Cell wall-localized BETA-XYLOSIDASE4 contributes to immunity of Arabidopsis against Botrytis cinerea.

Plant cell walls constitute physical barriers that restrict access of microbial pathogens to the contents of plant cells. The primary cell wall of multicellular plants predominantly consists of cellulose, hemicellulose, and pectin, and its composition can change upon stress. BETA-XYLOSIDASE4 (BXL4) belongs to a seven-member gene family in Arabidopsis (Arabidopsis thaliana), one of which encodes a protein (BXL1) involved in cell wall remodeling. We assayed the influence of BXL4 on plant immunity and investigated the subcellular localization and enzymatic activity of BXL4, making use of mutant and overexpression lines. BXL4 localized to the apoplast and was induced upon infection with the necrotrophic fungal pathogen Botrytis cinerea in a jasmonoyl isoleucine-dependent manner. The bxl4 mutants showed a reduced resistance to B. cinerea, while resistance was increased in conditional overexpression lines. Ectopic expression of BXL4 in Arabidopsis seed coat epidermal cells rescued a bxl1 mutant phenotype, suggesting that, like BXL1, BXL4 has both xylosidase and arabinosidase activity. We conclude that BXL4 is a xylosidase/arabinosidase that is secreted to the apoplast and its expression is upregulated under pathogen attack, contributing to immunity against B. cinerea, possibly by removal of arabinose and xylose side-chains of polysaccharides in the primary cell wall.

RUBY, a Putative Galactose Oxidase, Influences Pectin Properties and Promotes Cell-To-Cell Adhesion in the Seed Coat Epidermis of Arabidopsis.

Cell-to-cell adhesion is essential for establishment of multicellularity. In plants, such adhesion is mediated through a middle lamella composed primarily of pectic polysaccharides. The molecular interactions that influence cell-to-cell adhesion are not fully understood. We have used Arabidopsis (Arabidopsis thaliana) seed coat mucilage as a model system to investigate interactions between cell wall carbohydrates. Using a forward-genetic approach, we have discovered a gene, RUBY PARTICLES IN MUCILAGE (RUBY), encoding a protein that is annotated as a member of the Auxiliary Activity 5 (AA5) family of Carbohydrate-Active Enzymes (Gal/glyoxal oxidases) and is secreted to the apoplast late in the differentiation of seed coat epidermal cells. We show that RUBY is required for the Gal oxidase activity of intact seeds; the oxidation of Gal in side-chains of rhamnogalacturonan-I (RG-I) present in mucilage-modified2 (mum2) mucilage, but not in wild-type mucilage; the retention of branched RG-I in the seed following extrusion; and the enhancement of cell-to-cell adhesion in the seed coat epidermis. These data support the hypothesis that RUBY is a Gal oxidase that strengthens pectin cohesion within the middle lamella, and possibly the mucilage of wild-type seed coat epidermal cells, through oxidation of RG-I Gal side-chains.

Three novel transcription factors involved in cannabinoid biosynthesis in Cannabis sativa L.

KEY MESSAGE: Three novel transcription factors were successfully identified and shown to interact with the trichome-specific THCAS promoter regulatory region. Cannabinoids are important secondary metabolites present in Cannabis sativa L. (cannabis). One cannabinoid that has received considerable attention, 9-tetrahydrocannabinol (THC), is derived from Delta-9-Tetrahydrocannabinolic acid (THCA) and responsible for the mood-altering and pain-relieving effects of cannabis. A detailed understanding of transcriptional control of THCA synthase (THCAS) is currently lacking. The primary site of cannabinoid biosynthesis is the glandular trichomes that form on female flowers. Transcription factors (TFs) have been shown to play an important role in secondary-metabolite biosynthesis and glandular trichome formation in Artemisia annua, Solanum lycopersicum and Humulus lupulus. However, analogous information is not available for cannabis. Here, we characterize a 548 bp fragment of the THCAS promoter and regulatory region that drives trichome-specific expression. Using this promoter fragment in a yeast-one-hybrid screen, we identified 3 novel TFs (CsAP2L1, CsWRKY1 and CsMYB1) and provided evidence that these 3 TFs regulate the THCAS promoter in planta. The O-Box element within the proximal region of the THCAS promoter is necessary for CsAP2L1-induced transcriptional activation of THCAS promoter. Similar to THCAS, the genes for all three TFs have trichome-specific expression, and subcellular localization of the TFs indicates that all three proteins are in the nucleus. CsAP2L1 and THCAS exhibit a similar temporal, spatial and strain-specific gene expression profiles, while those expression patterns of CsWRKY1 and CsMYB1 are opposite from THCAS. Our results identify CsAP2L1 playing a positive role in the regulation of THCAS expression, while CsWRKY1 and CsMYB1 may serve as negative regulators of THCAS expression.

Pectin Modification in Seed Coat Mucilage by In Vivo Expression of Rhamnogalacturonan-I- and Homogalacturonan-Degrading Enzymes.

The cell wall is essential for plant survival. Determining the relationship between cell wall structure and function using mutant analysis or overexpressing cell wall-modifying enzymes has been challenging due to the complexity of the cell wall and the appearance of secondary, compensatory effects when individual polymers are modified. In addition, viability of the plants can be severely impacted by wall modification. A useful model system for studying structure-function relationships among extracellular matrix components is the seed coat epidermal cells of Arabidopsis thaliana. These cells synthesize relatively simple, easily accessible, pectin-rich mucilage that is not essential for plant viability. In this study, we expressed enzymes predicted to modify polysaccharide components of mucilage in the apoplast of seed coat epidermal cells and explored their impacts on mucilage. The seed coat epidermal-specific promoter TESTA ABUNDANT2 (TBA2) was used to drive expression of these enzymes to avoid adverse effects in other parts of the plant. Mature transgenic seeds expressing Rhamnogalacturonate lyase A (RglA) or Rhamnogalacturonate lyase B (RglB) that degrade the pectin rhamnogalacturonan-I (RG-I), a major component of mucilage, had greatly reduced mucilage capsules surrounding the seeds and concomitant decreases in the monosaccharides that comprise the RG-I backbone. Degradation of the minor mucilage component homogalacturonan (HG) using the HG-degrading enzymes Pectin lyase A (PLA) or ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE2 (ADPG2) resulted in developing seed coat epidermal cells with disrupted cell-cell adhesion and signs of early cell death. These results demonstrate the feasibility of manipulating the seed coat epidermal cell extracellular matrix using a targeted genetic engineering approach.

Seed Mucilage: Biological Functions and Potential Applications in Biotechnology.

In plants, the diaspore (seed dispersal unit) may include a seed coat and/or pericarp to protect the embryo and assist in dispersion. In many species, the seed coat and/or pericarp secrete a gelatinous mixture of cell wall polysaccharides known as mucilage. In several species, mucilage synthesis, secretion and modification have been studied extensively as model systems for the investigation of plant cell wall structure and function. Despite this, efforts toward understanding the role of mucilage have received less attention. Mucilage has been hypothesized to impact seed dispersal through interaction with soil, protecting the seed in the gut following ingestion by animals or affecting the ability of seeds to sink or float in water. Mucilage has been found to influence seed germination and seedling establishment, most often during abiotic stress, probably by maintaining seed hydration when water is scarce. Finally, mucilage has been documented to mediate interactions with various organisms. Advances in transgenic technology should enable the genetic modification of mucilage structure and function in crop plants. Cells synthesizing mucilage may also be a suitable platform for creating custom polysaccharides or proteins with industrial applications. Thus, in the near future, it is likely that research on seed mucilage will expand well beyond the current focus. Here we summarize our understanding of the biological functions of mucilage and provide an outlook on the future of mucilage research.

Expression Patterns and Functional Characterization of Arabidopsis Galactose Oxidase-Like Genes Suggest Specialized Roles for Galactose Oxidases in Plants.

Galactose oxidases (GalOxs) are well-known enzymes that have been identified in several fungal species and characterized using structural and enzymatic approaches. However, until very recently, almost no information on their biological functions was available. The Arabidopsis (Arabidopsis thaliana) gene ruby particles in mucilage (RUBY) encodes a putative plant GalOx that is required for pectin cross-linking through modification of galactose (Gal) side chains and promotes cell-cell adhesion between seed coat epidermal cells. RUBY is one member of a family of seven putative GalOxs encoded in the Arabidopsis genome. To examine the function(s) of GalOxs in plants, we studied the remaining six galactose oxidase-like (GOXL) proteins. Like RUBY, four of these proteins (GOXL1, GOXL3, GOXL5 and GOXL6) were found to localize primarily to the apoplast, while GOXL2 and GOXL4 were found primarily in the cytoplasm. Complementation and GalOx assay data suggested that GOXL1, GOXL3 and possibly GOXL6 have similar biochemical activity to RUBY, whereas GOXL5 only weakly complemented and GOXL2 and GOXL4 showed no activity. Members of this protein family separated into four distinct clades prior to the divergence of the angiosperms. There have been recent duplications in Brassicaceae resulting in two closely related pairs of genes that have either retained similarity in expression (GOXL1 and GOXL6) or show expression divergence (GOXL3 and RUBY). Mutant phenotypes were not detected when these genes were disrupted, but their expression patterns suggest that these proteins may function in tissues that require mechanical reinforcements in the absence of lignification.

Arabidopsis FLYING SAUCER 2 Functions Redundantly with FLY1 to Establish Normal Seed Coat Mucilage.

Following exposure to water, mature Arabidopsis seeds are surrounded by a gelatinous capsule, termed mucilage. The mucilage consists of pectin-rich polysaccharides, which are produced in epidermal cells of the seed coat. Although pectin is a major component of plant cell walls, its biosynthesis and biological functions are not fully understood. Previously, we reported that a transmembrane RING E3 ubiquitin ligase, FLYING SAUCER 1 (FLY1) regulates the degree of pectin methyl esterification for mucilage capsule formation. The Arabidopsis thaliana genome has a single FLY1 homolog, FLY2. In this study, we show that the FLY2 protein functions in mucilage modification together with FLY1. FLY2 was expressed in seed coat epidermal cells during mucilage synthesis, but its expression level was much lower than that of FLY1. While fly2 showed no obvious difference in mucilage capsule formation from wild type, the fly1 fly2 double mutants showed more severe defects in mucilage than fly1 alone. FLY2-EYFP that was expressed under the control of the FLY1 promoter rescued fly1 mucilage, showing that FLY2 has the same molecular function as FLY1. FLY2-EYFP colocalized with marker proteins of Golgi apparatus (sialyltransferase-mRFP) and late endosome (mRFP-ARA7), indicating that as FLY1, FLY2 controls pectin modification by functioning in these endomembrane organelles. Furthermore, phylogenetic analysis suggests that FLY1 and FLY2 originated from a common ancestral gene by gene duplication prior to the emergence of Brassicaceae. Taken together, our findings suggest that FLY2 functions in the Golgi apparatus and/or the late endosome of seed coat epidermal cells in a manner similar to FLY1.

ECERIFERUM11/C-TERMINAL DOMAIN PHOSPHATASE-LIKE2 Affects Secretory Trafficking.

Secretory trafficking is highly conserved in all eukaryotic cells and is required for secretion of proteins as well as extracellular matrix components. In plants, the export of cuticular waxes and various cell wall components relies on secretory trafficking, but the molecular mechanisms underlying their secretion are not well understood. In this study, we characterize the Arabidopsis (Arabidopsis thaliana) dwarf eceriferum11 (cer11) mutant and we show that it exhibits reduced stem cuticular wax deposition, aberrant seed coat mucilage extrusion, and delayed secondary cell wall columella formation, as well as a block in secretory GFP trafficking. Cloning of the CER11 gene revealed that it encodes a C-TERMINAL DOMAIN PHOSPHATASE-LIKE2 (CPL2) protein. Thus, secretory trafficking in plant cells in general, and secretion of extracellular matrix constituents in developing epidermal cells in particular, involves a dephosphorylation step catalyzed by CER11/CPL2.

Assessing the utility of seed coat-specific promoters to engineer cell wall polysaccharide composition of mucilage.

KEY MESSAGE: Polysaccharide composition of seed mucilage was successfully modified using three seed coat-specific promoters driving expression of genes encoding cell wall-modifying enzymes. Arabidopsis thaliana seed coat epidermal cells synthesize and secrete large quantities of mucilage, a specialized secondary cell wall composed of cellulose, hemicellulose, and pectin. The composition and structure of mucilage confers its unique properties of expansion, extrusion, and adherence. We are developing seed mucilage as a model to study the biochemical and biological consequences of manipulating cell wall polysaccharides in vivo using cell wall-modifying enzymes. To specifically engineer mucilage composition and avoid altering other cell types, seed coat-specific promoters are required. In this study, we investigated the ability of seed coat-specific promoters from three genes, TESTA-ABUNDANT2 (TBA2), PEROXIDASE36 (PER36), and MUCILAGE-MODIFIED4 (MUM4), to express the cell wall modifying ß-galactosidase (BGAL)-encoding gene MUCILAGE-MODIFIED2 (MUM2) and complement the mum2 mutant. The strength of the three promoters relative to one another was found to vary by two to 250 fold, and correlated with their ability to rescue the mum2 mutant phenotype. The strongest of the three promoters, TBA2p, was then used to examine the ability of three MUM2 homologs to complement the mum2 extrusion and cell wall composition phenotypes. The degree of complementation was variable and correlated with the amino acid sequence similarity between the homologous gene products and MUM2. These data demonstrate that all three seed coat-specific promoters can drive expression of genes encoding carbohydrate-active enzymes in a spatial and temporal pattern sufficiently to modify polysaccharide composition in seed mucilage without obvious negative consequences to the rest of the plant.

Identification and Characterization of Arabidopsis Seed Coat Mucilage Proteins.

Plant cell wall proteins are important regulators of cell wall architecture and function. However, because cell wall proteins are difficult to extract and analyze, they are generally poorly understood. Here, we describe the identification and characterization of proteins integral to the Arabidopsis (Arabidopsis thaliana) seed coat mucilage, a specialized layer of the extracellular matrix composed of plant cell wall carbohydrates that is used as a model for cell wall research. The proteins identified in mucilage include those previously identified by genetic analysis, and several mucilage proteins are reduced in mucilage-deficient mutant seeds, suggesting that these proteins are genuinely associated with the mucilage. Arabidopsis mucilage has both nonadherent and adherent layers. Both layers have similar protein profiles except for proteins involved in lipid metabolism, which are present exclusively in the adherent mucilage. The most abundant mucilage proteins include a family of proteins named TESTA ABUNDANT1 (TBA1) to TBA3; a less abundant fourth homolog was named TBA-LIKE (TBAL). TBA and TBAL transcripts and promoter activities were detected in developing seed coats, and their expression requires seed coat differentiation regulators. TBA proteins are secreted to the mucilage pocket during differentiation. Although reverse genetics failed to identify a function for TBAs/TBAL, the TBA promoters are highly expressed and cell type specific and so should be very useful tools for targeting proteins to the seed coat epidermis. Altogether, these results highlight the mucilage proteome as a model for cell walls in general, as it shares similarities with other cell wall proteomes while also containing mucilage-specific features.

Identification of a seed coat-specific promoter fragment from the Arabidopsis MUCILAGE-MODIFIED4 gene.

KEY MESSAGE: The Arabidopsis seed coat-specific promoter fragment described is an important tool for basic and applied research in Brassicaceae species. During differentiation, the epidermal cells of the Arabidopsis seed coat produce and secrete large quantities of mucilage. On hydration of mature seeds, this mucilage becomes easily accessible as it is extruded to form a tightly attached halo at the seed surface. Mucilage is composed mainly of pectin, and also contains the key cell wall components cellulose, hemicellulose, and proteins, making it a valuable model for studying numerous aspects of cell wall biology. Seed coat-specific promoters are an important tool that can be used to assess the effects of expressing biosynthetic enzymes and diverse cell wall-modifying proteins on mucilage structure and function. Additionally, they can be used for production of easily accessible recombinant proteins of commercial interest. The MUCILAGE-MODIFIED4 (MUM4) gene is expressed in a wide variety of plant tissues and is strongly up-regulated in the seed coat during mucilage synthesis, implying the presence of a seed coat-specific region in its promoter. Promoter deletion analysis facilitated isolation of a 308 base pair sequence (MUM4 0.3Pro ) that directs reporter gene expression in the seed coat cells of both Arabidopsis and Camelina sativa, and is regulated by the same transcription factor cascade as endogenous MUM4. Therefore, MUM4 0.3Pro is a promoter fragment that serves as a new tool for seed coat biology research.

HIGHLY METHYL ESTERIFIED SEEDS is a pectin methyl esterase involved in embryo development.

Homogalacturonan pectin domains are synthesized in a highly methyl-esterified form that later can be differentially demethyl esterified by pectin methyl esterase (PME) to strengthen or loosen plant cell walls that contain pectin, including seed coat mucilage, a specialized secondary cell wall of seed coat epidermal cells. As a means to identify the active PMEs in seed coat mucilage, we identified seven PMEs expressed during seed coat development. One of these, HIGHLY METHYL ESTERIFIED SEEDS (HMS), is abundant during mucilage secretion, peaking at 7 d postanthesis in both the seed coat and the embryo. We have determined that this gene is required for normal levels of PME activity and homogalacturonan methyl esterification in the seed. The hms-1 mutant displays altered embryo morphology and mucilage extrusion, both of which are a consequence of defects in embryo development. A significant decrease in the size of cells in the embryo suggests that the changes in embryo morphology are a consequence of lack of cell expansion. Progeny from a cross between hms-1 and the previously characterized PME inhibitor5 overexpression line suggest that HMS acts independently from other cell wall-modifying enzymes in the embryo. We propose that HMS is required for cell wall loosening in the embryo to facilitate cell expansion during the accumulation of storage reserves and that its role in the seed coat is masked by redundancy.

Unidirectional movement of cellulose synthase complexes in Arabidopsis seed coat epidermal cells deposit cellulose involved in mucilage extrusion, adherence, and ray formation.

Cellulose synthase5 (CESA5) synthesizes cellulose necessary for seed mucilage adherence to seed coat epidermal cells of Arabidopsis (Arabidopsis thaliana). The involvement of additional CESA proteins in this process and details concerning the manner in which cellulose is deposited in the mucilage pocket are unknown. Here, we show that both CESA3 and CESA10 are highly expressed in this cell type at the time of mucilage synthesis and localize to the plasma membrane adjacent to the mucilage pocket. The isoxaben resistant1-1 and isoxaben resistant1-2 mutants affecting CESA3 show defects consistent with altered mucilage cellulose biosynthesis. CESA3 can interact with CESA5 in vitro, and green fluorescent protein-tagged CESA5, CESA3, and CESA10 proteins move in a linear, unidirectional fashion around the cytoplasmic column of the cell, parallel with the surface of the seed, in a pattern similar to that of cortical microtubules. Consistent with this movement, cytological evidence suggests that the mucilage is coiled around the columella and unwinds during mucilage extrusion to form a linear ray. Mutations in CESA5 and CESA3 affect the speed of mucilage extrusion and mucilage adherence. These findings imply that cellulose fibrils are synthesized in an ordered helical array around the columella, providing a distinct structure to the mucilage that is important for both mucilage extrusion and adherence.

Flying saucer1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed mucilage.

Pectins are complex polysaccharides that form the gel matrix of the primary cell wall and are abundant in the middle lamella that holds plant cells together. Their degree of methylesterification (DM) impacts wall strength and cell adhesion since unesterified pectin regions can cross-link via Ca(2+) ions to form stronger gels. Here, we characterize flying saucer1 (fly1), a novel Arabidopsis thaliana seed coat mutant, which displays primary wall detachment, reduced mucilage extrusion, and increased mucilage adherence. These defects appear to result from a lower DM in mucilage and are enhanced by the addition of Ca(2+) or completely rescued using alkaline Ca(2+) chelators. FLY1 encodes a transmembrane protein with a RING-H2 domain that has in vitro E3 ubiquitin ligase activity. FLY1 is orthologous to TRANSMEMBRANE UBIQUITIN LIGASE1, a Golgi-localized E3 ligase involved in the quality control of membrane proteins in yeast. However, FLY1-yellow fluorescent protein (YFP) fusions are localized in punctae that are predominantly distinct from the Golgi and the trans-Golgi network/early endosome in the seed coat epidermis. Wortmannin treatment, which induces the fusion of late endosomes in plants, resulted in enlarged FLY1-YFP bodies. We propose that FLY1 regulates the DM of pectin in mucilage, potentially by recycling pectin methylesterase enzymes in the endomembrane system of seed coat epidermal cells.

SALT-OVERLY SENSITIVE5 Mediates Arabidopsis Seed Coat Mucilage Adherence and Organization through Pectins.

Interactions between cell wall polymers are critical for establishing cell wall integrity and cell-cell adhesion. Here, we exploit the Arabidopsis (Arabidopsis thaliana) seed coat mucilage system to examine cell wall polymer interactions. On hydration, seeds release an adherent mucilage layer strongly attached to the seed in addition to a nonadherent layer that can be removed by gentle agitation. Rhamnogalacturonan I (RG I) is the primary component of adherent mucilage, with homogalacturonan, cellulose, and xyloglucan constituting minor components. Adherent mucilage contains rays composed of cellulose and pectin that extend above the center of each epidermal cell. CELLULOSE SYNTHASE5 (CESA5) and the arabinogalactan protein SALT-OVERLY SENSITIVE5 (SOS5) are required for mucilage adherence through unknown mechanisms. SOS5 has been suggested to mediate adherence by influencing cellulose biosynthesis. We, therefore, investigated the relationship between SOS5 and CESA5. cesa5-1 seeds show reduced cellulose, RG I, and ray size in adherent mucilage. In contrast, sos5-2 seeds have wild-type levels of cellulose but completely lack adherent RG I and rays. Thus, relative to each other, cesa5-1 has a greater effect on cellulose, whereas sos5-2 mainly affects pectin. The double mutant cesa5-1 sos5-2 has a much more severe loss of mucilage adherence, suggesting that SOS5 and CESA5 function independently. Double-mutant analyses with mutations in MUCILAGE MODIFIED2 and FLYING SAUCER1 that reduce mucilage release through pectin modification suggest that only SOS5 influences pectin-mediated adherence. Together, these findings suggest that SOS5 mediates adherence through pectins and does so independently of but in concert with cellulose synthesized by CESA5.

Demethylesterification of cell wall pectins in Arabidopsis plays a role in seed germination.

The methylesterification status of cell wall homogalacturonans, mediated through the action of pectin methylesterases (PMEs), influences the biophysical properties of plant cell walls such as elasticity and porosity, important parameters for cell elongation and water uptake. The completion of seed germination requires cell wall extensibility changes in both the radicle itself and in the micropylar tissues surrounding the radicle. In wild-type seeds of Arabidopsis (Arabidopsis thaliana), PME activities peaked around the time of testa rupture but declined just before the completion of germination (endosperm weakening and rupture). We overexpressed an Arabidopsis PME inhibitor to investigate PME involvement in seed germination. Seeds of the resultant lines showed a denser methylesterification status of their cell wall homogalacturonans, but there were no changes in the neutral sugar and uronic acid composition of the cell walls. As compared with wild-type seeds, the PME activities of the overexpressing lines were greatly reduced throughout germination, and the low steady-state levels neither increased nor decreased. The most striking phenotype was a significantly faster rate of germination, which was not connected to altered testa rupture morphology but to alterations of the micropylar endosperm cells, evident by environmental scanning electron microscopy. The transgenic seeds also exhibited an apparent reduced sensitivity to abscisic acid with respect to its inhibitory effects on germination. We speculate that PME activity contributes to the temporal regulation of radicle emergence in endospermic seeds by altering the mechanical properties of the cell walls and thereby the balance between the two opposing forces of radicle elongation and mechanical resistance of the endosperm.

Arabidopsis glabra2 mutant seeds deficient in mucilage biosynthesis produce more oil.

Seed oil, one of the major seed storage compounds in plants, is of great economic importance for human consumption, as an industrial raw material and as a source of biofuels. Thus, improving the seed oil yield in crops is an important objective. The GLABRA2 (GL2) gene in Arabidopsis thaliana encodes a transcription factor that is required for the proper differentiation of several epidermal cell types. GL2 has also been shown to regulate seed oil levels, as a loss-of-function mutation in the GL2 gene results in plants with a higher seed oil content than wild-type. We have extended this observation by showing that loss-of-function mutations in several positive regulators of GL2 also result in a high seed oil phenotype. The GL2 gene is expressed in both the seed coat and embryo, but the embryo is the main site of seed oil accumulation. Surprisingly, our results indicate that it is loss of GL2 activity in the seed coat, not the embryo, that contributes to the high seed oil phenotype. One target of GL2 in the seed coat is the gene MUCILAGE MODIFIED 4 (MUM4), which encodes a rhamnose synthase that is required for seed mucilage biosynthesis. We found that mum4 mutant seeds, like those of gl2 mutants, have an increased seed oil content in comparison with wild-type. Therefore, GL2 regulates seed oil production at least partly through its influence on MUM4 expression in the seed coat. We propose that gl2 mutant seeds produce more oil due to increased carbon allocation to the embryo in the absence of seed coat mucilage biosynthesis.

Identification and analysis of an outer-seed-coat-specific promoter from Arabidopsis thaliana.

Differentiation of the Arabidopsis thaliana (Arabidopsis) seed coat epidermal cells involves pronounced changes highlighted by the synthesis and secretion of copious amounts of dispensable, pectinaceous mucilage followed by a thick cellulosic secondary cell wall. This cell type, therefore, represents an excellent molecular-genetic model to study the biosynthesis and modification of cell wall components, particularly pectin. To support such research, we sought to identify a promoter that drives expression specifically in the Arabidopsis seed coat epidermis. Arabidopsis seed coat microarray data was analysed for genes expressed in the wild type seed coat but not the seed coat of the apetala2 mutant where the epidermal cells fail to differentiate. Of 14 candidate genes, 9 showed a seed-specific expression pattern by reverse transcriptase-PCR. Transcriptional regulatory region-ß-glucuronidase (GUS) reporter gene fusions introduced into Arabidopsis identified one promoter, that of the DIRIGENT PROTEIN1 (DP1) gene, as seed coat specific. The specificity of the expression was confirmed using a second reporter gene, Citrine YFP. Expression of both reporter genes was limited to the epidermal and palisade cell layers of the seed coat. Quantitative PCR data using wild type seed coat RNA suggested that the promoter is particularly active at 7 days post anthesis. The DP1 promoter was able to direct transcription of GUS in a similar pattern in the Brassica napus seed coat. Thus, in addition to its application in studying the plant cell wall, this promoter will provide an experimental tool for expressing high-valued recombinant proteins as well as modifying seed coat traits in economically important crops.

Cell wall polysaccharides are mislocalized to the Vacuole in echidna mutants.

During cell wall biosynthesis, the Golgi apparatus is the platform for cell wall matrix biosynthesis and the site of packaging, of both matrix polysaccharides and proteins, into secretory vesicles with the correct targeting information. The objective of this study was to dissect the post-Golgi trafficking of cell wall polysaccharides using echidna as a vesicle traffic mutant of Arabidopsis thaliana and the pectin-secreting cells of the seed coat as a model system. ECHIDNA encodes a trans-Golgi network (TGN)-localized protein, which was previously shown to be required for proper structure and function of the secretory pathway. In echidna mutants, some cell wall matrix polysaccharides accumulate inside cells, rather than being secreted to the apoplast. In this study, live cell imaging of fluorescent protein markers as well as transmission electron microscopy (TEM)/immunoTEM of cryofixed seed coat cells were used to examine the consequences of TGN disorganization in echidna mutants under conditions of high polysaccharide production and secretion. While in wild-type seed coat cells, pectin is secreted to the apical surface, in echidna, polysaccharides accumulate in post-Golgi vesicles, the central lytic vacuole and endoplasmic reticulum-derived bodies. In contrast, proteins were partially mistargeted to internal multilamellar membranes in echidna. These results suggest that while secretion of both cell wall polysaccharides and proteins at the TGN requires ECHIDNA, different vesicle trafficking components may mediate downstream events in their secretion from the TGN.