Cell Wall Domains Research Articles

Plasmodesmata and intercellular molecular traffic control.

Plasmodesmata are plasma membrane-lined connections that join plant cells to their neighbours, establishing an intercellular cytoplasmic continuum through which molecules can travel between cells, tissues, and organs. As plasmodesmata connect almost all cells in plants, their molecular traffic carries information and resources across a range of scales, but dynamic control of plasmodesmal aperture can change the possible domains of molecular exchange under different conditions. Plasmodesmal aperture is controlled by specialised signalling cascades accommodated in spatially discrete membrane and cell wall domains. Thus, the composition of plasmodesmata defines their capacity for molecular trafficking. Further, their shape and density can likewise define trafficking capacity, with the cell walls between different cell types hosting different numbers and forms of plasmodesmata to drive molecular flux in physiologically important directions. The molecular traffic that travels through plasmodesmata ranges from small metabolites through to proteins, and possibly even larger mRNAs. Smaller molecules are transmitted between cells via passive mechanisms but how larger molecules are efficiently trafficked through plasmodesmata remains a key question in plasmodesmal biology. How plasmodesmata are formed, the shape they take, what they are made of, and what passes through them regulate molecular traffic through plants, underpinning a wide range of plant physiology.

The effects of osmotic stress on the cell wall-plasma membrane domains of the unicellular streptophyte, Penium margaritaceum.

Penium margaritaceum is a unicellular zygnematophyte (basal Streptophyteor Charophyte) that has been used as a model organism for the study of cell walls of Streptophytes and for elucidating organismal adaptations that were key in the evolution of land plants.. When Penium is incubated in sorbitol-enhance medium, i.e., hyperosmotic medium, 1000-1500 Hechtian strands form within minutes and connect the plasma membrane to the cell wall. As cells acclimate to this osmotic stress over time, further significant changes occur at the cell wall and plasma membrane domains. The homogalacturonan lattice of the outer cell wall layer is significantly reduced and is accompanied by the formation of a highly elongate, "filamentous" phenotype. Distinct peripheral thickenings appear between the CW and plasma membrane and contain membranous components and a branched granular matrix. Monoclonal antibody labeling of these thickenings indicates the presence of rhamnogalacturonan-I epitopes. Acclimatization also results in the proliferation of the cell's vacuolar networks and macroautophagy. Penium's ability to acclimatize to osmotic stress offers insight into the transition of ancient zygnematophytes from an aquatic to terrestrial existence.

Subcellular coordination of plant cell wall synthesis.

Organelles of the plant cell cooperate to synthesize and secrete a strong yet flexible polysaccharide-based extracellular matrix: the cell wall. Cell wall composition varies among plant species, across cell types within a plant, within different regions of a single cell wall, and in response to intrinsic or extrinsic signals. This diversity in cell wall makeup is underpinned by common cellular mechanisms for cell wall production. Cellulose synthase complexes function at the plasma membrane and deposit their product into the cell wall. Matrix polysaccharides are synthesized by a multitude of glycosyltransferases in hundreds of mobile Golgi stacks, and an extensive set of vesicle trafficking proteins govern secretion to the cell wall. In this review, we discuss the different subcellular locations at which cell wall synthesis occurs, review the molecular mechanisms that control cell wall biosynthesis, and examine how these are regulated in response to different perturbations to maintain cell wall homeostasis.

Laccases and Peroxidases Co-Localize in Lignified Secondary Cell Walls throughout Stem Development.

Lignin, a critical phenolic polymer in secondary cell walls of plant cells, enables strength in fibers and water transportation in xylem vessel elements. Secreted enzymes, namely laccases (LACs) and peroxidases (PRXs), facilitate lignin polymerization by oxidizing lignin monomers (monolignols). Previous work in Arabidopsis (Arabidopsis thaliana) demonstrated that AtLAC4 and AtPRX64 localized to discrete lignified cell wall domains in fibers, although the spatial distributions of other enzymes in these large gene families are unknown. Here, we show that characteristic sets of putative lignin-associated LACs and PRXs localize to precise regions during stem development, with LACs and PRXs co-occurring in cell wall domains. AtLAC4, AtLAC17, and AtPRX72 localized to the thick secondary cell wall of xylem vessel elements and fibers, whereas AtLAC4, AtPRX64, and AtPRX71 localized to fiber cell corners. Interestingly, AtLAC4 had a transient cell corner localization early in fiber development that disappeared in the mature stem. In contrast with these secondary cell wall localizations, AtLAC10, AtPRX42, AtPRX52, and AtPRX71 were found in nonlignified tissues. Despite ubiquitous PRX occurrence in cell walls, PRX oxidative activity was restricted to lignifying regions during development, which suggested regulated production of apoplastic hydrogen peroxide. Relative amounts of apoplastic reactive oxygen species differed between lignified cell types, which could modulate PRX activity. Together, these results indicate that precise localization of oxidative enzymes and differential distribution of oxidative substrates, such as hydrogen peroxide, provide mechanisms to control spatiotemporal deposition of lignin during development.

Coordination of five class III peroxidase-encoding genes for early germination events of Arabidopsis thaliana

The Class III peroxidases (CIII Prxs) belong to a plant-specific multigene family. Thanks to their double catalytic cycle they can oxidize compounds or release reactive oxygen species (ROS). They are either involved in different cell wall stiffening processes such as lignification and suberization, in cell wall loosening or defense mechanisms. Germination is an important developmental stage requiring specific peroxidase activity. However, little is known about which isoforms are involved. Five CIII Prx encoding genes: AtPrx04, AtPrx16, AtPrx62, AtPrx69, and AtPrx71 were identified from published microarray data mining. Delayed or induced testa and endosperm rupture were observed for the corresponding CIII Prx mutant lines indicating either a gene-specific inducing or repressing role during germination, respectively. Via in situ hybridization AtPrx16, AtPrx62, AtPrx69 and AtPrx71 transcripts were exclusively localized to the micropylar endosperm facing the radicle, and transcriptomic data analysis enabled positioning the five CIII Prxs in a co-expression network enriched in germination, cell wall, cell wall proteins and xyloglucan hits. Evidence were produced showing that the five CIII Prxs were cell wall-targeted proteins and that the micropylar endosperm displayed a complex cell wall domain topochemistry. Finally, we drew a spatio-temporal model highlighting the fine sequential gene expression and the possible involvement of micropylar endosperm cell wall domains to explain the non-redundant cell wall stiffening and loosening functions of the CIII Prxs in a single cell type. We also highlighted the necessity of a peroxidase homeostasis to accurately control the micropylar endosperm cell wall dynamics during Arabidopsis germination events.

Arabidopsis Trichome Contains Two Plasma Membrane Domains with Different Lipid Compositions Which Attract Distinct EXO70 Subunits

Plasma membrane (PM) lipid composition and domain organization are modulated by polarized exocytosis. Conversely, targeting of secretory vesicles at specific domains in the PM is carried out by exocyst complexes, which contain EXO70 subunits that play a significant role in the final recognition of the target membrane. As we have shown previously, a mature Arabidopsis trichome contains a basal domain with a thin cell wall and an apical domain with a thick secondary cell wall, which is developed in an EXO70H4-dependent manner. These domains are separated by a cell wall structure named the Ortmannian ring. Using phospholipid markers, we demonstrate that there are two distinct PM domains corresponding to these cell wall domains. The apical domain is enriched in phosphatidic acid (PA) and phosphatidylserine, with an undetectable amount of phosphatidylinositol 4,5-bisphosphate (PIP2), whereas the basal domain is PIP2-rich. While the apical domain recruits EXO70H4, the basal domain recruits EXO70A1, which corresponds to the lipid-binding capacities of these two paralogs. Loss of EXO70H4 results in a loss of the Ortmannian ring border and decreased apical PA accumulation, which causes the PA and PIP2 domains to merge together. Using transmission electron microscopy, we describe these accumulations as a unique anatomical feature of the apical cell wall—radially distributed rod-shaped membranous pockets, where both EXO70H4 and lipid markers are immobilized.

A Rho-actin signaling pathway shapes cell wall boundaries in Arabidopsis xylem vessels

Patterned cell wall deposition is crucial for cell shapes and functions. In Arabidopsis xylem vessels, ROP11 GTPase locally inhibits cell wall deposition through microtubule disassembly, inducing pits in cell walls. Here, we show that an additional ROP signaling pathway promotes cell wall growth at pit boundaries. Two proteins, Boundary of ROP domain1 (BDR1) and Wallin (WAL), localize to pit boundaries and regulate cell wall growth. WAL interacts with F-actin and promotes actin assembly at pit boundaries while BDR1 is a ROP effector. BDR1 interacts with WAL, suggesting that WAL could be recruited to the plasma membrane by a ROP-dependent mechanism. These results demonstrate that BDR1 and WAL mediate a ROP-actin pathway that shapes pit boundaries. The study reveals a distinct machinery in which two closely associated ROP pathways oppositely regulate cell wall deposition patterns for the establishment of tiny but highly specialized cell wall domains.

Pectin Demethylesterification Generates Platforms that Anchor Peroxidases to Remodel Plant Cell Wall Domains

Plant cell walls are made of polysaccharidic-proteinaceous complex matrices. Molecular interactions governing their organization remain understudied. We take advantage of the highly dynamic cell walls of Arabidopsis seed mucilage secretory cells to propose a hierarchical multi-molecular interaction model within a cell wall domain. We show that the PECTINMETHYLESTERASE INHIBITOR6 activity creates a partially demethylesterified pectin pattern acting as a platform allowing positioning of PEROXIDASE36 in a remote primary cell wall domain during early development. This allows triggering the loosening of this domain during later development, in turn leading to proper physiological function upon mature seed imbibition and germination. We anticipate that this pioneer example of molecular scaffold within a cell wall domain is more widespread through other combinations of the individual molecular players all belonging to large multigenic families. These results highlight the role of cell wall polysaccharide-protein interactions in the organization of cell wall domains.

Characterization of CRISPR Mutants Targeting Genes Modulating Pectin Degradation in Ripening Tomato.

Tomato (Solanum lycopersicum) is a globally important crop with an economic value in the tens of billions of dollars, and a significant supplier of essential vitamins, minerals, and phytochemicals in the human diet. Shelf life is a key quality trait related to alterations in cuticle properties and remodeling of the fruit cell walls. Studies with transgenic tomato plants undertaken over the last 20 years have indicated that a range of pectin-degrading enzymes are involved in cell wall remodeling. These studies usually involved silencing of only a single gene and it has proved difficult to compare the effects of silencing these genes across the different experimental systems. Here we report the generation of CRISPR-based mutants in the ripening-related genes encoding the pectin-degrading enzymes pectate lyase (PL), polygalacturonase 2a (PG2a), and β-galactanase (TBG4). Comparison of the physiochemical properties of the fruits from a range of PL, PG2a, and TBG4 CRISPR lines demonstrated that only mutations in PL resulted in firmer fruits, although mutations in PG2a and TBG4 influenced fruit color and weight. Pectin localization, distribution, and solubility in the pericarp cells of the CRISPR mutant fruits were investigated using the monoclonal antibody probes LM19 to deesterified homogalacturonan, INRA-RU1 to rhamnogalacturonan I, LM5 to β-1,4-galactan, and LM6 to arabinan epitopes, respectively. The data indicate that PL, PG2a, and TBG4 act on separate cell wall domains and the importance of cellulose microfibril-associated pectin is reflected in its increased occurrence in the different mutant lines.

Distribution, mobility, and anchoring of lignin-related oxidative enzymes in Arabidopsis secondary cell walls.

Lignin is an important phenolic biopolymer that provides strength and rigidity to the secondary cell walls of tracheary elements, sclereids, and fibers in vascular plants. Lignin precursors, called monolignols, are synthesized in the cell and exported to the cell wall where they are polymerized into lignin by oxidative enzymes such as laccases and peroxidases. In Arabidopsis thaliana, a peroxidase (PRX64) and laccase (LAC4) are shown to localize differently within cell wall domains in interfascicular fibers: PRX64 localizes to the middle lamella whereas LAC4 localizes throughout the secondary cell wall layers. Similarly, laccases localized to, and are responsible for, the helical depositions of lignin in protoxylem tracheary elements. In addition, we tested the mobility of laccases in the cell wall using fluorescence recovery after photobleaching. mCHERRY-tagged LAC4 was immobile in secondary cell wall domains, but mobile in the primary cell wall when ectopically expressed. A small secreted red fluorescent protein (sec-mCHERRY) was engineered as a control and was found to be mobile in both the primary and secondary cell walls. Unlike sec-mCHERRY, the tight anchoring of LAC4 to secondary cell wall domains indicated that it cannot be remobilized once secreted, and this anchoring underlies the spatial control of lignification.

Laccases direct lignification in the discrete secondary cell wall domains of protoxylem.

Plants precisely control lignin deposition in spiral or annular secondary cell wall domains during protoxylem tracheary element (TE) development. Because protoxylem TEs function to transport water within rapidly elongating tissues, it is important that lignin deposition is restricted to the secondary cell walls in order to preserve the plasticity of adjacent primary wall domains. The Arabidopsis (Arabidopsis thaliana) inducible VASCULAR NAC DOMAIN7 (VND7) protoxylem TE differentiation system permits the use of mutant backgrounds, fluorescent protein tagging, and high-resolution live-cell imaging of xylem cells during secondary cell wall development. Enzymes synthesizing monolignols, as well as putative monolignol transporters, showed a uniform distribution during protoxylem TE differentiation. By contrast, the oxidative enzymes LACCASE4 (LAC4) and LAC17 were spatially localized to secondary cell walls throughout protoxylem TE differentiation. These data support the hypothesis that precise delivery of oxidative enzymes determines the pattern of cell wall lignification. This view was supported by lac4lac17 mutant analysis demonstrating that laccases are necessary for protoxylem TE lignification. Overexpression studies showed that laccases are sufficient to catalyze ectopic lignin polymerization in primary cell walls when exogenous monolignols are supplied. Our data support a model of protoxylem TE lignification in which monolignols are highly mobile once exported to the cell wall, and in which precise targeting of laccases to secondary cell wall domains directs lignin deposition.

Synthesis of the plant cell wallˈs most complex glycan: pectin ‐ surprises in glycosyltransferase processing and anchoring in the Golgi

Plant cell walls impart structure and identity to plant cells, have roles in growth, development and defense against microbes, and constitute renewal biomass for biofuel and biomaterial production. Plant walls are comprised of cellulose, the matrix polysaccharides pectin and hemicellulose, cell wall proteins and in many secondary walls, lignin. Pectin is the most structurally complex polysaccharide. The Arabidopsis GAUT1‐related gene family encodes 15 GAUT (GAlactUronosylTransferase) and 10 GATL (GAUT‐Like) proteins that are members of CAZy GT8. GAUT1 is a pectin biosynthetic homogalacturonan (HG):galacturononosyltransferase (GalAT) that is part of a Golgilocalized HG:GalAT GAUT1:GAUT7 core complex. GAUT1 is proteolytically processed and its transmembrane domain removed, leaving the question of how GAUT1 is retained in the Golgi lumen. Biochemical and fluorescent fusion‐protein studies show that GAUT1 is anchored in the Golgi lumen by a reducing agent‐sensitive association with GAUT7. Immunoadsorption of the GAUT1:GAUT7 complex and mass spectrometry identified 12 additional putative GAUT protein complex members whose identities suggest that pectin synthesis may occur as domains. The recent identification of a novel wall glycoconjugate structure is consistent with this view. The data support a role for GAUTs in synthesizing diverse pectic cell wall domains in primary and secondary walls. Funding from USDA AFRI 2010‐65115‐20396; NSF‐MCB 0646109, DOE DE‐FG02‐09ER20097 and BioEnergy Science Center grant DE‐PS02‐06ER64304. BioEnergy Science Center is a U.S. Department of Energy Bioenergy Research Center supported by Office of Biological and Environmental Research in DOE Office of Science.

Probing native lignin macromolecular configuration in Arabidopsis thaliana in specific cell wall types: Further insights into limited substrate degeneracy and assembly of the lignins of ref8, fah 1–2 and C4H::F5H lines

The interest in renewable, plant-derived, bioenergy/biofuels has resulted in a renaissance of plant cell-wall/lignin research. Herein, effects of modulating lignin monomeric compositions in a single plant species, Arabidopsis, are described. The earliest stage of putative "AcBr/Klason lignin" deposition was apparently unaffected by modulating p-coumarate 3-hydroxylase or ferulate 5-hydroxylase activities. This finding helps account for the inability of many other studies to fully suppress the reported putative levels of lignin deposition through monolignol biosynthesis manipulation, and also underscores limitations in frequently used lignin analytical protocols. The overall putative lignin content was greatly reduced (circa 62%) in a plant line harboring an H-(p-hydroxyphenyl) enriched lignin phenotype. This slightly increased H-monomer deposition level apparently occurred in cell-wall domains normally harboring guaiacyl (G) and/or syringyl (S) lignin moieties. For G- and S-enriched lignin phenotypes, the overall lignification process appeared analogous to wild type, with only xylem fiber and interfascicular fiber cells forming the S-enriched lignins. Laser microscope dissection of vascular bundles and interfascicular fibers, followed by pyrolysis GC/MS, supported these findings. Some cell types, presumably metaxylem and possibly protoxylem, also afforded small amounts of benzodioxane (sub)structures due to limited substrate degeneracy (i.e. utilizing 5-hydroxyconiferyl alcohol rather than sinapyl alcohol). For all plant lines studied, the 8-O-4' inter-unit frequency of cleavable H, G and/or S monomers was essentially invariant of monomeric composition for a given (putative) lignin content. These data again underscore the need for determination of lignin primary structures and identification of all proteins/enzymes involved in control of lignin polymer assembly/macromolecular configuration.

Posttranslational modifications required for cell surface localization and function of the fungal adhesin Aga1p.

Adherence of fungal cells to host substrates and each other affects their access to nutrients, sexual conjugation, and survival in hosts. Adhesins are cell surface proteins that mediate these different cell adhesion interactions. In this study, we examine the in vivo functional requirements for specific posttranslational modifications to these proteins, including glycophosphatidylinositol (GPI) anchor addition and O-linked glycosylation. The processing of some fungal GPI anchors, creating links to cell wall beta-1,6 glucans, is postulated to facilitate postsecretory traffic of proteins to cell wall domains conducive to their functions. By studying the yeast sexual adhesin subunit Aga1p, we found that deletion of its signal sequence for GPI addition eliminated its activity, while deletions of different internal domains had various effects on function. Substitution of the Aga1p GPI signal domain with those of other GPI-anchored proteins, a single transmembrane domain, or a cysteine capable of forming a disulfide all produced functional adhesins. A portion of the cellular pool of Aga1p was determined to be cell wall resident. Aga1p and the alpha-agglutinin Agalpha1p were shown to be under glycosylated in cells lacking the protein mannosyltransferase genes PMT1 and PMT2, with phenotypes manifested only in MATalpha cells for single mutants but in both cell types when both genes are absent. We conclude that posttranslational modifications to Aga1p are necessary for its biogenesis and activity. Our studies also suggest that in addition to GPI-glucan linkages, other cell surface anchorage mechanisms, such as transmembrane domains or disulfides, may be employed by fungal species to localize adhesins.

A (1-->3,6)-beta-d-galactosyl epitope containing uronic acids associated with bioactive pectins occurs in discrete cell wall domains in hypocotyl and root tissues of flax seedlings.

We have used a well-characterized antibody specific for an epitope consisting of (1-->3,6)-beta-d-galactosyl residues with terminal glucuronic or 4-O-methylglucuronic acids of a bioactive pectin and immunocytochemistry to investigate its secretion and wall distribution in the hypocotyl and root tissues of flax seedlings. Our results show that this antigenic epitope is associated with flax pectins and is expressed by all the cells of the hypocotyl and root tissues. In the hypocotyl, it is abundant in the primary wall of epidermal cells as well as in the secondary wall of fiber cells, and is relatively less abundant in parenchyma cell walls. In contrast, the epitope is not detected in the middle lamellae and cell junction regions. In the root tip cells, immunogold electron microscopy shows that the cell walls of peripheral, columella, meristematic, cortical, and epidermal cells contain significant amounts of this epitope and that the distribution patterns are distinct. Together, these findings show that the antigenic epitope occurs in discrete domains of the wall implying a strict spatial regulation of the epitope-containing molecules. The results also show that, in root cells, the epitope is present within Golgi cisternae and is predominantly assembled in the trans and the trans-Golgi network compartments.

Stop-and-go movements of plant Golgi stacks are mediated by the acto-myosin system.

The Golgi apparatus in plant cells consists of a large number of independent Golgi stack/trans-Golgi network/Golgi matrix units that appear to be randomly distributed throughout the cytoplasm. To study the dynamic behavior of these Golgi units in living plant cells, we have cloned a cDNA from soybean (Glycine max), GmMan1, encoding the resident Golgi protein alpha-1,2 mannosidase I. The predicted protein of approximately 65 kD shows similarity of general structure and sequence (45% identity) to class I animal and fungal alpha-1,2 mannosidases. Expression of a GmMan1::green fluorescent protein fusion construct in tobacco (Nicotiana tabacum) Bright Yellow 2 suspension-cultured cells revealed the presence of several hundred to thousands of fluorescent spots. Immuno-electron microscopy demonstrates that these spots correspond to individual Golgi stacks and that the fusion protein is largely confined to the cis-side of the stacks. In living cells, the stacks carry out stop-and-go movements, oscillating rapidly between directed movement and random "wiggling." Directed movement (maximal velocity 4.2 microm/s) is related to cytoplasmic streaming, occurs along straight trajectories, and is dependent upon intact actin microfilaments and myosin motors, since treatment with cytochalasin D or butanedione monoxime blocks the streaming motion. In contrast, microtubule-disrupting drugs appear to have a small but reproducible stimulatory effect on streaming behavior. We present a model that postulates that the stop-and-go motion of Golgi-trans-Golgi network units is regulated by "stop signals" produced by endoplasmic reticulum export sites and locally expanding cell wall domains to optimize endoplasmic reticulum to Golgi and Golgi to cell wall trafficking.

The range of mobility of the non‐cellulosic polysaccharides is similar in primary cell walls with different polysaccharide compositions

The molecular mobility of the non‐cellulosic polysaccharides in hydrated primary cell walls of three monocotyledons (Italian ryegrass, pineapple and onion) and one dicotyledon (cabbage) was studied using solid‐state 13C NMR spectroscopy. These cell walls were chosen as they have different non‐cellulosic polysaccharide compositions. By exploiting proton rotating‐frame and spin‐spin relaxation time constants three different cell wall domains which responded to cross‐polarization experiments were identified. Most of the non‐cellulosic polysaccharides occupied a mobile domain (C), but some occupied a partly rigid domain (B). Crystalline cellulose occupied a highly rigid domain (A). In the cell walls of Italian ryegrass and pineapple, domain C contained mainly glucuronoarabinoxylans and small amounts of rhamnogalacturonans; domain B contained small amounts of xyloglucans and galacturonans. However, in the cell walls of onion and cabbage, domain C contained mainly rhamnogalacturonans with galactans (in onion) or arabinans (in cabbage) as side chains; domain B contained galacturonans and xyloglucans. Single‐pulse excitation was used on Italian ryegrass and cabbage cell walls to reveal signals from a highly mobile fourth domain (D). In Italian ryegrass cell walls domain D contained glucuronoarabinoxylans and small amounts of rhamnogalacturonan, whereas in cabbage cell walls it contained arabinan side chains of rhamnogalacturonans. A novel feature of the research was the use of solid‐state 13C NMR spectroscopy to examine the molecular mobilities of the polysaccharides in monocotyledon cell walls that contain glucuronoarabinoxylans.

Pectin immunolocalization and calcium visualization in differentiating derivatives from poplar cambium

Calcium distribution and pectin esterification patterns in the cambial zone of poplar branches were studied with ionic microscopy and immunological tools respectively. Dynamic changes correlating with cell growth and cell differentiation were observed both on the xylem and on the phloem sides. In expanding cell walls of xylem derivatives, unesterified pectins were restricted to cell junctions and middle lamellae, occasionally accompanied by calcium ions. In contrast, in differentiating and mature phloem cells, acidic pectins and Ca2+ were present all over the walls leading to early stiffening of the polysaccharide network. Significant labelling was detected with JIM5 antibodies in some dictyosomes suggesting exocytosis of low methylated polymers towards the cell walls. At cell junctions, unesterified pectins might originate from the activity of pectinmethylesterases localized in these areas. Thus un- and deesterified pectins might be located in different cell wall domains whose distribution, varying with cell type, will confer specific extensibility to the wall matrix.

Spatial organization of the assembly pathways of glycoproteins and complex polysaccharides in the Golgi apparatus of plants.

The Golgi apparatus of plant cells is the site of assembly of glycoproteins, proteoglycans, and complex polysaccharides, but little is known about how the different assembly pathways are organized within the Golgi stacks. To study these questions we have employed immunocytochemical techniques and antibodies raised against the hydroxyproline-rich cell wall glycoprotein, extensin, and two types of complex polysaccharides, an acidic pectic polysaccharide known as rhamnogalacturonan I (RG-I), and the neutral hemicellulose, xyloglucan (XG). Our micrographs demonstrate that individual Golgi stacks can process simultaneously glycoproteins and complex polysaccharides. O-linked arabinosylation of the hydroxyproline residues of extensin occurs in cis-cisternae, and glycosylated molecules pass through all cisternae before they are packaged into secretory vesicles in the monensin-sensitive, trans-Golgi network. In contrast, in root tip cortical parenchyma cells, the anti-RG-I and the anti-XG antibodies are shown to bind to complementary subsets of Golgi cisternae, and several lines of indirect evidence suggest that these complex polysaccharides may also exit from different cisternae. Thus, RG-I type polysaccharides appear to be synthesized in cis- and medial cisternae, and have the potential to leave from a monensin-insensitive, medial cisternal compartment. The labeling pattern for XG suggests that it is assembled in trans-Golgi cisternae and departs from the monensin-sensitive trans-Golgi network. This physical separation of the synthesis/secretion pathways of major categories of complex polysaccharides may prevent the synthesis of mixed polysaccharides, and provides a means for producing secretory vesicles that can be targeted to different cell wall domains.