabilizing its final shape after coiling but also generating the tensile strength responsible for the coiling. This theory is further substantiated by the absence of gelatinous layers in the fibers of the rare tendrils that fail to coil. These data indicate that gelatinous-type fibers are responsible for the coiling of redvine tendrils and a number of other tendrils and vines.     

Immunolocalization of beta-D-glucans,pectins, and arabinogalactan-proteins during intrusive growth and elongation of nonarticulated laticifers in Asclepias speciosa Torr

Nonarticulated laticifers are latex-containing cells that elongate indefinitely and grow intrusively between the walls of meristematic cells. To identify biochemical mechanisms involved in the growth of nonarticulated laticifers, we have analyzed the distribution of various polysaccharides and proteoglycans in walls of meristematic cells in contact with laticifers, nonadjacent to laticifers, and in laticifer walls. In the shoot apex of Asclepias speciosa, the levels of callose and a (1-->4)-beta-galactan epitope are lower in meristematic walls in contact with laticifers than in nonadjacent walls. In contrast, we did not detect a decline in xyloglucan, homogalacturonan, and arabinogalactan-protein epitopes upon contact of meristematic cells with laticifers. Laticifer elongation is also associated with the development of a homogalacturonan-rich middle lamella between laticifers and their neighboring cells. Furthermore, laticifers lay down walls that differ from those of their surrounding cells. This is particularly evident for epitopes in rhamnogalacturonan I. A (1-->5)-alpha-arabinan epitope in this pectin is more abundant in laticifers than meristematic cells, while the opposite is observed for a (1-->4)-beta-galactan epitope. Also, different cell wall components exhibit distinct distribution patterns within laticifer walls. The (1-->5)-alpha-arabinan epitope is distributed throughout the laticifer walls while certain homogalacturonan and arabinogalactan-protein epitopes are preferentially located in particular regions of laticifer walls. Taken together, our results indicate that laticifer penetration causes changes in the walls of meristematic cells and that there are differences in wall composition within laticifer walls and between laticifers and their surrounding cells.     

CELL WALL CARBOHYDRATE EPITOPES IN THE GREEN ALGA OEDOGONIUM BHARUCHAE F. MINOR (OEDOGONIALES,CHLOROPHYTA)1

Cell wall changes in vegetative and suffultory cells (SCs) and in oogonial structures from Oedogonium bharuchae N. D. Kamat f. minor Vélez were characterized using monoclonal antibodies against several carbohydrate epitopes. Vegetative cells and SCs develop only a primary cell wall (PCW), whereas mature oogonial cells secrete a second wall, the oogonium cell wall (OCW). Based on histochemical and immunolabeling results, (1→4)‐β‐glucans in the form of crystalline cellulose together with a variable degree of Me‐esterified homogalacturonans (HGs) and hydroxyproline‐rich glycoprotein (HRGP) epitopes were detected in the PCW. The OCW showed arabinosides of the extensin type and low levels of arabinogalactan‐protein (AGP) glycans but lacked cellulose, at least in its crystalline form. Surprisingly, strong colabeling in the cytoplasm of mature oogonia cells with three different antibodies (LM‐5, LM‐6, and CCRC‐M2) was found, suggesting the presence of rhamnogalacturonan I (RG‐I)–like structures. Our results are discussed relating the possible functions of these cell wall epitopes with polysaccharides and O‐glycoproteins during oogonium differentiation. This study represents the first attempt to characterize these two types of cell walls in O. bharuchae, comparing their similarities and differences with those from other green algae and land plants. This work represents a contribution to the understanding of how cell walls have evolved from simple few‐celled to complex multicelled organisms.     

Intra- and Extracellular Localization of Lignin Peroxidase during the Degradation of Solid Wood and Wood Fragments by Phanerochaete chrysosporium by Using Transmission Electron Microscopy and Immuno-Gold Labeling

The distribution of lignin peroxidase during degradation of both wood and woody fragments by the white rot fungus Phanerochaete chrysosporium was investigated by using anti-lignin peroxidase in conjunction with postembedding transmission electron microscopy and immuno-gold labeling techniques. The enzyme was localized in the peripheral regions of the fungal cell cytoplasm in association with the cell membrane, fungal cell wall, and extracellular slime materials. In solid wood, lignin peroxidase was detected in low concentrations associated with both superficial and degradation zones within secondary cell walls undergoing fungal attack. A similar but much greater level of extracellular peroxidase activity was associated with wood fragments degraded by the fungus grown under liquid culture conditions optimal for production of the enzyme. Efforts to infiltrate degraded wood pieces with high levels of lignin peroxidase showed the enzyme to be restricted to superficial regions of wood decay and to penetrate wood cell walls only where the wall structure had been modified. In this respect the enzyme was able to penetrate characteristic zones of degradation within the secondary walls of fibers to sites of lignin attack. This suggests a possibility for a close substrate-enzyme association during wood cell wall degradation.     

Domain-specific and cell type-specific localization of two types of cell wall matrix polysaccharides in the clover root tip

Using immunocytochemical techniques and antibodies that specifically recognize xyloglucan (anti-XG), polygalacturonic acid/rhamnogalacturonan I (anti-PGA/RG-I), and methylesterified pectins (JIM 7), we have shown that these polysaccharides are differentially synthesized and localized during cell development and differentiation in the clover root tip. In cortical cells XG epitopes are present at a threefold greater density in the newly formed cross walls than in the older longitudinal walls, and PGA/RG-I epitopes are detected solely in the expanded middle lamella of cortical cell corners, even after pretreatment of sections with pectinmethylesterase to uncover masked epitopes. These results suggest that in cortical cells XG and PGA/RG-I are differentially localized not only to particular wall domains, but also to particular cell walls. In contrast to their nonoverlapping distribution in cortical cells, XG epitopes and PGA/RG-I epitopes largely colocalize in the epidermal cell walls. The results also demonstrate that the middle lamella of the longitudinal walls shared by epidermal cells and by epidermal and cortical cells constitutes a barrier to the diffusion of cell wall and mucilage molecules. Synthesis of XG and PGA/RG-I epitope-containing polysaccharides also varies during cellular differentiation in the root cap. The differentiation of gravitropic columella cells into mucilage-secreting peripheral cells is marked by a dramatic increase in the synthesis and secretion of molecules containing XG and PGA/RG-I epitopes. In contrast, JIM 7 epitopes are present at abundant levels in columella cell walls, but are not detectable in peripheral cell walls or in secreted mucilage. There were also changes in the cisternal labeling of the Golgi stacks during cellular differentiation in the root tip. Whereas PGA/RG-I epitopes are detected primarily in cis- and medial Golgi cisternae in cortical cells (Moore, P. J., K. M. M. Swords, M. A. Lynch, and L. A. Staehelin. 1991. J. Cell Biol. 112:589-602), they are localized predominantly in the trans-Golgi cisternae and the trans-Golgi network in epidermal and peripheral root cap cells. These observations suggest that during cellular differentiation the plant Golgi apparatus can be both structurally and functionally reorganized.     

Subcellular localization of the Streptococcus mutans P1 protein C terminus.

To determine the subcellular location of the Streptococcus mutans P1 protein C-terminal anchor, cell envelope fractionation experiments were conducted in combination with Western immunoblotting, using monoclonal antibody MAb 6-8C specific for an epitope that maps near the C terminus of P1 protein and also a polyclonal antibody preparation directed against the P1 C-terminal 144 amino acids (P1COOH). P1 protein was detected in cell walls but not the membrane purified from S. mutans cells by the monoclonal antibody. In contrast, P1 protein was not detected in the same cell wall preparation using the anti-P1COOH polyclonal antibody. However, proteins released from the cell walls by treatment with mutanolysin contained antigen that was recognized by the anti-P1COOH antibody, suggesting that the epitopes recognized by the antibody were masked by peptidoglycan in the cell wall preparations. When cell walls were treated with boiling trichloroacetic acid to solubilize cell-wall-associated carbohydrate, P1 antigen could not be detected in either the solubilized carbohydrate, or in the remaining peptidoglycan, regardless of whether polyclonal or monoclonal antibody was used. However, when the peptidoglycan was treated with mutanolysin, P1 antigen could be detected in the mutanolysin solubilized fraction by MAb 6-8C. Collectively, these data suggest that the C-terminal 144 amino acids of the P1 protein are embedded within the cell wall, and associated exclusively with the peptidoglycan. Furthermore, the ability of the anti-P1COOH antibody to recognize P1 antigen only after mutanolysin treatment of cell walls suggests these C-terminal 144 amino acids are tightly intercalated within the peptidoglycan strands.     

Ultrastructure of fibre and parenchyma cell walls during early stages of culm development in Dendrocalamus asper

BACKGROUND AND AIMS: The anatomy of bamboo culms and the multilayered structure of fibre cell walls are known to be the main determinant factors for its physical and mechanical properties. Studies on the bamboo cell wall have focussed mainly on fully elongated and mature fibres. The main aim of this study was to describe the ultrastructure of primary and secondary cell walls in culm tissues of Dendrocalamus asper at different stages of development. METHODS: The development of fibre and parenchyma tissues was classified into four stages based on light microscopy observations made in tissues from juvenile plants. The stages were used as a basis for transmission electron microscopy study on the ultrastructure of the cell wall during the process of primary and early secondary cell wall formation. Macerations and phloroglucinol-HCl staining were employed to investigate fibre cell elongation and fibre cell wall lignification, respectively. KEY RESULTS: The observations indicated that the primary wall is formed by the deposition of two distinct layers during the elongation of the internode and that secondary wall synthesis may begin before the complete cessation of internode and fibre elongation. Elongation was followed by a maturation phase characterized by the deposition of multiple secondary wall layers, which varied in number according to the cell type, location in the culm tissue and stage of shoot development. Lignification of fibre cell walls started at the period prior to the cessation of internode elongation. CONCLUSIONS: The structure of the primary cell wall was comprised of two layers. The fibre secondary cell wall began to be laid down while the cells were still undergoing some elongation, suggesting that it may act to cause the slow-down and eventual cessation of cell elongation.     

Changes in biochemical composition of the cell wall of the cotton fiber during development

The composition of the cell wall of the cotton fiber (Gossypium hirsutum L. Acala SJ-1) has been studied from the early stages of elongation (5 days postanthesis) through the period of secondary wall formation, using cell walls derived both from fibers developing on the plant and from fibers obtained from excised, cultured ovules. The cell wall of the elongating cotton fiber was shown to be a dynamic structure. Expressed as a weight per cent of the total cell wall, cellulose, neutral sugars (rhamnose, fucose, arabinose, mannose, galactose, and noncellulosic glucose), uronic acids, and total protein undergo marked changes in content during the elongation period. As a way of analyzing absolute changes in the walls with time, data have also been expressed as grams component per millimeter of fiber length. Expressed in this way for plant-grown fibers, the data show that the thickness of the cell wall is relatively constant until about 12 days postanthesis; after this time it markedly increases until secondary wall cellulose deposition is completed. Between 12 and 16 days postanthesis increases in all components contribute to total wall increase per millimeter fiber length. The deposition of secondary wall cellulose begins at about 16 days postanthesis (at least 5 days prior to the cessation of elongation) and continues until about 32 days postanthesis. At the time of the onset of secondary wall cellulose deposition, a sharp decline in protein and uronic acid content occurs. The content of some of the individual neutral sugars changes during development, the most prominent change being a large increase in noncellulosic glucose which occurs just prior to the onset of secondary wall cellulose deposition. Methylation analyses indicate that this glucose, at least in part, is 3-linked. In contrast to the neutral sugars, no significant changes in cell wall amino acid composition are observed during fiber development.