Immunolocalization of hemicelluloses in Arabidopsis thaliana stem. Part I: temporal and spatial distribution of xylans

We investigated the microdistribution of xylans in different cell types of Arabidopsis stem using immunolocalization methods with LM10 and LM11 antibodies. Xylan labeling in xylary fibers (fibers) was initially detected at the cell corner of the S(1) layer and increased gradually during fiber maturation, showing correlation between xylan labeling and general secondary cell wall formation processes in fibers. Metaxylem vessels (vessels) showed earlier development of secondary cell walls than fibers, but revealed almost identical labeling patterns to fibers during maturation. No difference in labeling patterns and intensity was detected in the cell wall of fibers, vessels and protoxylem vessels (proto-vessels) between LM10 and LM11, indicating that vascular bundle cells may be chemically composed of a highly homogeneous xylan type. Interestingly, interfascicular fibers (If-fibers) showed different labeling patterns between the two antibodies and also between different developmental stages. LM10 showed no labeling in primary cell walls and intercellular layers of If-fibers at the S(1) formation stage, but some labeling was detected in middle lamella cell corner regions at the S(2) formation stage. In contrast, LM11 revealed uniform labeling across the If-fiber cell wall during all developmental stages. These results suggest that If-fibers have different xylan deposition processes and patterns from vascular bundle cells. The presence of xylan was also confirmed in parenchyma cells following pectinase treatment. Together our results indicate that there are temporal and spatial differences in xylan labeling between cell types in Arabidopsis stem. Differences in xylan labeling between Arabidopsis stem and poplar are also discussed.     

Spatial and temporal variability of xylan distribution in differentiating secondary xylem of hybrid aspen

Xylans occupy approximately one-third of the cell wall components in hardwoods and their chemical structures are well understood. However, the microdistribution of xylans (O-acetyl-4-O-methylglucuronoxylans, AcGXs) in the cell wall and their correlation with functional properties of cells in hardwood xylem is poorly understood. We demonstrate here the spatial and temporal distribution of xylans in secondary xylem cells of hybrid aspen using immunolocalization with LM10 and LM11 antibodies. Xylan labeling was detected earliest in fibers at the cell corner of the S? layer, and then later in vessels and ray cells respectively. Fibers showed a heterogeneous labeling pattern in the mature cell wall with stronger labeling of low substituted xylans (lsAcGXs) in the outer than inner cell wall. In contrast, vessels showed uniform labeling in the mature cell wall with stronger labeling of lsAcGXs than fibers. Xylan labeling in ray cells was detected much later than that in fibers and vessels, but was also detected at the beginning of secondary cell wall formation as in fibers and vessels with uniform labeling in the cell wall regardless of developmental stage. Interestingly, pit membranes including fiber-, vessel- and ray-vessel pits showed strong labeling of highly substituted xylans (hsAcGXs) during differentiation, although this labeling gradually disappeared during pit maturation. Together our observations indicate that there are temporal and spatial variations of xylan deposition and chemical structure of xylans between cells in aspen xylem. Differences in xylan localization between aspen (hardwood) and cedar (softwood) are also discussed.     

Secondary Cell Wall Deposition in Developing Secondary Xylem of Poplar

Although poplar is widely used for genomic and biotechnological manipulations of wood, the cellular basis of wood development in poplar has not been accurately documented at an ultrastructural level. Developing secondary xylem cells from hybrid poplar (Populus deltoides × P. trichocarpa), which were actively making secondary cell walls, were preserved with high pressure freezing/freeze substitution for light and electron microscopy. The distribution of xylans and mannans in the different cell types of developing secondary xylem were detected with immunofiuorescence and immuno-gold labeling. While xylans, detected with the monoclonal antibody LM10, had a general distribution across the secondary xylem, mannans were enriched in the S2 secondary cell wall layer of fibers. To observe the cellular structures associated with secondary wall production, cryofixed fibers were examined with transmission electron microscopy during differentiation. There were abundant cortical microtubules and endomembrane activity in cells during the intense phase of secondary cell wall synthesis. Microtubule-associated small membrane compartments were commonly observed, as well as Golgi and secretory vesicles fusing with the plasma membrane.     

Distribution of glucomannans and xylans in poplar xylem and their changes under tension stress

Present work investigated glucomannan (GM) and xylan distribution in poplar xylem cells of normal- (NW), opposite- (OW) and tension wood (TW) with immunolocalization methods. GM labeling was mostly detected in the middle- and inner S(2) (+S(3)) layer of NW and OW fibers, while xylan labeling was observed in the whole secondary cell wall. GM labeling in vessels of NW and OW was much weaker than in fibers and mostly detected in the S(2) layer, whereas slightly stronger xylan labeling than fibers was detected in the whole secondary cell wall of vessels. Ray cells in NW and OW showed no GM labeling, but strong xylan labeling. These results indicate that GMs and xylans are spatially distributed in poplar xylem cells with different concentrations present in different cell types. Surprisingly, TW showed significant decrease of GM labeling in the normal secondary cell wall of gelatinous (G) fibers compared to NW and OW, while xylan labeling was almost identical indicating that the GM and xylan synthetic pathways in fibers have different reaction mechanisms against tension stress. Unlike fibers, no notable changes in GM labeling were detected in vessels of TW, suggesting that GM synthesis in vessels may not be affected by tension stress. GM and xylan was also detected in the G-layer with slightly stronger and much weaker labeling than the normal secondary cell wall of G-fibers. Differences in GM and xylan distribution are also discussed for the same functional cells found in hardwoods and softwoods.     

Ultrastructural Localization of a Bean Glycine-Rich Protein in Unlignified Primary Walls of Protoxylem Cells

A polyclonal antibody was used to localize a glycine-rich cell wall protein (GRP 1.8) in French bean hypocotyls with the indirect immunogold method. GRP 1.8 could be localized mainly in the unlignified primary cell walls of the oldest protoxylem elements and also in cell corners of both proto- and metaxylem elements. In addition, GRP 1.8 was detected in phloem using tissue printing. The labeled primary walls of dead protoxylem cells showed a characteristically dispersed ultrastructure, resulting from the action of hydrolases during the final steps of cell maturation and from mechanical stress due to hypocotyl growth. Primary walls of living protoxylem and adjacent parenchyma cells were only weakly labeled. This was true also for the secondary walls of proto- and metaxylem cells, which in addition showed high background labeling. Inhibition of lignification with a specific and potent inhibitor of phenylalanine ammonia-lyase did not lead to enhanced labeling of secondary walls, showing that lignin does not mask the presence of GRP 1.8 in these walls. Dictyosomes of living proto- and metaxylem cells were not labeled, but dictyosomes of xylem parenchyma cells without secondary walls, adjacent to strongly labeled protoxylem elements, were clearly labeled. These observations suggest that GRP 1.8 is not produced by xylem vessels but by xylem parenchyma cells that export the protein to the wall of protoxylem vessels.     

Immunolocalization and structural variations of xylan in differentiating earlywood tracheid cell walls of Cryptomeria japonica

We investigated the spatial and temporal distribution of xylans in the cell walls of differentiating earlywood tracheids of Cryptomeria japonica using two different types of monoclonal antibodies (LM10 and LM11) combined with immunomicroscopy. Xylans were first deposited in the corner of the S₁ layer in the early stages of S₁ formation in tracheids. Cell corner middle lamella also showed strong xylan labeling from the early stage of cell wall formation. During secondary cell wall formation, the innermost layer and the boundary between the S₁ and S₂ layers (S₁/S₂ region) showed weaker labeling than other parts of the cell wall. However, mature tracheids had an almost uniform distribution of xylans throughout the entire cell wall. Xylan localization labeled with LM10 antibody was stronger in the outer S₂ layer than in the inner layer, whereas xylans labeled with LM11 antibody were almost uniformly distributed in the S₂ layer. In addition, the LM10 antibody showed almost no xylan labeling in the S₁/S₂ region, whereas the LM11 antibody revealed strong xylan labeling in the S₁/S₂ region. These findings suggest that structurally different types of xylans may be deposited in the tracheid cell wall depending on the developmental stage of, or location in, the cell wall. Our study also indicates that deposition of xylans in the early stages of tracheid cell wall formation may be spatially consistent with the early stage of lignin deposition in the tracheid cell wall.     

Localisation and characterisation of cell wall mannan polysaccharides in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

Polysaccharides containing -1,4-mannosyl residues (mannans) are abundant in the lignified secondary cell walls of gymnosperms, and are also found as major seed storage polysaccharides in some plants, such as legume species. Although they have been found in a variety of angiosperm tissues, little is known about their presence and tissue localisation in the model angiosperm, Arabidopsis thaliana (L.) Heynh. In this study, antibodies that specifically recognised mannans in competitive ELISA experiments were raised in rabbits. Using these antibodies, we showed that Golgi-rich vesicles derived from Arabidopsis callus were able to synthesise mannan polysaccharides in vitro. Immunofluorescence light microscopy and immunogold electron microscopy of Arabidopsis inflorescence stem sections revealed that the mannan polysaccharide epitopes were localised in the thickened secondary cell walls of xylem elements, xylem parenchyma and interfascicular fibres. Similarly, mannan epitopes were present in the xylem of the leaf vascular bundles. Surprisingly, the thickened epidermal cell walls of both leaves and stems also contained abundant mannan epitopes. Low levels were observed in most other cell types examined. Thus, mannans are widespread in Arabidopsis tissues, and may be of particular significance in both lignified and non-lignified thickened cell walls. Polysaccharide analysis using carbohydrate gel electrophoresis (PACE) of cell wall preparations digested with a specific mannanase showed that there is glucomannan in inflorescence stems. The findings show that Arabidopsis can be used as a model plant in studies of the synthesis and functions of mannans.Abbreviations BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - PACE polysaccharide analysis by carbohydrate gel electrophoresis     

Cell wall modifications in Arabidopsis plants with altered alpha-L-arabinofuranosidase activity

Although cell wall remodeling is an essential feature of plant growth and development, the underlying molecular mechanisms are poorly understood. This work describes the characterization of Arabidopsis (Arabidopsis thaliana) plants with altered expression of ARAF1, a bifunctional alpha-L-arabinofuranosidase/beta-D-xylosidase (At3g10740) belonging to family 51 glycosyl-hydrolases. ARAF1 was localized in several cell types in the vascular system of roots and stems, including xylem vessels and parenchyma cells surrounding the vessels, the cambium, and the phloem. araf1 T-DNA insertional mutants showed no visible phenotype, whereas transgenic plants that overexpressed ARAF1 exhibited a delay in inflorescence emergence and altered stem architecture. Although global monosaccharide analysis indicated only slight differences in cell wall composition in both mutant and overexpressing lines, immunolocalization experiments using anti-arabinan (LM6) and anti-xylan (LM10) antibodies indicated cell type-specific alterations in cell wall structure. In araf1 mutants, an increase in LM6 signal intensity was observed in the phloem, cambium, and xylem parenchyma in stems and roots, largely coinciding with ARAF1 expression sites. The ectopic overexpression of ARAF1 resulted in an increase in LM10 labeling in the secondary walls of interfascicular fibers and xylem vessels. The combined ARAF1 gene expression and immunolocalization studies suggest that arabinan-containing pectins are potential in vivo substrates of ARAF1 in Arabidopsis.     

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.     

Immunolocalization of β-1-4-galactan and its relationship with lignin distribution in developing compression wood of Cryptomeria japonica

Compression wood (CW) contains higher quantities of β-1-4-galactan than does normal wood (NW). However, the physiological roles and ultrastructural distribution of β-1-4-galactan during CW formation are still not well understood. The present work investigated deposition of β-1-4-galactan in differentiating tracheids of Cryptomeria japonica during CW formation using an immunological probe (LM5) combined with immunomicroscopy. Our immunolabeling studies clearly showed that differences in the distribution of β-1-4-galactan between NW (and opposite wood, OW) and CW are initiated during the formation of the S₁ layer. At this stage, CW was strongly labeled in the S₁ layer, whereas no label was observed in the S₁ layer of NW and OW. Immunogold labeling showed that β-1-4-galactan in the S₁ layer of CW tracheids significantly decreased during the formation of the S₂ layer. Most β-1-4-galactan labeling was present in the outer S₂ region in mature CW tracheids, and was absent in the inner S₂ layer that contained helical cavities in the cell wall. In addition, delignified CW tracheids showed significantly more labeling of β-1-4-galactan in the secondary cell wall, suggesting that lignin is likely to mask β-1-4-galactan epitopes. The study clearly showed that β-1-4-galactan in CW was mainly deposited in the outer portion of the secondary cell wall, indicating that its distribution may be spatially consistent with lignin distribution in CW tracheids of Cryptomeria japonica.     

Examination of morphological changes in the first formed protoxylem in Arabidopsis seedlings

We examined morphological changes in the first-formed protoxylem vessels in Arabidopsis seedlings. Between 2.5 and 8 days after imbibition, mean hypocotyl and root length increased 1.52 and 23.3 times, respectively. In the 2.5-day-old seedlings, two continuous protoxylem vessels were present in the hypocotyl-root axis. In the 8-day-old upper hypocotyls, six protoxylem vessels were observed, and in the lower hypocotyls, four protoxylem vessels and one or two metaxylem vessels were observed. In the 8-day-old roots, there were two protoxylem vessels and two or three metaxylem vessels. Two protoxylem vessels in the hypocotyls connected to two metaxylem vessels in the roots of 8-day-old seedlings. At the 0.3-mm part below the hypocotyl-root boundary, the mean intervals of neighboring annular secondary wall thickenings in protoxylem vessels in 8-day-old roots were 12.9% larger than those in 2.5-day-old roots. In more apical parts of 8-day-old roots, the mean intervals fluctuated between 1.71 and 2.29 microm. In 8-day-old seedlings, metaxylem vessels were formed between 0.4 mm above the hypocotyl-root boundary and 17 mm below the boundary. The intervals in these regions were not extended so much as protoxylem vessels were collapsed. The first-formed protoxylem vessels presumably retain their water-conductive function after metaxylem formation.     

Structural analysis of K<Superscript>+</Superscript> dependence in <Emphasis Type="SmallCaps">l</Emphasis>-asparaginases from <Emphasis Type="Italic">Lotus japonicus</Emphasis>

Wood is composed of various types of cells and each type of cell has different structural and functional properties. However, the temporal and spatial diversities of cell wall components in the cell wall between different cell types are rarely understood. To extend our understanding of distributional diversities of cell wall components among cells, we investigated the immunolabeling of mannans (O-acetyl-galactoglucomannans, GGMs) and xylans (arabino-4-O-methylglucuronoxylans, AGXs) in ray cells and pits. The labeling of GGMs and AGXs was temporally different in ray cells. GGM labeling began to be detected in ray cells at early stages of S₁ formation in tracheids, whereas AGX labeling began to be detected in ray cells at the S₂ formation stage in tracheids. The occurrence of GGM and AGX labeling in ray cells was also temporally different from that of tracheids. AGX labeling began to be detected much later in ray cells than in tracheids. GGM labeling also began to be detected in ray cells either slightly earlier or later than in tracheids. In pits, GGM labeling was detected in bordered and cross-field pit membranes at early stages of pit formation, but not observed in mature pits, indicating that enzymes capable of GGM degradation may be involved in pit membrane formation. In contrast to GGMs, AGXs were not detected in pit membranes during the entire developmental process of bordered and cross-field pits. AGXs showed structural and depositional variations in pit borders depending on the developmental stage of bordered and cross-field pits.     

Occurrence of xylan and mannan polysaccharides and their spatial relationship with other cell wall components in differentiating compression wood tracheids of <Emphasis Type="Italic">Cryptomeria japonica</Emphasis>

Compression wood (CW) tracheids have different cell wall components than normal wood (NW) tracheids. However, temporal and spatial information on cell wall components in CW tracheids is poorly understood. We investigated the distribution of arabino-4-O-methylglucuronoxylans (AGXs) and O-acetyl-galactoglucomannans (GGMs) in differentiating CW tracheids. AGX labeling began to be detected in the corner of the S₁ layer at the early S₁ formation stage. Subsequently, the cell corner middle lamella (ccML) showed strong AGX labeling when intercellular spaces were not fully formed. AGX labeling was uniformly distributed in the S₁ layer, but showed uneven distribution in the S₂ layer. AGX labeling was mainly detected in the inner S₂ layer after the beginning of the helical cavity formation. The outer S₂ layer showed almost no labeling of low substituted AGXs. Only a very small amount of high substituted AGXs was distributed in the outer S₂ layer. These patterns of AGX labeling in the S₂ layer opposed the lignin and β-1-4-galactan distribution in CW tracheids. GGM labeling patterns were almost identical to AGX labeling in the early stages of CW tracheids, and GGM labeling was detected in the entire S₂ layer from the early S₂ formation stage of CW tracheids with some spatial differences in labeling density depending on developmental stage. Compared with NW tracheids, CW tracheids showed significantly different AGX distributions in the secondary cell wall but similar GGM labeling patterns. No significant differences were observed in labeling after delignification of CW tracheids.     

The MeJA-inducible copper amine oxidase AtAO1 is expressed in xylem tissue and guard cells

Copper amine oxidases oxidize the polyamine putrescine to 4-aminobutanal with the production of the plant signal molecule hydrogen peroxide (H₂O₂) and ammonia. The Arabidopsis (Arabidopsis thaliana) gene At4g14940 (AtAO1, previously referred to as ATAO1) encodes an apoplastic copper amine oxidase expressed in lateral root cap cells and developing xylem, especially in root protoxylem and metaxylem precursors. In our recent study, we demonstrated that AtAO1 expression is strongly induced in the root vascular tissues by the wound-signal hormone methyl jasmonate (MeJA). Furthermore, we also demonstrated that the H₂O₂ derived by the AtAO1-driven oxidation of putrescine, mediates the MeJA–induced early protoxylem differentiation in Arabidopsis roots. H₂O₂ may contribute to protoxylem differentiation by signaling developmental cell death and by acting as co-substrate in peroxidase-mediated cell wall stiffening and lignin polymerization. Here, by the means of AtAO1 promoter::green fluorescent protein-β-glucuronidase (AtAO1::GFP-GUS) fusion analysis, we show that a strong AtAO1 gene expression occurs also in guard cells of leaves and flowers. The high expression levels of AtAO1 in tissues or cell types regulating water supply and water loss may suggest a role of the encoded protein in water balance homeostasis, by modulating coordinated adjustments in anatomical and functional features of xylem tissue and guard cells during acclimation to adverse environmental conditions.     

PROCAMBIUM VS. CAMBIUM AND PROTOXYLEM VS. METAXYLEM IN POPULUS DELTOIDES SEEDLINGS

The concept of a procambium-cambium continuum was examined in Populus deltoides by following its development in serially sectioned bud and stem tissues. As in other species, the term cambium is used to refer to that part of the continuum associated with the formation of secondary vascular tissues; i.e., with secondary growth. However, that part of the continuum associated with the formation of primary vascular tissues is subdivided to facilitate interpretation of the consecutive stages of primary xylem differentiation. Thus, the procambium as envisioned by other authors is subdivided into procambium, initiating layer, and metacambium, all of which develop acropetally and in complete continuity. The procambium is derived from the residual meristem in the form of acropetally developing strands and traces. The initiating layer is represented by the first, tangentially separated, periclinal divisions that delineate the position of the prospective cambium. The metacambium is a later stage during which additional periclinally dividing cells unite the initiating layer into a tangentially continuous meristem within a trace bundle. After establishment of the initiating layer, the procambial trace is completely phloem dominated. Protoxylem differentiation begins in an originating center at the base of the leaf primordium and it progresses basipetally to form the protoxylem pole. Cells of the initiating layer do not contribute to the formation of either protoxylem or protophloem. However, those cells of the initiating layer directly opposite the protoxylem pole divide precociously and later differentiate to metaxylem, thus forming a radial file of protoxylem-metaxylem elements. Protoxylem elements of lateral traces are longitudinally continuous with the protoxylem of their parent traces, whereas those of a central trace are longitudinally continuous with the metaxylem of its parent trace. Metaxylem is formed later than protoxylem and it is derived from the metacambium. Metaxylem does not form a continuous system with protoxylem of the same trace because of the different temporal and spatial origins of the two kinds of xylem. Rather, metaxylem is longitudinally continuous with secondary xylem of older traces below. An attempt was made to determine the functional significance of the pattern of protoxylem and metaxylem differentiation in relation to primary and secondary plant development.     

Protoxylem: the deposition of a network containing glycine-rich cell wall proteins starts in the cell corners in close association with the pectins of the middle lamella

Antibodies were used to localise polysaccharide and protein networks in the protoxylem of etiolated soybean (Glycine max L.) hypocotyls. The deposition of glycine-rich proteins (GRPs) starts in the cell corners between protoxylem elements and xylem parenchyma cells. Finally, the GRPs form a network between two mature protoxylem elements. The network also interconnects the ring- and spiral-shaped secondary wall thickenings, as well as the thickenings with the middle lamellae of living xylem parenchyma cells. In addition to the GRP network, a polysaccharide network composed mainly of pectins is involved in the attachment of the secondary wall thickenings to the middle lamellae of xylem parenchyma cells.     

The poplar glycosyltransferase GT47C is functionally conserved with Arabidopsis Fragile fiber8

Xylan is the major hemicellulose in dicot wood. Unraveling genes involved in the biosynthesis of xylan will be of importance in understanding the process of wood formation. In this report, we investigated the possible role of poplar GT47C, a glycosyltransferase belonging to family GT47, in the biosynthesis of xylan. PoGT47C from the hybrid poplar Populus alba x tremula exhibits 84% sequence similarity to Fragile fiber8 (FRA8), which is involved in the biosynthesis of glucuronoxylan in Arabidopsis. Phylogenetic analysis of glycosyltransferase family GT47 in the Populus trichocarpa genome revealed that GT47C is the only close homolog of FRA8. In situ hybridization showed that the PoGT47C gene was expressed in developing primary xylem, secondary xylem and phloem fibers of stems, and in developing secondary xylem of roots. Sequence analysis suggests that PoGT47C is a type II membrane protein, and study of the subcellular localization demonstrated that fluorescent protein-tagged PoGT47C was located in the Golgi. Immunolocalization with a xylan monoclonal antibody LM10 revealed a nearly complete loss of xylan signals in the secondary walls of fibers and vessels in the Arabidopsis fra8 mutant. Expression of PoGT47C in the fra8 mutant restored the secondary wall thickness and xylan content to the wild-type level. Together, these results suggest that PoGT47C is functionally conserved with FRA8 and it is probably involved in xylan synthesis during wood formation.     

Structural cell-wall proteins in protoxylem development: evidence for a repair process mediated by a glycine-rich protein

Polyclonal antibodies were used to localize structural cell-wall proteins in differentiating protoxylem elements in etiolated bean and soybean hypocotyls at the light- and electron-microscopic level. A proline-rich protein was localized in the lignified secondary walls, but not in the primary walls of protoxylem elements, which remain unlignified, as shown with lignin-specific antibodies. Secretion of the proline-rich protein was observed during lignification in different cell types. A glycine-rich protein (GRP1.8) was specifically localized in the modified primary walls of mature protoxylem elements and in cell corners between xylem elements and xylem parenchyma cells. The protein was secreted by Golgi bodies both in protoxylem cells after the lignification of their secondary walls and in the surrounding xylem parenchyma cells. The modified primary walls of protoxylem elements were visualized under the light microscope as filaments or sheets staining distinctly with the protein stain Coomassie blue. Electron micrographs of these walls show that they are composed of an amorphous material of moderate electron-density and of polysaccharide microfibrils. These materials form a three-dimensional network, interconnecting the ring- or spiral-shaped secondary wall thickenings of protoxylem elements and xylem parenchyma cells. The results demonstrate that the modified primary walls of protoxylem cells are not simply breakdown products due to partial hydrolysis and passive elongation, as believed until now. Extensive repair processes produce cell walls with unique staining properties. It is concluded that these walls are unusually rich in protein and therefore have special chemical and physical properties.