Plant Physiology Preview. Published on December 21, 2016, as DOI:10.1104/pp.16.01600
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Running head: Identification of seed coat mucilage proteins
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Corresponding author: George W. Haughn
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Department of Botany, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada
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+1 604 822-9089
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[email protected]
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Title: Identification and characterization of seed coat mucilage proteins1
Allen Yi-Lun Tsai2, Tadashi Kunieda3, Jason Rogalski, Leonard J. Foster, Brian E. Ellis, George
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W. Haughn*
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Department of Botany (A.Y.-L.T., T.K., G.W.H), Michael Smith Laboratories (A.Y.-L.T., J.R.,
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L.J.F., B.E.E.), and Department of Biochemistry & Molecular Biology (L.J.F.), University of
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British Columbia, Vancouver, British Columbia, Canada V6T 1Z4
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1
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Discovery Grants to B.E.E. and G.W.H.), the BC Proteomic Network Graduate/Postdoctoral
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Training Grant (A.Y.-L.T.), the NSERC Collaborative Research and Training Experience
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(CREATE) Program Working on Walls (A.Y.-L.T., G.W.H., B.E.E.), and the Japan Society for
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the Promotion of Science (JSPS) Postdoctoral Fellowship for Research Abroad (T.K.).
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2
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Kurokami, Chuo-ku, Kumamoto 860-8555, Japan
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3
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8-9-1 Okamoto, Higashinada-ku, Kobe 658-8501, Japan
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*Address correspondence to [email protected]
This work was supported by the National Sciences and Engineering Research Council (NSERC,
Present address: Graduate School of Science and Technology, Kumamoto University, 2-39-1
Present address: Department of Biology, Faculty of Science and Engineering, Konan University
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Summary: Arabidopsis seed coat mucilage, an extracellular matrix composed of cell wall
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carbohydrates, contains a proteome functionally similar to that of cell wall but also include
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proteins unique to mucilage.
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List of author contributions: A.Y.-L.T. designed the research; A.Y.-L.T., T.K. and J.R.
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performed the research; A.Y.-L.T., L.J.F., B.E.E. and G.W.H. analyzed the data and wrote the
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article.
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Abstract
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Plant cell wall proteins are important regulators of cell wall architecture and function. However,
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because cell wall proteins are difficult to extract and analyze, they are generally poorly
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understood. Here we describe the identification and characterization of proteins integral to the
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Arabidopsis (Arabidopsis thaliana) seed coat mucilage, a specialized layer of the extracellular
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matrix composed of plant cell wall carbohydrates that is used as a model for cell wall research.
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The proteins identified in mucilage include those previously identified by genetic analysis, and
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several mucilage proteins are reduced in mucilage-deficient mutant seeds, suggesting that these
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proteins are genuinely associated with the mucilage. Arabidopsis mucilage has both non-
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adherent and adherent layers. Both layers have similar protein profiles except for proteins
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involved in lipid metabolism, which are present exclusively in the adherent mucilage. The most
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abundant mucilage proteins include a family of proteins named TESTA ABUNDANT (TBA)
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1~3; a less abundant fourth homologue was named TBA-LIKE (TBAL). TBA and TBAL
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transcripts and promoter activities were detected in developing seed coats, and their expression
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requires seed coat differentiation regulators. TBA proteins are secreted to the mucilage pocket
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during differentiation. Although reverse genetics failed to identify a function for TBAs/TBAL,
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the TBA promoters are highly expressed and cell-type specific and so should be very useful tools
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for targeting proteins to the seed coat epidermis. Altogether, these results highlight the mucilage
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proteome as a model for cell walls in general as it shares similarities with other cell wall
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proteomes while also containing mucilage-specific features.
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Introduction
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The plant cell wall plays key roles in structural support, cell-cell cohesion and interaction
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of the cell with the environment. It is a dynamic structure and can be strengthened or loosened in
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response to environmental or developmental cues (Fry, 2004; Passardi et al., 2004). Plant cell
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walls typically contain cellulose and hemicellulose, and may include pectin or lignin depending
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on the type of wall. In addition to these carbohydrate components, 5~10% of the cell wall
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biomass consists of proteins (Cassab and Varner, 1988; Burton et al., 2010). Despite being a
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relatively minor component in terms of cell wall biomass, these proteins are critical regulators of
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the cell wall architecture and, therefore, its physical properties. For example, structural proteins
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can cross-link various cell wall polysaccharides (Showalter, 1993) while carbohydrate-active
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enzymes modify polysaccharide structure.
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Since cell wall proteins are generally difficult to extract and analyze, they remain a
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relatively poorly understood component of the cell wall. Several factors complicate the analysis
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of cell wall proteins. First, they often undergo extensive post-translational modifications such as
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proline hydroxylation, glycosylation and addition of glycosylphosphatidylinositol (GPI) anchors
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(Jamet et al., 2008b; Albenne et al., 2013). These modifications not only alter protein mass,
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thereby complicating protein identification, but they can also anchor the proteins in the apoplast
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by covalent or non-covalent interactions (Kieliszewski and Lamport, 1994; Spiro, 2002) which
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make cell wall protein extraction and identification more challenging. Extraction of cell wall
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proteins typically requires harsh conditions (Lee et al., 2004, Jamet et al., 2008b) that often lead
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to protein degradation and contamination with cytoplasmic proteins, with a resulting decrease in
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the quality of proteomic data. In addition, the cell wall resides in extracellular space and abuts
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the perimeters of adjacent cells. Since a variety of cell types with distinctive cells walls are found
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in most tissues and organs, it is common that cell wall extracts typically include carbohydrate
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and proteins derived from multiple cell types, and the relative contribution of specific cell types
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is difficult to assess. Despite these problems, several studies have characterized cell wall
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proteomes from different tissue types in various plant species, including the model plant
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Arabidopsis (reviewed in Albenne et al., 2013). Out of the ~5000 Arabidopsis genes that encode
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a predicted signal peptide to allow a protein to enter the secretory pathway, 1000~2000 are
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thought to be cell wall proteins (Jamet et al., 2006). However, currently most published cell wall
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proteomes contain less than 100 proteins each and are contaminated by cytoplasmic proteins to a
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variable extent depending on the tissue type and extraction techniques (Albenne et al., 2013).
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This suggests that many cell wall proteins remain to be discovered and characterized, and
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emphasizes the need for better models and more robust methodologies.
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Arabidopsis seed coat mucilage is a specialized layer of the extracellular matrix
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composed of cell wall carbohydrates arranged in a distinct structure (reviewed in Arsovski et al.,
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2010; Western, 2012; Haughn and Western, 2012; North et al., 2014, Voiniciuc et al., 2015a)
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that is used as a model to study cell wall structure and function. It contains cellulose and
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hemicellulose (Macquet et al., 2007b; Young et al., 2008; Harpaz-Saad et al., 2011; Mendu et al.,
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2011; Sullivan et al., 2011; Griffiths et al., 2014; Yu et al., 2014; Voiniciuc et al., 2015b;
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Voiniciuc et al., 2015c; Hu et al., 2016a; Hu et al., 2016b) but is particularly rich in pectin, with
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unsubstituted rhamnogalacturonan I (RGI) making up ~85% of the total mucilage carbohydrate
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(Western et al., 2000, Western et al., 2001; Willats et al., 2001; Dean et al., 2007; Macquet al.,
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2007b; Young et al., 2008). Similar to cell walls, Arabidopsis seed coat mucilage also contains
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proteins. Forward and reverse genetics studies have identified several loci required for proper
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mucilage synthesis, secretion and extrusion (reviewed in Haughn and Chaudhury, 2005;
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Arsovski et al., 2010; Western, 2012; Haughn and Western, 2012; North et al., 2014; Francoz et
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al., 2015). Several of these gene products are believed to be secreted to the mucilage pocket or
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adjacent primary wall in the developing seed coat. For example, the mucilage-modifying enzyme
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MUCILAGE MODIFIED 2 (MUM2) is secreted to the mucilage pocket during mucilage
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synthesis (Western et al., 2001; Dean et al., 2007; Macquet et al., 2007a). PEROXIDASE 36
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(PER36) has been shown to localize to the radial and tangential primary cell wall adjacent to the
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mucilage pocket (Kunieda et al., 2013). Two other genes that encode proteins needed for normal
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mucilage, SUBTILISIN-LIKE SERINE PROTEASE 1.7 (SBT1.7; Rautengarten et al. 2008) and
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arabinofuranosidase BETA-XYLOSIDASE 1 (BXL1; Arsovski et al., 2009) contain signal
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peptides and modify mucilage carbohydrates. However, a thorough analysis of mucilage proteins
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has not been described.
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The deposition of seed coat mucilage is known as myxospermy, and is common in
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angiosperms (Young and Evans, 1973; Grubert, 1974). During differentiation, Arabidopsis seed
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coat epidermal cells synthesize mucilage components and deposit them between the plasma
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membrane and primary wall at the junction between the radial and tangential cell walls, forming
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a ring-shaped mucilage pocket surrounding a volcano-shaped cytoplasmic column (Western et
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al., 2000; Windsor et al., 2000). A cellulose-rich secondary cell wall, the columella, is
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subsequently deposited beneath the mucilage, gradually replacing the cytoplasm (Western et al.,
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2000; Windsor et al., 2000). Upon exposure of mature seeds to water, the pectin-rich mucilage
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swells rapidly, ruptures the primary wall and extrudes to encapsulate the seed. The extruded
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Arabidopsis seed mucilage has at least two distinct layers, non-adherent and adherent (Western
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et al., 2000; Macquet et al., 2007b). The outermost layer (non-adherent layer) is amorphous in
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appearance, composed primarily of pectin and, as its name suggests, easily separated from the
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seed by gentle shaking. The layer of mucilage adjacent to the seed coat has a distinct ray-like
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structure, has cellulose and hemicellulose in addition to pectin and is strongly adherent to the
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seed surface. Relative to cell wall preparations from most other tissue types, seed coat mucilage
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can be easily extracted in large amounts without contamination with cell wall material from other
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cell types (Haughn and Chaudhury, 2005; Haughn and Western, 2012; North et al., 2014). These
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advantages suggest seed coat mucilage can yield cell wall proteomes that are potentially of
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higher quality than cell wall proteomes derived from other tissues. Here we describe the
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extraction and proteomic analysis of the mature Arabidopsis seed coat mucilage, and discuss the
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protein profiles of the mucilage in comparison with other cell wall proteomes. In addition, we
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characterize a family of unknown proteins that are particularly abundant in seed coat mucilage
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and strongly expressed in the developing seed coat.
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Results
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Proteins are a component of mucilage extracted from Arabidopsis seeds
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In order to identify and characterize proteins integral to the seed coat mucilage, a
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protocol was developed to extract seed coat mucilage for protein analyses (Figure 1). Upon
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hydration, Arabidopsis seed coat epidermal cells extrude mucilage as two distinct layers – an
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outer non-adherent layer that detaches easily from the hydrated seed and a dense halo-like
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adherent layer that is bound tightly to the seed coat (Figure 1A, Western et al., 2000). We took
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advantage of the different physical properties of these two layers to separate them by sequential
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extraction (Figure 1B). Seeds imbibed in water were shaken gently to separate the non-adherent
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mucilage. The seeds were then shaken at high speed for several hours to remove the adherent
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mucilage (Figure 1B). Ruthenium Red and calcofluor staining showed that the pectin and
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cellulosic components of seed coat mucilage were almost completely removed by the sequential
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extraction (Figure 1B, Supplemental Figure S1). The harvested adherent and non-adherent
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mucilage samples were chemically de-glycosylated, trypsin-digested and analyzed by mass
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spectrometry to identify mucilage-associated proteins in these samples (Figure 1B; Supplemental
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Datasets S1 and S2). Of those proteins detected by this process, only the ones identified in more
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than one biological replicates with MASCOT scores > 40 (where score ≥ 25 corresponds to 5%
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false-discovery rate), and at least once with multiple peptides, were considered for further
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analyses. Based on these criteria, 30 proteins were considered to be robustly identified from
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mucilage, all contain predicted signal peptides (Supplemental Dataset S3). Cruciferin A1 and
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cruciferin C were discarded from further analyses, since they are not known to be secreted to the
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apoplast. This leaves a total 28 potential mucilage proteins identified (Supplemental Dataset S3,
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Table 1). 1 protein was found only in the non-adherent layer, 15 only in the adherent layer, while
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the remaining 12 proteins were found in both mucilage layers (Table 1).
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Mucilage proteins are functionally similar to other cell wall proteins
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When proteins found in each mucilage layer were sorted by their predicted functions
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(Table 1, Figure 2A), most fell within the various functional categories of cell wall proteins as
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defined by Albenne et al., 2013. These categories include carbohydrate-active enzymes, oxido-
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reductases, proteases, proteins involved in lipid metabolism and arabinogalactan proteins
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(AGPs), as well as miscellaneous proteins and proteins with unknown functions. The fact that
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seed coat mucilage-associated proteins appear to be functionally similar to proteins from other
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Arabidopsis cell wall proteomes reinforces the concept that seed coat mucilage is a specialized
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type of cell wall (Haughn and Western, 2012). On the other hand, nearly half of the specific
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mucilage-associated protein isoforms were unique to mucilage and not identified in other cell
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wall proteomes to date (Figure 2B) including a family of unknown proteins (discussed below),
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RmlC-like cupins superfamily proteins and GDSL lipases (Table 1) . Homologues of RmlC-like
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cupins superfamily proteins and GDSL lipases are commonly found in other cell walls (Albenne
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et al., 2013), suggesting these proteins may represent mucilage-specific isoforms.
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In general, the adherent layer displays a richer and more diverse protein profile compared
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to the non-adherent layer, including 15 proteins that are unique to the adherent layer (Table 1).
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Interestingly, this adherent-specific group includes a number of proteins involved in lipid
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metabolism (Figure 2A). Otherwise, the numbers of proteins belong to each predicted functional
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class are more or less comparable between the two mucilage layers (Figure 2A). This suggests
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that the types of protein-mediated biological processes associated with mucilage modification in
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the apoplast are comparable within the two layers.
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Since the seeds from which mucilage was obtained in these experiments had not been
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processed in any way prior to hydration and mucilage extraction, the possibility remains that the
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proteins we identified originate from sources other than the mucilage. In order to address this
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concern, the non-adherent mucilage protein profiles obtained from Col-0 seeds were compared
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with protein profiles obtained from the seed surface extracts from the mucilage mutants mucilage
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modified 2-10 (mum2-10) and apetala 2-7 (ap2-7) (Figure 3A). mum2-10 seeds synthesize
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mucilage, but do not extrude it when hydrated (Dean et al., 2007; Macquet et al., 2007a),
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whereas ap2-7 seed coat epidermal cells fail to differentiate, and therefore do not synthesize
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mucilage (Jofuku et al., 1994; Western et al., 2001; Dean et al., 2011). Since mucilage can only
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be extracted from hydrated Col-0 seeds, proteins that are significantly over-represented in Col-0
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non-adherent mucilage compared to mum2-10 and/or ap2-7 seed surface extracts would be
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predicted to be derived from the extruded mucilage. Several mucilage proteins identified were
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indeed found at much higher levels in Col-0 compared to mum2-10 and ap2-7 (Figure 3B,
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Supplemental Datasets S4 and S5). Overall, the recovery of mucilage-associated protein was
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reduced by ~90% when mum2-10 seed was used, and by ~99% when ap2-7 seed was used as
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compared to Col-0 seed (Figure 3B). These data support the hypothesis that proteins identified in
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this study are derived from extruded mucilage of seed coat epidermal cells and not from the
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primary wall.
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The identity of many mucilage proteins is consistent with a role in mucilage/cell wall
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modification
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The collection of enzymes identified by our proteomics analyses includes all the secreted
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enzymes required for normal mucilage extrusion that have been previously identified by genetic
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analysis: MUM2 (At5g63800/Q9FFN4; Dean et al., 2007), BXL1 (At5g49360/Q9FGY1;
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Arsovski et al., 2009), PER36 (At3g50990/Q9SD46; Kunieda et al., 2013) and SBT1.7
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(At5g67360/O65351; Rautengarten et al., 2008) (Table 1). Their identification here thus
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validates the robustness of the proteomic analysis.
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In an attempt to determine the roles of other mucilage proteins we identified, plant lines
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with T-DNA insertions in the genes BETA-GLUCOSIDASE 44 (BGLU44, At3g18080/Q9LV33),
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BETA-HEXOSAMINIDASE 3 (HEXO3, At1g65590/Q8L7S6), ASPARTIC PROTEASE IN
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GUARD CELL 1 (ASPG1, At3g18490/Q9LS40), AUXIN INDUCIBLE IN ROOTS 12 (AIR12,
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At3g07390/Q94BT2), RESPONSIVE TO DEHYDRATION 1a (RD21a, At1g47128/P43297) and
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SERINE CARBOXYPEPTIDASE-LIKE 35 (SCPL35, At5g08260/Q9LEY1) were characterized
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(Supplemental Figure S2, Supplemental Table S1). These genes were chosen because they don’t
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appear to have homologues that are also expressed in seed coat epidermal cells, thus decreasing
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the possibility of functional redundancy obscuring mutant phenotypes. In each case, the T-DNA
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insertion decreased or eliminated the steady state levels of transcript in homozygous lines
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(Supplemental Figure S2). Seeds of each insertional mutant were imbibed in either water, 0.05 M
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ethylenediaminetetraacetic acid (EDTA), 0.05 M CaCl2, or 0.5 M Na2CO3, stained with
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Ruthenium Red and examined for seed mucilage abnormalities. EDTA is believed to loosen
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mucilage by disrupting the homogalacturonan salt bridges through Ca2+ chelation (Western et al.,
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2001; Rautengarten et al., 2008; Saez-Aguayo et al., 2013; Voiniciuc et al., 2013). Na2CO3
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treatment also loosens mucilage, possibly by cleaving cross-linking ester bonds between
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homogalacturonan polymers (Selvendran and Ryden, 1990; Fry, 2000; McCartney and Knox
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2002). In contrast, mucilage extruded in a CaCl2 solution is more compact and stains more
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intensely with Ruthenium Red than mucilage extruded in water, presumably by enhancing Ca2+
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salt bridging between mucilage homogalacturonan molecules. However, no clear mucilage
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defects were found in any the mucilage protein mutant lines under the conditions tested
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(Supplemental Figure S3), suggesting that if the corresponding gene products have a role in
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mucilage modification, the mutant phenotype must be relatively subtle or conditional.
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TBA proteins were found to be highly abundant in seed coat mucilage
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Among the mucilage proteins identified, three proteins of the unknown protein family
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0540 (UPF0540) At1g62000/Q39168, At1g62060/O04573 and At1g62080/O04575 were of
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particular interest because they were consistently identified as the most abundant proteins in
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almost all samples (Table 1, Supplemental Dataset S4). Consistent with these protein data, the
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corresponding genes were found to be expressed in the seed coat at very high levels
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(Supplemental Figure S4, Schmid et al., 2005; Winter et al., 2007; Dean et al., 2011; Le et al.,
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2010). However, the proteins encoded by these genes do not contain known functional domains
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other than putative signal peptides, so no function has been ascribed to them to date. Members of
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the UPF0540 protein family are strongly conserved, as they share 79% amino acid sequence
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identity and 81% similarity with one another (Figure 4). Furthermore, the loci that encode these
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proteins are tightly clustered on chromosome 1, suggesting the gene family may have expanded
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through tandem duplication events. Due to the abundance of the UPF0540 proteins in the seed
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coat, these genes were named TESTA ABUNDANT 1 (TBA1, At1g62000/Q39168), TBA2
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(At1g62060/O04573) and TBA3 (At1g62080/O04575). Interestingly, a peptide from a 4th
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member of the UPF0540 family, At1g62220/O04587, was also detected in adherent mucilage
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(Supplemental Dataset S1 and S3). However, At1g62220/O04587 was identified with only one
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peptide with a score below the cut-off for statistical significance. Due to the strong similarities in
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amino acid sequences and expression patterns between At1g62220/O04587 and the TBA
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proteins, At1g62220/O04587 was named TBA-LIKE (TBAL).
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TBAs are small proteins (~150 aa) with many conserved Ser and Thr residues predicted
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by NetOGlyc to be O-glycosylated (Steentoft et al., 2013) (Figure 4). These characteristics
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suggest TBAs and TBAL may function as structural proteins that interact with various
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polysaccharides in seed coat mucilage.
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TBA proteins are synthesized in the developing seed coat epidermis and secreted to the
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apoplast
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Public microarray data suggests that TBA and TBAL are expressed uniquely in the seed
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coat, and reverse transcription polymerase chain reaction (RT-PCR) results are consistent with
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this pattern (Figure 5). TBA and TBAL transcripts could only be detected in siliques (Figure 5A)
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and more specifically in the 7- and 10-days post-anthesis (DPA) seed coat (Figure 5B). TBA2
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expression levels are by far the highest, and peaked at 7 DPA (coinciding with mucilage
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synthesis; Figure 5B), whereas the expression levels of the remaining genes were lower and
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peaked at 10 DPA (coinciding with columella synthesis; Figure 5B).
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To verify the expression pattern of TBA and TBAL, reporter assays were performed on
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tissues of plants carrying chimeric genes encoding the β-glucuronidase gene (GUS) under the
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control of the TBA and TBAL native promoters. Consistent with RT-PCR data, GUS activity
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could be detected in developing seeds 7 and 10 DPA (Figure 6B) but not in seedlings, leaves,
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stems (Figure 6A) and embryo (Figure 6B). GUS under the control of TBA2p appeared earlier
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compared to other promoters, though in general all TBA and TBAL promoters were active by 10
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DPA (Figure 6B). These data support the hypothesis that all four promoters are primarily active
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in the seed coat. The fact that the TBA-promoter-GUS patterns mirror the presence of TBA
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transcripts implies that the expression of the TBA genes is largely regulated by their upstream
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cis-regulatory elements.
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Several transcription factors are known to regulate the differentiation of seed coat
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epidermis and the synthesis of seed coat mucilage. Some of these master regulators include
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NAC-REGULATED SEED MORPHOLOGY 1 (NARS1), NARS2, MUCILAGE MODIFIED 1
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(MUM1), MYELOBLASTOSIS 61 (MYB61) and TRANSPARENT TESTA GLABRA 1
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(TTG1) (Koornneef, 1981; Penfield et al., 2001; Kunieda et al., 2008; Huang et al., 2011). Since
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the TBA genes are expressed exclusively in the seed coat, we asked whether they are under
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control of these transcription factors. RT-PCR results showed that TBA and TBAL transcripts
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were absent in developing seeds of both the nars1 nars2 double mutant and ttg1-1 (Figure 7),
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which suggests that NARS1/NARS2 and TTG1 are all required for TBA and TBAL expression.
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Intriguingly, TBA2 transcripts appear to be absent in the Landsberg erecta ecotype, suggesting
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there may be TBA expression variation among different natural accessions.
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The sub-cellular localization of TBAs was characterized using C-terminally tagged
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citrine-yellow fluorescent protein (cYFP) TBA translational fusion constructs driven by their
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endogenous promoters. In agreement with the expression data, cYFP signals were detected in the
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developing seed coat. No cYFP signal was observed in 4 DPA seeds (Figure 8). By 6 DPA,
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TBA1-cYFP and TBA2-cYFP could be detected in the seed coat epidermal lateral cell walls and
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the developing mucilage pockets. All three TBAs could be detected in the mucilage pockets by 8
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DPA (Figure 8). By 10 DPA, the cYFP signal was absent from the mucilage pocket, but was
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observed in the developing columella (Figure 8). This expression pattern coincides
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spatiotemporally with the TBA transcript and promoter activity patterns (Figures 5 and 6), and
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reinforces the characterization of TBA proteins as mucilage proteins. Furthermore, cYFP
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fluorescence could only be detected in the outer epidermal layer of seed coat (Figure 8),
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suggesting that TBA proteins may only be synthesized in mucilage-secretory cells.
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Despite the fact that TBAs were initially identified in mature mucilage, TBA-cYFP
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fluorescence was absent from mucilage pockets by 10 DPA, which raised the possibility that
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TBAs might be unstable proteins. To test this idea, immunoblotting experiments were performed
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to try to detect TBA-cYFP extracted from developing siliques. Interestingly, full-length TBA-
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cYFP proteins were difficult to detect in the siliques of TBA-cYFP transgenic plants by
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immunoblotting. The abundance of TBA-cYFP was quite variable among different lines, and
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TBA3-cYFP appears to be partially insoluble (Supplemental Figure S5). These results suggest
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TBA-cYFP proteins are likely unstable and may undergo proteolysis or other post-translational
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modification within the mucilage pocket.
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TBA proteins are likely functionally redundant
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In an attempt to determine the function of the TBA proteins, we characterized tba loss-of-
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function mutants. Since the three TBA proteins are highly conserved and share a similar
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expression pattern, we anticipated that they might be functionally redundant. Further, because all
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these genes are closely linked on the chromosome, making the construction of double, triple and
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quadruple tba mutants relatively difficult. To overcome these problems, an artificial microRNA
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(amiRNA) driven by the UBIQUITIN EXTENSION PROTEIN 1 promoter (UBQ1p) was
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designed to knock-down all three TBA homologues and TBAL simultaneously. When TBA1
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transcript levels in developing siliques were quantified in 12 UBQ1p:TBA-amiRNA transgenic
313
lines, four lines showed significant down-regulation of TBA1, but only line #5 showed
314
significant down-regulation in all three TBA genes and TBAL (Figure 9A). However, none of the
315
UBQ1p:TBA-amiRNA lines showed any mucilage defects when their seeds were imbibed in
316
water, 0.05 M EDTA, 0.05 M CaCl2 or 0.5 M Na2CO3 followed by Ruthenium Red staining
317
(Figure 9B). These results suggest that either the TBA genes are functionally redundant even at
318
low levels of expression, or that the amiRNA knock-down lines possess mucilage phenotypes
319
that are not clearly discernable with Ruthenium Red staining.
320
321
Discussion
322
Proteins are an integral part of the Arabidopsis seed coat mucilage
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323
Arabidopsis seed coat mucilage is a specialized layer of the extracellular matrix
324
composed of cell wall carbohydrates arranged in a distinct structure in the apoplast of seed coat
325
epidermal cells. Because of its accessibility and dispensability, mucilage has been used as a
326
genetic model for studying structure and function of the plant cell wall (Arsovski et al., 2009;
327
Haughn and Western, 2012). Forward genetic analysis has enabled the identification of several
328
proteins that are secreted with mucilage and required for normal mucilage structure. To more
329
comprehensively define the array of proteins involved in mucilage structure and modification,
330
we have used proteomic analysis to examine mucilage extruded by mature Arabidopsis seeds.
331
The mucilage extracted by extensive shaking yielded protein preparations that possessed a
332
consistent array of secreted polypeptides relatively free of intracellular proteins. The 28 proteins
333
identified in Col-0 seed coat mucilage by this approach may not be very numerous, but they
334
represent the same classes of proteins found in the cell wall proteomes of other tissue types,
335
which generally contain less than 100 proteins (Albenne et al., 2013). This suggests the mucilage
336
protein extraction protocol is at least comparable with other cell wall proteome studies in terms
337
of protein recovery rate. Furthermore, the mucilage proteins identified include all proteins
338
believed to be secreted to the mucilage pocket that are not membrane-anchored: MUM2,
339
SBT1.7, BXL1 and PER36 (Dean et al., 2007; Macquet et al., 2007a; Rautengarten et al., 2008;
340
Arsovski et al., 2009; Kunieda et al., 2013). In addition, proteomic analysis of mucilage extracts
341
from the seeds of ap2 mutants that fail to differentiate a seed coat epidermis showed decreases
342
among the most abundant mucilage proteins with the exception of PER36 (see below) which
343
unlike the others, localizes to the primary cell wall surrounding the mucilage. Therefore, we
344
believe our method is sufficiently robust in characterizing the mature mucilage proteome.
345
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346
Seed coat mucilage is a suitable model for cell wall protein analyses
347
Most of the proteins of mature seed coat mucilage are functionally similar to proteins
348
found in primary cell walls from other tissues (Albenne et al., 2013; Figure 2), consistent with
349
the idea that mucilage and cell walls share many biosynthetic and functional processes. Seed coat
350
mucilage has the experimental advantage over other types of cell walls in that it is actively
351
extruded and can be extracted without tissue homogenization and associated cytoplasmic
352
contamination (Supplemental Datasets S1-S3). All of the mucilage proteins identified contain
353
predicted signal peptides, while proteins without signal peptides detected in mucilage generally
354
scored poorly (Supplemental Dataset S3), suggesting this method indeed strongly favours
355
apoplastic proteins. This re-enforces mucilage as a strong model to study cell wall proteins, as it
356
has markedly reduced cytoplasmic contamination compare to other cell wall proteomes, while
357
retaining comparable protein recovery rate (Albenne et al., 2013). However, because the
358
extraction of mucilage requires its hydration-induced extrusion, the proteomic profile established
359
for mature mucilage may not include proteins that are normally present only in early
360
developmental stages. Characterizing the mucilage proteins from developing seeds will require
361
other analytical strategies.
362
363
Seed coat mucilage proteome is a specialized cell wall proteome
364
Carbohydrate-active enzymes identified in the seed mucilage proteome include two
365
enzymes previously detected by molecular genetic analyses. Mucilage proteins MUM2 and
366
BXL1 are required for the removal of arabinogalactan side chains of cell wall polysaccharides
367
and for proper mucilage extrusion (Dean et al., 2007; Macquet et al., 2007a; Arsovski et al.,
368
2009). Four other carbohydrate-active enzymes not previously known to be mucilage-associated
17
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369
were also identified, including BETA-HEXOSAMINIDASE 3 (HEXO3, At1g65590/Q8L7S6).
370
HEXO3 has been shown to be involved in the removal of N-acetylglucosamine residues from
371
glycoproteins and in the formation of paucimannosidic N-glycan (Gutternigg et al., 2007;
372
Liebminger et al., 2011; this study). HEXO3 localizes primarily to the plasma membrane,
373
although a minor fraction has been suggested to be soluble in the apoplast, which would be
374
consistent with our findings (Liebminger et al., 2011). However, the overall biological role of
375
HEXO3 and its orthologue HEXO1 remains unknown, as no defects in growth and stress
376
responses were found in their respective mutants (Liebminger et al., 2011).
377
Proteases are commonly found in cell walls, and four were detected in the mucilage
378
proteome. SBT1.7 has been previously identified by its mutant mucilage phenotype of defective
379
extrusion and altered homogalacturonan methylation state. It has been suggested that SBT1.7
380
participates in the removal of the inhibitor domain from a pectin methylesterase (Rautengarten et
381
al., 2008). Two other proteases detected in mucilage have been connected to roles in other
382
tissues. Ectopic expression of ASPARTIC PROTEASE IN GUARD CELL 1 (ASPG1,
383
At3g18490/Q9LS40) enhances ABA-induced stomata closure, reactive oxygen species (ROS)
384
production and drought resistance (Yao et al., 2012), and ASPG1 expression is also induced by
385
ABA (Yao et al., 2012). RESPONSIVE TO DEHYDRATION 21a (RD21a, At1g47128/P43297)
386
is up-regulated during drought stress, suggesting it may also be connected to ABA-regulated
387
processes (Koizumi et al., 1993). In addition, RD21a is known to facilitate apoptosis, which the
388
outer seed coat epidermal cells eventually undergo at the end of seed development (Lampl et al.,
389
2013). However, no obvious mucilage defects were observed in either aspg1 or rd21a seeds.
390
Oxido-reductases can potentially modify cell wall components either by regulating the
391
production and turnover of ROS, or by participating in oxidative modification of other cellular
18
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392
metabolites. Mucilage proteins of this category include PER36, which was previously shown to
393
facilitate mucilage extrusion by weakening the primary cell wall (Kunieda et al., 2013). PER36
394
localizes to the radial and tangential primary walls of the mucilage pockets but was not detected
395
in the mucilage itself (Kunieda et al., 2013). Therefore, it is likely that PER36 is not part of the
396
mucilage proteome but rather, a contaminant from the primary wall. Consistent with this, PER36
397
was the only protein found to be more abundant in mum2-10 seed surface extracts compared to
398
Col-0 (Figure 3B). Primary cell wall proteins such as PER36 would be expected to be over-
399
represented in the proteome extracted from mum2-10 seeds, since mucilage extrudes very poorly
400
from this mutant.
401
Proteins involved in lipid metabolism are also common in cell wall proteomes (Albenne
402
et al., 2013). These proteins are likely involved in the synthesis and modification of cuticles
403
deposited outside the cell wall. In seed coat mucilage, proteins involved in lipid metabolism
404
associate exclusively with the adherent layer, making it the only obvious distinction between the
405
proteomes of the two mucilage layers (Figure 2A). Since the seed coat epidermis likely has a
406
cuticle (Watanabe et al., 2004; Panikashvili et al., 2009) and the primary wall remains attached
407
to the top of the columella embedded in the adherent layer, these proteins may be involved in the
408
synthesis/modification of a seed coat cuticle. The fact that proteins involved in lipid metabolism
409
are found only in the adherent layer suggests they are very strongly bound to the primary cell
410
wall, either covalently cross-linked with other cell wall polymers or perhaps anchored by
411
hydrophobic interactions with the cuticle. In contrast, PER36 was observed in the primary cell
412
wall, but was found in both adherent and non-adherent mucilage in our analysis, suggesting it is
413
less strongly bound to the cell wall than the proteins involved in lipid metabolism.
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414
One difference between the mucilage and other cell wall proteomes is the apparent lack
415
of structural proteins in mucilage. No extensins and hydroxyproline-rich glycoproteins (HPRGs),
416
typically major components of cell wall proteomes, were observed (Jamet et al., 2008a; Albenne
417
et al., 2013), although the leucine-rich repeat family protein At3g24480/Q9LHF1 might play a
418
structural role. Further, the TBA proteins structure (short, no identifiable protein domains,
419
potential glycosylation sites) and abundance (see also below) are characteristics consistent with
420
those of structural proteins, although we have no direct evidence supporting this hypothesis.
421
AGPs may also function as structural proteins, as has been suggested for ARABINOXYLAN
422
PECTIN ARABINOGALACTAN PROTEIN1 (APAP1) (Tan et al., 2013). Two fasciclin-like
423
arabinogalactan
424
(At5g06390/Q66GR0) were identified in mucilage but their loss-of-function phenotypes did not
425
provide any insight into a possible structural role. Surprisingly, SALT OVERLY SENSITIVE 5
426
(SOS5/FLA4, At2g46550), the only FLA known to be required for normal mucilage structure
427
(Harpaz-Saad et al., 2011; Griffiths et al., 2014), was not identified as a component of the mature
428
mucilage proteome. It may be possible that that SOS5 impacts mucilage structure indirectly,
429
perhaps by facilitating matrix polysaccharide biosynthesis in the Golgi or acting as a GPI-
430
anchored carrier of carbohydrates to the apoplast.
proteins
(FLAs)
FLA10
(At3g60900/Q9LZX4)
and
FLA17
431
Despite the identification of numerous new mucilage proteins, in addition to the four
432
proteins (MUM2, BXL1, PER36 and SBT1.7) previously known to regulate mucilage extrusion
433
and structure (Dean et al., 2007; Macquet et al., 2007a; Arsovski et al., 2009; Kunieda et al.,
434
2013; Rautengarten et al., 2008), the biological roles of the new proteins in mucilage remain
435
unknown. Analysis of loss-of-function mutants for many of the genes encoding these proteins
436
failed to identify mucilage extrusion and morphology defects. This may reflect functional
20
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
437
redundancy. Alternatively, it may indicate that these mucilage proteins play roles that do not
438
directly impact extrusion, adherence or structure of the mature mucilage, and that therefore do
439
not generate a loss-of-function phenotype readily detectable by Ruthenium Red staining and light
440
microscopy.
441
442
TBA proteins are seed coat-specific proteins with potential roles in seed coat differentiation
443
Among the mucilage proteins most frequently identified in this study are a family of three
444
uncharacterized proteins designated TESTA ABUNDANT (TBA), which are highly and
445
specifically expressed in the seed coat epidermis during late seed development (Figures 5 and 6).
446
The expression of TBAs requires seed coat differentiation master regulators NARS1, NARS2 and
447
TTG1 (Figure 7), and coincides temporally and spatially with mucilage synthesis and secretion,
448
while ectopically expressed TBAs were shown to localize to the seed coat mucilage and the
449
columella (Figure 8). Since TBAs are abundant, and are predicted to be heavily glycosylated
450
while lacking known functional domains, they may function as structural proteins and cross-link
451
other cell wall polysaccharides (Figure 4). However, our attempts to use amiRNA to knock-
452
down all of the TBA homologues simultaneously failed to produce a Ruthenium Red stained
453
mucilage mutant phenotype (Figure 9) so this hypothesis remains to be validated. It is interesting
454
to note that, following the completion of mucilage synthesis during columella formation, cYFP
455
fluorescence in the mucilage pocket abruptly disappears (Figure 8, 10 DPA) leaving fluorescence
456
only in the columella. These observations suggest that the TBA-cYFP proteins may be actively
457
degraded, although we cannot rule out the possibility that fluorescence of the intact protein is
458
being quenched due to changes in the chemical environment of the mucilage pocket.
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459
The seed coat-specificity and relative strength of the TBA promoters make them valuable
460
tools for seed coat mucilage studies. Since the seed coat mucilage is not essential to plant fitness
461
under laboratory conditions, it can tolerate genetic perturbation, which has made it a powerful
462
genetic model for cell wall analysis. The specificity of the TBA promoters makes them suitable
463
tools to genetically manipulate the seed coat epidermis in general, and seed mucilage
464
specifically. Another Arabidopsis seed coat-specific promoter, from the DIRIGENT PROTEIN 1
465
gene (DP1), differs from expression of the TBA promoters in that DP1 is expressed in both the
466
epidermal and palisade cell layers and peaks in expression during mid-seed development
467
(Esfandiari et al., 2013). Since the TBA promoters are active only in the epidermis late in seed
468
development, the TBA and DP1 promoters complement each other by covering different
469
temporal and spatial domains.
470
In summary, a novel method was developed to extract and detect proteins integral to the
471
Arabidopsis seed coat mucilage. 28 proteins were identified in mature seed coat mucilage,
472
mostly with predicted functions consistent with a cell wall proteome. The protein profiles are
473
largely similar between the adherent and non-adherent mucilage, with the exception of lipid
474
metabolism proteins that occur exclusively in the adherent layer mucilage. Three homologous,
475
previously undescribed proteins we named TBA were highly abundant in seed coat mucilage.
476
Although their functions remain to be determined, their seed coat epidermis-specific promoters
477
should prove to be useful tools for targeted gene expression.
478
479
Materials and Methods
480
Plant materials and growth conditions
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
481
Most Arabidopsis (Arabidopsis thaliana) plants used in this study were derived from the Col-0
482
ecotype, except for ttg1-1 which is derived from the Ler ecotype. Seeds were germinated on
483
plates with Arabidopsis thaliana medium (Haughn and Somerville, 1986) at 7% agar and
484
transferred to soil SunshineMix 4 (SunGro). Plants were grown with continuous fluorescent
485
illumination of 80 to 140 µEm-2 s-1 at 20°C to 22°C. T-DNA insertion lines used in this study
486
were obtained from ABRC and are listed in Supplemental Table S1. T-DNA insertion lines were
487
selected with a PCR-based assay using primers listed in Supplemental Table S2.
488
489
Seed coat mucilage extraction
490
Dry seeds (40 mg) were imbibed with 800 µl of ddH2O in a microcentrifuge tube. The seeds
491
were gently shaken on a tabletop shaker for 1h at ~120 rpm. Supernatants that contain the non-
492
adherent mucilage were collected. The seeds were washed once with 200 µl of ddH2O, which
493
was pooled with the supernatant to form the non-adherent mucilage fraction. To obtain the
494
adherent mucilage, 800 µl of ddH2O were added to the seeds after extracting the non-adherent
495
mucilage, and the seeds were secured horizontally to a tabletop vortex and shaken at top speed
496
for 3h. The supernatants containing the adherent mucilage were collected. The seeds were
497
washed once with 200 µl ddH2O, which was pooled with the supernatant as the adherent layer
498
fraction. The mucilage samples were freeze-dried overnight then chemically de-glycosylated as
499
described by Edge et al., 1981. Briefly, 15 µl of anisole (Sigma-Aldrich) and 135 µl of
500
trifluoromethanesulfonic acid (Sigma-Aldrich) were added to one freeze-dried mucilage sample
501
in a Reacti-vial. The samples were sealed and incubated in 4°C for 2 h. 4 µl of 0.2%
502
bromophenol blue were added to each sample, and 60% pyridine (Sigma-Aldrich) were added
503
drop-wise to each mucilage sample on ice until the solution turned to light blue. The neutralized
23
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
504
mucilage samples were dialyzed overnight in ddH2O with dialysis tubing pore sizes 3500-5000
505
Da then freeze-dried overnight.
506
507
Mass spectrometry
508
Mucilage protein samples were re-suspended in SDS-PAGE sample buffer and separated briefly
509
by 10% SDS-PAGE gel until all of the molecular weight markers just entered the resolving gel.
510
The proteins were stained with blue silver (Candiano et al., 2004) and the entire lane excised
511
from the gel as one gel slice. Protein samples were analyzed by tandem mass spectrometry
512
(MS/MS) at the Centre for High-Throughput Biology (CHiBi) Proteomics Core Facility at the
513
University of British Columbia. In brief, samples were subjected to reduction/alkylation with
514
dithiothreitol/iodoacetamide followed by digestion with trypsin essentially as described by
515
Shevchenko et al., 1996. The resulting peptides were desalted and concentrated with STAGE tips
516
(Rappsilber et al., 2003) and analyzed by LC-MS/MS on a linear-trapping quadrupole - Orbitrap
517
mass spectrometer LTQ Orbitrap Velos on-line coupled to an Agilent 1290 Series HPLC using a
518
nanospray ionization source (ThermoFisher Scientific) including a 2-cm-long, 100-μm-inner
519
diameter fused silica trap column, 20-cm-long 50-μm-inner diameter fused silica fritted
520
analytical column and a 20-μm-inner diameter fused silica gold coated spray tip (6-μm-diameter
521
opening, pulled on a P-2000 laser puller from Sutter Instruments, coated on Leica EM SCD005
522
Super Cool Sputtering Device). The trap column is packed with 5 μm-diameter Aqua C-18
523
beads (Phenomenex, www.phenomenex.com) while the analytical column is packed with 1.9
524
μm-diameter Reprosil-Pur C-18-AQ beads (Dr. Maisch, www.Dr-Maisch.com). Standard 90 min
525
gradients were run from 10-32% buffer B (0.5% acetic acid, 80% acetonitrile) over 51 min, then
526
from 32-40% in the next 5 min, then increased to 100% over 2 min period, held at 100% for 2.5
24
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
527
min, and then dropped to 0% for another 20 min. The HPLC system included Agilent 1290 series
528
Pump and Autosampler with Thermostat. The thermostat temperature was set at 6°C. The
529
sample was loaded on the trap column at 5 μL/min and the analysis was performed at 0.1
530
μL/min. The LTQ-Orbitrap was set to acquire a full-range scan at 60,000 resolution from 350 to
531
1600 Th in the Orbitrap to simultaneously fragment the top ten peptide ions by CID and top 5 by
532
HCD (resolution 7500) in each cycle in the LTQ (minimum intensity 1000 counts). Parent ions
533
were then excluded from MS/MS for the next 30 sec. Singly charged ions were excluded since
534
in ESI mode peptides usually carry multiple charges.
535
recalibrated using lock-mass function. Mass accuracy: error of mass measurement is typically
536
within 5 ppm and is not allowed to exceed 10 ppm.
537
For quantitative analyses, Col-0 mucilage along with mum2-10 and ap2-7 seed surface extracts
538
were prepared from 80 mg of seeds using the protocol described above for non-adherent
539
mucilage. All protein samples were reduced/alkylated and digested as described above, and
540
dimethylated with light, medium and heavy formaldehyde. Col-0 samples were labelled with
541
light formaldehyde for all replicates. mum2-10 samples were labelled with medium
542
formaldehyde for replicates 1 and 2, and heavy formaldehyde for replicate 3. ap2-7 samples were
543
labelled with heavy formaldehyde for replicates 1 and 2, and medium formaldehyde for replicate
544
3. Samples from all three genotypes were pooled before the MS analysis. For replicates 1 and 2,
545
samples were analyzed using the LTQ Orbitrap Velo as described above. For replicate 3, samples
546
were analyzed using the Impact II quadrupole – time of flight mass spectrometer (Bruker
547
Daltonics). on-line coupled to an Easy nano LC 1000 HPLC (ThermoFisher Scientific) using a
548
Captive spray nanospray ionization source (Bruker Daltonics) including a 2-cm-long, 100-μm-
549
inner diameter fused silica fritted trap column, 75-μm-inner diameter fused silica analytical
The Orbitrap was continuously
25
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
550
column with an integrated spray tip (6 – 8 μm-diameter opening, pulled on a P-2000 laser puller
551
from Sutter Instruments). The trap column is packed with 5 μm Aqua C-18 beads (Phenomenex,
552
www.phenomenex.com) while the analytical column is packed with 1.9 μm-diameter Reprosil-
553
Pur C-18-AQ beads (Dr. Maisch, www.Dr-Maisch.com). The analytical column was held at
554
50°C by an in-house constructed column heater. Samples were re-suspended and loaded in buffer
555
A (0.1% aqueous formic acid). Standard 45 min gradients were run from 10-60% buffer B
556
(0.1% formic acid, 80% acetonitrile) over 28 min, then increased to 100% over 2 min period,
557
held at 100% for 15 min. The LC thermostat temperature was set at 7°C. The sample was
558
loaded on the trap column at 850 Bar and the analysis was performed at 0.25 μL/min flow rate.
559
The Impact II was set to acquire in a data-dependent auto-MS/MS mode with inactive focus
560
fragmenting the 20 most abundant ions (one at the time at 18 Hz rate) after each full-range scan
561
from m/z 200 Th to m/z 2000 Th (at 5 Hz rate). The isolation window for MS/MS was 2 to 3 Th
562
depending on parent ion mass to charge ratio and the collision energy ranged from 23 to 65 eV
563
depending on ion mass and charge. Parent ions were then excluded from MS/MS for the next
564
0.4 min and reconsidered if their intensity increased more than 5 times. Singly charged ions
565
were excluded since in ESI mode peptides usually carry multiple charges. Strict active exclusion
566
was applied. Mass accuracy: error of mass measurement is typically within 5 ppm and is not
567
allowed to exceed 10 ppm. The nano ESI source was operated at 1700 V capillary voltage, 0.20
568
Bar nano buster pressure, 3 L/min drying gas and 150°C drying temperature.
569
570
MS data analyses
571
For all qualitative and replicates 1 and 2 of the quantitative analyses, LC-MS/MS data were
572
processed with Proteome Discoverer v.1.2 (ThermoFisher Scientific) then searched against the
26
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
573
Uniprot-Swissprot Arabidopsis thaliana database using the MASCOT algorithm v.2.4 (Perkins et
574
al., 1999; http://www.matrixscience.com). Database contains 12069 sequences; no contaminants
575
were added in the search space. The following parameters were applied: peptide mass accuracy
576
10 parts per million; fragment mass accuracy 0.6 Da; trypsin enzyme specificity, fixed
577
modifications - carbamidomethyl, variable modifications - methionine oxidation, deamidated N,
578
Q and N-acetyl peptides, ESI-TRAP fragment characteristics.
579
IonScores exceeding the individually calculated 99% confidence limit (as opposed to the average
580
limit for the whole experiment) were considered as accurately identified. Proteome Discoverer
581
parameters – Event Detector: mass precision 4 ppm (corresponds to extracted ion chromatograms
582
at ±12 ppm max error), S/N threshold 1; Quantitation Method – Ratio Calculation – Replace
583
Missing Quantitation Values with Minimum Intensity – yes, Use Single Peak Quantitation
584
Channels – yes, - Protein Quantification – Use All Peptides – yes. In order for a protein to be
585
considered a true mucilage protein in qualitative analysis, it must be identified in at least two out
586
of the three biological replicates with MASCOT protein scores > 40 (score ≥ 25 correspond to
587
false-discovery rate ≤ 5%), and identified in at least one out of the three biological replicates
588
with two or more unique peptides.
589
For replicate 3 of the quantitative analysis, data analysis was performed using MaxQuant
590
1.5.3.30 (Cox and Mann, 2008) with the Arabidopsis thaliana protein sequence database plus
591
common contaminants. The search was performed using the following parameters: peptide mass
592
accuracy 10 parts per million; fragment mass accuracy 0.05 Da; trypsin enzyme specificity, fixed
593
modifications - carbamidomethyl, variable modifications - methionine oxidation, and N-acetyl
594
proteins. Only those peptides exceeding the individually calculated 99% confidence limit (as
595
opposed to the average limit for the whole experiment) were considered as accurately identified.
Only those peptides with
27
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
596
Relative protein levels from mum2-10 and ap2-7 seed surface extracts were normalized to Col-0
597
non-adherent mucilage. Proteins that could be detected and quantified in all three replicates were
598
analyzed. 2-tailed Student’s T-test was used to determine the statistical significance of relative
599
protein level differences between mum2-10, ap2-7 and Col-0.
600
601
Expression analyses
602
RNA was extracted from various plant tissues using Trizol reagent (Life Technologies), except
603
that siliques were processed with the RNAqueous Total RNA Isolation Kit (Ambion), while
604
developing seed coats and embryos were processed with the RNAqueous-Micro Total RNA
605
Isolation Kit (Ambion) according to the manufacturer’s instructions. cDNA synthesis was carried
606
out using the Superscript II reverse transcriptase (Life Technologies) according to the
607
manufacturer’s instructions. qPCR was performed using iQ SYBR Green Supermix (BioRad)
608
and the primers listed in Supplemental Table S3. The qPCR reactions were assayed with the
609
BioRad iQ5 Multicolor Real-Time PCR Detection System (BioRad). CYTOSOLIC
610
GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPC) transcripts were used as
611
internal control. 2-tailed Student’s T-test was used to determine the statistical significance of
612
differences in TBA and TBAL expression levels between the amiRNA lines and WT.
613
For gene expression analysis of TBAs in transcription factor mutants, RNA extraction and cDNA
614
synthesis were performed as described previously (Kunieda et al., 2013). PCR was performed
615
using Mango-Taq polymerase (Bioline) and the same primers as were used for qPCR.
616
617
Generation of transgenic plants
28
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
618
The TBAp:GUS constructs were generated using the pBI101 vector, with the promoter fragments
619
amplified from Col-0 genomic DNA. The TBAp:TBA-cYFP translational fusion constructs were
620
assembled in the citrine-pCambia1300 vector as described by Debono et al., 2009, with the
621
promoter and coding region fragments amplified from Col-0 genomic DNA. The TBA amiRNA
622
constructs were designed and built as described by Schwab et al., 2006, using the UBQ1-
623
pCambia1300 vector as described by Ambrose et al., 2011. Primers used for these constructs are
624
listed in Supplemental Table S3. The TBA and TBAL promoters were defined as the DNA
625
sequence extending upstream of the TBA start codon to the next annotated gene, not including
626
pseudogenes, to capture as much promoter region as possible without introducing another gene.
627
TBA1p is ~0.5 kilobases (kB), TBA2p is ~1.3 kB, TBA3p is ~1.8 kB and TBALp is ~1.4 kB long.
628
Col-0 Arabidopsis plants were transformed using the floral dip method (Clough and Bent, 1998),
629
at least 20 independent transgenic lines were selected for each construct. Results from at least 3
630
representative lines are shown.
631
632
Microscopy
633
For seed coat mucilage staining with Ruthenium Red, ~20 dry seeds were imbibed in 1 ml of
634
either ddH2O, 0.05 M EDTA, 0.05 M CaCl2, or 0.5 M Na2CO3, for 1 h and washed twice with
635
ddH2O. The seeds were then stained with 0.01% (w/v) Ruthenium Red (Sigma-Aldrich) for 1 h
636
then washed once with ddH2O. Seeds were imaged with a DFC450 C camera (Leica) on an
637
Axioskop 2 upright light microscope (Carl Zeiss AG).
638
Histochemical GUS assays were performed essentially as described in Esfandiari et al., 2013.
639
Tissue samples were vacuum-infiltrated with GUS staining solution (0.5 mM potassium
640
ferricyanide, 0.5 mM potassium ferrocyanide, 20 mM Na2EDTA, 0.1 % (v/v) Triton X-100
29
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
641
supplemented
with
1
mg/mL
5-bromo-4-chloro-3-indolyl-β-D-glucuronide
(Gold
642
BioTechnology) in 100 mM phosphate buffer (pH 7), incubated at 37°C for 16 h, and then
643
washed several times with 75% ethanol. Tissues were imaged with a DP72 camera (Olympus)
644
mounted on a SZX10 stereomicroscope (Olympus).
645
All confocal images were acquired from an Ultraview VoX Spinning Disk Confocal System
646
(PerkinElmer).
647
For cellulose staining, seeds after mucilage extraction were stained with 0.1% (w/v) Calcofluor
648
White for 5 min and then washed twice with ddH2O. The seeds were inspected under ultraviolet
649
light with the Axioskop 2 microscope.
650
651
Immunoblot analyses
652
Developing siliques were ground in liquid nitrogen and added to extraction buffer containing 50
653
mM Tris–HCl (pH 8), 150 mM NaCl, 1 mM EDTA, 10% (v/v) glycerol, 1% Triton X-100, 1
654
mM PMSF and protease inhibitor cocktail (Roche). The homogenates were centrifuged at 15,000
655
rpm for 10 min in 4°C and the supernatant was collected. Protein concentrations were
656
determined by Bradford assay (BioRad), and 100 µg protein samples were electrophoretically
657
resolved by 12% SDS-PAGE. cYFP fusion proteins were detected using a mouse anti-GFP
658
polyclonal antibody (Roche) and horseradish peroxidase-conjugated goat anti-mouse polyclonal
659
antibody (Santa Cruz Biotechnology). ECL Prime Western blotting detection reagent (GE
660
Healthcare) was used for target detection.
661
662
Supplemental Materials
663
Supplemental Figure S1. Mucilage extraction removes pectin and cellulose from seed coat
30
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
664
Supplemental Figure S2. Expression (RT-PCR) of genes mutated by T-DNA insertion
665
Supplemental Figure S3. Imbibed seeds from plants homozygous for a T-DNA insertion in
666
genes encoding proteins found in seed mucilage
667
Supplemental Figure S4. TBA and TBAL expression pattern in various plant tissues
668
Supplemental Figure S5. Detection TBA-cYFP by immunoblotting
669
Supplemental Table S1. T-DNA insertion lines used in this study
670
Supplemental Table S2. Primers used for mucilage protein T-DNA line analyses in this
671
study
672
Supplemental Table S3. Primers used for TBA and TBAL constructs in this study
673
Supplemental Dataset S1. Col-0 adherent mucilage protein MS data
674
Supplemental Dataset S2. Col-0 non-adherent mucilage protein MS data
675
Supplemental Dataset S3. Summary of Col-0 mucilage protein identification
676
Supplemental Dataset S4. mum2-10 and ap2-7 mucilage proteins quantification MS data
677
relative to Col-0
678
Supplemental Dataset S5. Summary of mum2-10 and ap2-7 mucilage proteins
679
quantification relative to Col-0
680
681
682
Acknowledgements
683
We thank Suzanne Perry, Jamie Hackworth and Jenny Hyung-Mee Moon of the UBC Centre for
684
High-Throughput Biology Proteomics Core Facility for technical assistance, data analyses and
685
equipment use; the UBC Bioimaging Facility for technical assistance and equipment use; Prof.
31
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
686
Ikuko Hara-Nishimura and Dr. Tomoo Shimada (Department of Botany, Kyoto University) for
687
providing the nars1 nars2 mutant.
32
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
688
Table 1. Total proteins identified in mature Col-0 seed coat mucilage
Accession
O04575
Q39168
O04573
ATG #
At1g62080
At1g62000
At1g62060
Name
TESTA ABUNDANT (TBA3)
TESTA ABUNDANT (TBA1)
TESTA ABUNDANT (TBA2)
RmlC-like cupins superfamily
protein
GDSL-motif
esterase/acyltransferase/lipase
Location
Both
Both
Both
Q9M8X3
At3g04170
Q9FK75
At5g45670
Q9FGY1
At5g49360
Adherent
At5g67360
BETA-XYLOSIDASE 1 (BXL1)
GDSL-motif
esterase/acyltransferase/lipase
SUBTILISIN-LIKE SERINE
PROTEASE 1.7 (Sbt1.7, ARA12)
Q9LU14
At3g16370
O65351
Q9LV33
At3g18080
B-S GLUCOSIDASE 44 (BGLU44)
Both
Q9SD46
At3g50990
Both
Q94CH6
At1g75900
Q9FFN4
At5g63800
Q8L7S6
At1g65590
Q9SCV4
At2g28470
Q9SUS0
At4g23560
Q9M8X6
At3g04200
Q9LLR6
At5g59310
Q9LS40
At3g18490
Q8VY93
At4g26790
Q9FMK9
At5g63140
Q9LDB4
At3g08770
Q9LEY1
At5g08260
Q94BT2
At3g07390
Q9LZX4
At3g60900
PEROXIDASE 36 (PER36)
GDSL-motif
esterase/acyltransferase/lipase
MUCILAGE MODIFIED 2
(MUM2)
BETA-HEXOSAMINIDASE 3
(HEXO3)
BETA-GALACTOSIDASE 8
(BGAL8)
Glycosyl hydrolase 9B15
(GH9B15)
RmlC-like cupins superfamily
protein
LIPID TRANSFER PROTEIN 4
(LTP4)
ASPARTIC PROTEASE IN GUARD
CELL 1 (ASPG1)
GDSL-motif
esterase/acyltransferase/lipase
PURPLE ACID PHOSPHATASE 29
(PAP29)
LIPID TRANSFER PROTEIN 6
(LTP6)
SERINE CARBOXYPEPTIDASELIKE 35 (SCPL35)
AUXIN-INDUCED IN ROOT
CULTURES 12 (AIR12)
FASCICLIN-LIKE
ARABINOGALACTAN-PROTEIN
Adherent
Adherent
Adherent
Both
Adherent
Both
Both
Both
Adherent
Both
Adherent
Both
Adherent
Adherent
Adherent
Both
Adherent
Adherent
Function
Unknown
Unknown
Unknown
Oxidoreductase
Lipid
metabolism
Carbohydrateactive enzyme
Lipid
metabolism
Protease
Carbohydrateactive enzyme
Oxidoreductase
Lipid
metabolism
Carbohydrateactive enzyme
Carbohydrateactive enzyme
Carbohydrateactive enzyme
Carbohydrateactive enzyme
Oxidoreductase
Lipid
metabolism
Protease
Lipid
metabolism
WallProtDB
N/A
N/A
N/A
N/A
N/A
Multiple
tissues
Leaves
Multiple
tissues
Multiple
tissues
Hypocotyl
N/A
Multiple
tissues
Multiple
tissues
Leaves
N/A
N/A
N/A
Multiple
tissues
N/A
Miscellaneous
Lipid
metabolism
Roots
Protease
Oxidoreductase
Arabinogalactan
protein
N/A
Multiple
tissues
33
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Roots
Cell culture
Q9LHF1
At3g24480
P43297
At1g47128
Q9SVU5
At4g28780
Q66GR0
At5g06390
10 (FLA10)
Leucine-rich repeat (LRR) family
protein
RESPONSIVE TO DEHYDRATION
21A (RD21a)
GDSL-motif
esterase/acyltransferase/lipase
FASCICLIN-LIKE
ARABINOGALACTAN PROTEIN
17 (FLA17)
Multiple
tissues
Multiple
tissues
Adherent
Structural
Adherent
Adherent
Protease
Lipid
metabolism
Nonadherent
Arabinogalactan
protein
N/A
689
34
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
N/A
690
Figure Legends
691
Figure 1. Strategy to isolate and identify seed coat mucilage proteins
692
A) Col-0 seed coat mucilage stained with Ruthenium Red. Double-headed arrows depict the two
693
mucilage layers. Bar = 100 µm.
694
B) Schematic depiction of the extraction and identification of mucilage proteins. The non-
695
adherent mucilage and adherent mucilage were extracted sequentially. Proteins in each mucilage
696
layer were identified by mass spectrometry after chemical de-glycosylation and trypsin digest.
697
698
Figure 2. Mucilage proteins are functionally similar to other cell wall proteins
699
A) Numbers of seed coat mucilage proteins from each mucilage layer sorted by the cell wall
700
protein functional categories.
701
B) Proportions of mucilage proteins previously identified in cell walls from other tissue types as
702
documented by WallProtDB. Numbers denote the number of proteins in each category, while the
703
percentages denote the proportion of proteins that occupy each category.
704
705
Figure 3. Proteins identified are genuinely associated with mucilage
706
A) Schematic depiction of the mucilage protein quantification in mum2-10 and ap2-7 seed
707
surface extracts relative to Col-0 non-adherent mucilage.
708
B) Relative levels of mucilage proteins in Col-0 non-adherent mucilage, mum2-10 seed surface
709
extract and ap2-7 seed surface extract. Values are normalized to Col-0. Averages ± SD are
710
shown, N= 3, * P<0.01, ** P<0.001.
711
712
Figure 4. Amino acid sequences of the TBA proteins
35
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
713
Amino acid sequence alignments of TBA1, TBA2 and TBA3. Dark grey highlights amino acid
714
residues that are identical, and light grey highlights amino acid residues that are similar.
715
Underlined
716
(http://www.cbs.dtu.dk/services/SignalP/). Bolded S and T residues are predicted by NetOGlyc
717
(http://www.cbs.dtu.dk/services/NetOGlyc/) to be O-glycosylated.
residues
denote
signal
peptides
predicted
by
SignalP
718
719
Figure 5. TBA and TBAL transcripts are found predominantly in the developing seed coat
720
A) RT-PCR detection of TBA1, TBA2, TBA3 and TBAL transcripts in seedlings, roots, rosette and
721
cauline leaves, stem, inflorescence and siliques. GAPC transcripts are shown as cDNA loading
722
controls.
723
B) Quantitative RT-PCR results showing the relative expression levels of TBA1, TBA2, TBA3
724
and TBAL in empty silique valves, 4 DPA seeds, 7 DPA seed coats, 7 DPA embryos, 10 DPA
725
seed coats and 10 DPA embryos. Expression levels were relative to GAPC transcript levels. N =
726
3, error bars denote SD. A second biological replicate was processed with similar results.
727
728
Figure 6. TBA and TBAL promoters are active exclusively in the seed coat
729
A) TBA1p:GUS, TBA2p:GUS, TBA3p:GUS, TBALp:GUS and Col-0 seedlings, rosette leaves,
730
stems, and inflorescences stained for GUS activities. Bar = 500 µm.
731
B) TBA1p:GUS, TBA2p:GUS, TBA3p:GUS, TBALp:GUS and Col-0 siliques and developing
732
seeds at 4, 7 and 10 DPA stained for GUS activities. 10 DPA seed coats and embryos were
733
dissected and stained separately as shown on the two columns on the right. Bar = 500 µm for
734
siliques, and 100 µm for dissected seed coats and embryos.
735
36
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
736
Figure 7. TBA and TBAL expression requires NARS1, NARS2 and TTG1
737
RT-PCR detection of TBA and TBAL transcripts in 7 DPA seeds of nars1 nars2, mum1-1,
738
myb61-1, ttg1-1 and their respective ecotype backgrounds. GAPC transcripts are shown as cDNA
739
loading controls.
740
741
Figure 8. TBA proteins are secreted to the seed coat epidermis apoplast
742
Confocal microscopy images denoting the localization of cYFP-tagged TBA1, TBA2 and TBA3
743
in developing seed coats driven by their respective endogenous promoters. M: mucilage pockets,
744
CC: cytoplasmic column, and C: columella. Bar = 10 µm.
745
746
Figure 9. Down-regulation of TBA and TBAL does not affect mucilage extrusion
747
A) qRT-PCR analysis of expression of TBA and TBAL in 10 DPA siliques from four independent
748
UBQ1p:TBA-amiRNA lines and Col-0. All expression levels were relative to GAPC, and were
749
then normalized to Col-0 expression levels. N = 3, error bars denote SD. * denotes transcript
750
level significantly different from WT where P<0.05, ** denotes transcript level significantly
751
different from WT where P<0.01.
752
B) Ruthenium Red-stained seed coat mucilage from the 4 independent UBQ1p:TBA-amiRNA
753
lines shown in (A). Seeds were imbibed in water, 0.05 M EDTA, 0.5 M Na2CO3, or 0.05 M
754
CaCl2 prior to staining. Bar = 100 µm.
755
756
Supplemental Figure S1. Mucilage extraction removes pectin and cellulose from seed coat
757
Col-0 seeds after non-adherent mucilage extraction (A and B) and after extraction of all mucilage
37
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
758
layers (C and D) stained by Ruthenium Red for pectin (A and C) and Calcofluor White (B and
759
D) for cellulose in the mucilage. Bar = 500 µm.
760
761
Supplemental Figure S2. Expression (RT-PCR) of genes mutated by T-DNA insertion
762
763
Supplemental Figure S3. Imbibed seeds from plants homozygous for a T-DNA insertion in
764
genes encoding proteins found in seed mucilage
765
Bar = 100 µM.
766
767
Supplemental Figure S4. TBA and TBAL expression pattern in various plant tissues
768
TBA and TBAL expression in various plant tissues (A), developing seed coat (B) and developing
769
seeds (C) as shown by the Arabidopsis eFP browser (http://bar.utoronto.ca/efp/cgi-
770
bin/efpWeb.cgi).
771
772
Supplemental Figure S5. Detection TBA-cYFP by immunoblotting
773
Immunoblots using anti-GFP antibody to probe TBA1-cYFP, TBA2-cYFP and TBA3-cYFP
774
from 7 DPA siliques, 10 DPA siliques and mature seeds. Ponceau staining of total protein shown
775
to confirm equal loading.
776
777
Supplemental Datasets S1-S2. Col-0 adherent and non-adherent mucilage proteins MS data
778
Mass-spectrometry results for the mucilage sample identification processed by Proteome
779
Discoverer. Dataset S1 contains data for Col-0 adherent mucilage, S2 contains data for Col-0
780
non-adherent mucilage. 3 replicates were performed for each, and data from each replicate are
38
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
781
separated as individual sheets for each dataset. Accession: UniProt identifier code for each
782
protein. Coverage: percentage of protein detected as peptides. #PSMs: peptide spectrum matches,
783
number of spectra that match any peptide from each protein. #Peptides: total number of peptides
784
identified for each protein. #AAs: sequence length of protein in amino acid residues. MW[kDa]:
785
mass of protein in kDa, not accounting for post-translational modifications. Calc.pI: theoretically
786
calculated isoelectric point. Score: total MASCOT score for each protein, the sum of all peptide
787
scores. Description: brief description for the protein exclusively associated with the accession.
788
Peptides detected for a protein can be expanded by clicking the “+” icon. Confidence icon:
789
confidence level associated with the peptide sequence. Sequence: amino acid sequence of the
790
peptide. Protein accession: Uniprot identifiers for all proteins that contain the sequence.
791
#Proteins: number of proteins that contain the peptide. #Protein groups: number of protein
792
groups which the peptide is found. Activation type: activation type of the spectrum which the
793
peptide was found. Modifications: static and dynamic modification identified on the peptide.
794
IonScore: MASCOT score of the peptide. Exp value: number of matches that score equal or
795
better than this peptide by chance alone. Δ Score: a measure of difference between the top two
796
scores for peptides identified by that spectrum. Rank: ordering or peptides by rank. Identity
797
High: threshold that determines whether peptides are ranked as high confidence when
798
performing a decoy search to calculate false discovery rates. Homology Threshold: threshold that
799
determines whether the proposed peptide sequence and the real sequence are considered similar.
800
Charge: total charge of the peptide. m/z [Da]: mass-to-charge ratio of the precursor ion. MH+
801
[Da]: protonated monoisotopic mass of the peptide. ΔM [ppm]: difference between the
802
theoretical mass of the peptide and the experimental mass of the precursor ion. RT [min]:
803
retention time when the peptide was observed. First scan: number of the first scan used to
39
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
804
identify the peptide. Last scan: the number of the last scan used to identify the peptide. MS
805
order: order of the MS spectrum, MS2 is an MS/MS scan. Ions Matched: percentage of ions
806
matched by the search engine. Spectrum File: name of the file containing the spectra.
807
Annotation: other notes associated with the peptides. More information are available in the
808
Xcalibur Proteome Discoverer User Guide version 1.3 (Thermo Scientific).
809
810
Supplemental Dataset S3. Summary of Col-0 mucilage protein identification
811
List of proteins detected in Col-0 mucilage summarized from Supplemental Datasets S1-S2 in
812
descending total MASCOT score. Proteins detected in different mucilage layers are shown in
813
individual sheets in the dataset. Score 1-3: MASCOT scores from replicates 1-3, respectively.
814
#Peptides 1-3: number of unique peptides detected in replicates 1-3, respectively. Signal peptide:
815
presence/absence
816
(http://www.cbs.dtu.dk/services/SignalP/). Proteins detected in more than one biological
817
replicate with scores > 40 are bolded; proteins selected for further analyses are highlighted in
818
yellow and listed in Table 1.
of
N-terminal
signal
peptide
as
predicted
by
SignalP
4.1
819
820
Supplemental Dataset S4. mum2-10 and ap2-7 mucilage proteins quantification MS data
821
relative to Col-0
822
Mass-spectrometry results for the mucilage protein quantification as processed by Proteome
823
Discoverer, parameters shown are essentially the same as Supplemental Datasets S1 and S2.
824
Other parameters shown include: Heavy/Light, Medium/Light: ratios of protein quantification
825
values for the respective labels. Heavy/Light count, Medium/Light count: numbers of peptide
826
ratios used to calculate protein ratios. Heavy/Light variability, Medium/Light variability:
40
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
827
variability of the peptide ratios used to calculate protein ratios. Area: average area of the 3
828
unique peptides with the largest areas. Quan Channel: channel name of the peptide that is used
829
for quantification. Quan Info: why a peptide was/wasn’t used in quantification. Quan Usage:
830
whether the peptide was used to quantify the protein. More information are available in the
831
Xcalibur Proteome Discoverer User Guide version 1.3 (Thermo Scientific). Replicate 3 was
832
processed by MaxQuant but contain essentially the same information, but does not include
833
individual peptide information.
834
835
Supplemental Dataset S5. Summary of mum2-10 and ap2-7 mucilage proteins
836
quantification relative to Col-0
837
List of mucilage protein quantities from mum2-10 and ap2-7 seed surface relative to Col-0
838
adherent mucilage summarized from Supplemental Dataset S4, in descending order of relative
839
quantity. #peptides 1-3: numbers of unique peptides detected for each protein from replicates 1
840
to 3, respectively. mum2-10:Col-0 ratios 1-3 and ap2-7:Col-0 ratios 1-3: protein abundance from
841
mum2-10 and ap2-7 relative to Col-0, from replicates 1 to 3, respectively. Proteins quantifiable
842
for all 3 replicates are bolded; mucilage proteins listed in Table 1 are highlighted yellow. Results
843
of proteins that fit both criteria are shown in Figure 3B.
41
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
844
References
845
Albenne C, Canut H, Jamet E (2013) Plant cell wall proteomics: the leadership of Arabidopsis
846
thaliana. Front Plant Sci 4: 111
847
848
Ambrose C, Allard JF, Cytrynbaum EN, Wasteneys GO (2011) A CLASP-modulated cell
849
edge barrier mechanism drives cell-wide cortical microtubule organization in Arabidopsis. Nat
850
Commun 2: 430
851
852
Arsovski AA, Haughn GW, Western TL (2010) Seed coat mucilage cells of Arabidopsis
853
thaliana as a model for plant cell wall research. Plant Signal Behav 5: 796-801
854
855
Arsovski AA, Popma TM, Haughn GW, Carpita NC, McCann MC, Western TL (2009)
856
AtBXL1 encodes a bifunctional beta-D-xylosidase/alpha-L-arabinofuranosidase required for
857
pectic arabinan modification in Arabidopsis mucilage secretory cells. Plant Physiol 150: 1219-
858
1234
859
860
Burton RA, Gidley MJ, Fincher GB (2010) Heterogeneity in the chemistry, structure and
861
function of plant cell walls. Nat Chem Biol 6: 724-732
862
863
Candiano G, Bruschi M, Musante L, Santucci L, Ghiggeri GM, Carnemolla B, Orecchia P,
864
Zardi L, Righetti PG (2004) Blue silver: a very sensitive colloidal Coomassie G-250 staining
865
for proteome analysis. Electrophoresis 25: 132713-132733
866
42
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
867
Cassab GI, Varner JE (1988) Cell wall proteins. Annu Rev Plant Physiol Plant Mol Biol 39:
868
321-353
869
870
Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated
871
transformation of Arabidopsis thaliana. Plant J 16: 735-743
872
873
Cox, J., Mann, M (2008) MaxQuant enables high peptide identification rates, individualized
874
p.p.b.-range mass accuracies and proteome-wide quantitation. Nature Biotechnol 26: 1367-1372
875
876
Dean G, Cao Y, Xiang D, Provart NJ, Ramsay L, Ahad A, White R, Selvaraj G, Datla R,
877
Haughn G (2011) Analysis of gene expression patterns during seed coat development in
878
Arabidopsis. Mol Plant 4: 1074-1091
879
880
Dean GH, Zheng H, Tewari J, Huang J, Young DS, Hwang YT, Western TL, Carpita NC,
881
McCann MC, Mansfield SD, Haughn GW (2007) The Arabidopsis MUM2 gene encodes a
882
beta-galactosidase required for the production of seed coat mucilage with correct hydration
883
properties. Plant Cell 19: 4007-4021
884
885
Debono A, Yeats TH, Rose JK, Bird D, Jetter R, Kunst L, Samuels L (2009) Arabidopsis
886
LTPG is a glycosylphosphatidylinositol-anchored lipid transfer protein required for export of
887
lipids to the plant surface. Plant Cell 21: 1230-1238
888
43
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
889
Edge AS, Faltynek CR, Hof L, Reichert LE Jr, Weber P (1981) Deglycosylation of
890
glycoproteins by trifluoromethanesulfonic acid. Anal Biochem 118: 131-137
891
892
Esfandiari E, Jin Z, Abdeen A, Griffiths JS, Western TL, Haughn GW (2013) Identification
893
and analysis of an outer seed coat-specific promoter from Arabidopsis thaliana. Plant Mol Biol
894
81: 93-104
895
896
Francoz E, Ranocha P, Burlat V, Dunand C (2015) Arabidopsis seed mucilage secretory cells:
897
regulation and dynamics. Trends Plant Sci 20: 515-524
898
899
Fry SC (2000) The Growing Plant Cell Wall: Chemical and Metabolic Analysis. (Caldwell, NJ:
900
The Blackburn Press)
901
902
Griffiths JS, Tsai AY, Xue H, Voiniciuc C, Sola K, Seifert GJ, Mansfield SD, Haughn GW
903
(2014) SALT-OVERLY SENSITIVE5 mediates Arabidopsis seed coat mucilage adherence and
904
organization through pectins. Plant Physiol 165: 991-1004
905
906
Grubert M (1974) Studies on the distribution of myxospermy among seeds and fruits of
907
Angiospermae and its ecological importance. Acta Biol Venez 8: 315-551
908
909
Gutternigg M, Kretschmer-Lubich D, Paschinger K, Rendić D, Hader J, Geier P, Ranftl R,
910
Jantsch V, Lochnit G, Wilson IB (2007) Biosynthesis of truncated N-linked oligosaccharides
44
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
911
results from non-orthologous hexosaminidase-mediated mechanisms in nematodes, plants, and
912
insects. J Biol Chem 282: 27825-277840
913
914
Harpaz-Saad S, McFarlane HE, Xu S, Divi UK, Forward B, Western TL, Kieber JJ (2011)
915
Cellulose synthesis via the FEI2 RLK/SOS5 pathway and cellulose synthase 5 is required for the
916
structure of seed coat mucilage in Arabidopsis. Plant J 68: 941-953
917
918
Haughn GW, Western TL (2012) Arabidopsis seed coat mucilage is a specialized cell wall that
919
can be used as a model for genetic analysis of plant cell wall structure and function. Front Plant
920
Sci 3: 64
921
922
Haughn G, Chaudhury A (2005) Genetic analysis of seed coat development in Arabidopsis.
923
Trends Plant Sci 10: 472-477
924
925
Haughn GW, Somerville C (1986) Sulfonylurea-resistant mutants of Arabidopsis thaliana. Mol
926
Gen Genet 204: 430-434
927
928
Huang J, DeBowles D, Esfandiari E, Dean G, Carpita NC, Haughn GW (2011) The
929
Arabidopsis transcription factor LUH/MUM1 is required for extrusion of seed coat mucilage.
930
Plant Physiol 156: 491-502
931
932
Hu R, Li J, Wang X, Zhao X, Wang Z, Yang X, Tang Q, He G, Zhou G, Kong Y (2016a)
933
Xylan synthesized by Irregular Xylem 14 (IRX14) maintains the structure of seed coat mucilage
45
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
934
in Arabidopsis. J Exp Bot 67: 1243-1257
935
936
Hu R, Li J, Yang X, Zhao X, Wang X, Tang Q, He G, Zhou G, Kong Y (2016b). Irregular
937
xylem 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis. Plant Mol Biol 92:
938
25-38
939
940
Jamet E, Albenne C, Boudart G, Irshad M, Canut H, Pont-Lezica R (2008a) Recent
941
advances in plant cell wall proteomics. Proteomics 8: 893-908
942
943
Jamet E, Boudart G, Borderies G, Charmont S, Lafitte C, Rossignol M, Canut H, Pont-
944
Lezica R (2008b) Isolation of plant cell wall proteins. Methods Mol Biol 425: 187-201
945
946
Jamet E, Canut H, Boudart G, Pont-Lezica RF (2006) Cell wall proteins: a new insight
947
through proteomics. Trends Plant Sci 11: 33-39
948
949
Jofuku KD, den Boer BG, Van Montagu M, Okamuro JK (1994) Control of Arabidopsis
950
flower and seed development by the homeotic gene APETALA2. Plant Cell 6: 1211-1225
951
952
Kieliszewski MJ, Lamport DTA (1994) Extensin: repetitive motifs, functional sites, post-
953
translational codes, and phylogeny. Plant J 5: 157-172
954
46
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
955
Koizumi M, Yamaguchi-Shinozaki K, Tsuji H, Shinozaki K (1993) Structure and expression
956
of two genes that encode distinct drought-inducible cysteine proteinases in Arabidopsis thaliana.
957
Gene 129: 175-182
958
959
Koornneef M (1981) The complex syndrome of ttg mutants. Arabid Inf Serv 18: 45-51
960
961
Kunieda T, Shimada T, Kondo M, Nishimura M, Nishitani K, Hara-Nishimura I (2013)
962
Spatiotemporal secretion of PEROXIDASE36 is required for seed coat mucilage extrusion in
963
Arabidopsis. Plant Cell 25: 1355-1367
964
965
Kunieda T, Mitsuda N, Ohme-Takagi M, Takeda S, Aida M, Tasaka M, Kondo M,
966
Nishimura M, Hara-Nishimura I (2008) NAC family proteins NARS1/NAC2 and
967
NARS2/NAM in the outer integument regulate embryogenesis in Arabidopsis. Plant Cell 20:
968
2631-2642
969
970
Lampl N, Alkan N, Davydov O, Fluhr R (2013) Set-point control of RD21 protease activity by
971
AtSerpin1 controls cell death in Arabidopsis. Plant J 74: 498-510
972
973
Le BH, Cheng C, Bui AQ, Wagmaister JA, Henry KF, Pelletier J, Kwong L, Belmonte M,
974
Kirkbride R, Horvath S, Drews GN, Fischer RL, Okamuro JK, Harada JJ, Goldberg RB
975
(2010) Global analysis of gene activity during Arabidopsis seed development and identification
976
of seed-specific transcription factors. Proc Natl Acad Sci USA 107: 8063-8070
977
47
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
978
Lee SJ, Saravanan RS, Damasceno CM, Yamane H, Kim BD, Rose JK (2004) Digging
979
deeper into the plant cell wall proteome. Plant Physiol Biochem 42: 979-988
980
981
Liebminger E, Veit C, Pabst M, Batoux M, Zipfel C, Altmann F, Mach L, Strasser R (2011)
982
Beta-N-acetylhexosaminidases HEXO1 and HEXO3 are responsible for the formation of
983
paucimannosidic N-glycans in Arabidopsis thaliana. J Biol Chem 286: 10793-10802
984
985
Macquet A, Ralet MC, Loudet O, Kronenberger J, Mouille G, Marion-Poll A, North HM
986
(2007a) A naturally occurring mutation in an Arabidopsis accession affects a beta-D-
987
galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed mucilage.
988
Plant Cell 19: 3990-4006
989
990
Macquet A, Ralet MC, Kronenberger J, Marion-Poll A, North HM (2007b) In-situ, chemical
991
and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage. Plant
992
Cell Physiol 48: 984-999
993
994
McCartney L, Knox JP (2002) Regulation of pectic polysaccharide domains in relation to cell
995
development and cell properties in the pea testa. J Exp Bot 53: 707-713
996
997
Mendu V, Griffiths J, Persson S, Stork J, Downie B, Vioniciuc C, Haughn G, Debolt S
998
(2011) Subfunctionalization of cellulose synthases in seed coat epidermal cells mediate
999
secondary radial wall synthesis and mucilage attachment. Plant Physiol 157: 441–453
1000
48
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
1001
North HM, Berger A, Saez-Aguayo S, Ralet MC (2014) Understanding polysaccharide
1002
production and properties using seed coat mutants: future perspectives for the exploitation of
1003
natural variants. Ann Bot 114: 1251-1263
1004
1005
Panikashvili D, Shi JX, Schreiber L, Aharoni A (2009) The Arabidopsis DCR encoding a
1006
soluble BAHD acyltransferase is required for cutin polyester formation and seed hydration
1007
properties. Plant Physiol 151: 1773-1789
1008
1009
Passardi F, Penel C, Dunand C (2004) Performing the paradoxical: how plant peroxidases
1010
modify the cell wall. Trends Plant Sci 9: 534-540
1011
1012
Penfield S, Meissner RC, Shoue DA, Carpita NC, Bevan MW (2001) MYB61 is required for
1013
mucilage deposition and extrusion in the Arabidopsis seed coat. Plant Cell 13: 2777-2791
1014
1015
Perkins DN, Pappin DJ, Creasy DM, Cottrell JS (1999) Probability-based protein
1016
identification by searching sequence databases using mass spectrometry data. Electrophoresis
1017
20: 3551-3567
1018
1019
Rappsilber J, Ishihama Y, Mann M (2003) Stop and go extraction tips for matrix-assisted laser
1020
desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal
1021
Chem 75: 663-670
1022
49
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
1023
Rautengarten C, Usadel B, Neumetzler L, Hartmann J, Büssis D, Altmann T (2008) A
1024
subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coats. Plant J
1025
54: 466-480
1026
1027
Saez-Aguayo S, Ralet MC, Berger A, Botran L, Ropartz D, Marion-Poll A, North HM
1028
(2013) PECTIN METHYLESTERASE INHIBITOR6 promotes Arabidopsis mucilage release by
1029
limiting methylesterification of homogalacturonan in seed coat epidermal cells. Plant Cell 25:
1030
308-323
1031
1032
Schmid M, Davison TS, Henz SR, Pape UJ, Demar M, Vingron M, Schölkopf B, Weigel D,
1033
Lohmann JU (2005) A gene expression map of Arabidopsis thaliana development. Nat Genet
1034
37: 501-506
1035
1036
Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D (2006) Highly specific gene
1037
silencing by artificial microRNAs in Arabidopsis. Plant Cell 18: 1121-1133
1038
1039
Selvendran RR, Ryden P (1990) Isolation and analysis of plant cell walls. In: Methods in Plant
1040
Biochemistry, Vol. 2: Carbohydrates, P.M. Dey and J.B. Harbourne, eds (San Diego, CA:
1041
Academic Press), 549-575
1042
1043
Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins
1044
silver-stained polyacrylamide gels. Anal Chem 68: 850-858
1045
50
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
1046
Showalter AM (1993) Structure and function of plant cell wall proteins. Plant Cell 5: 9-23
1047
1048
Spiro RG (2002) Protein glycosylation: nature, distribution, enzymatic formation, and disease
1049
implications of glycopeptide bonds. Glycobiology 12: 43R-56R
1050
1051
Steentoft C, Vakhrushev SY, Joshi HJ, Kong Y, Vester-Christensen MB, Schjoldager KT,
1052
Lavrsen K, Dabelsteen S, Pedersen NB, Marcos-Silva L, Gupta R, Bennett EP, Mandel U,
1053
Brunak S, Wandall HH, Levery SB, Clausen H (2013) Precision mapping of the human O-
1054
GalNAc glycoproteome through SimpleCell technology. EMBO J 32:1478-1488
1055
1056
Sullivan S, Ralet M-C, Berger A, Diatloff E, Bischoff V, Gonneau M, Marion-Poll A, North
1057
HM (2011) CESA5 is Required for the Synthesis of Cellulose with a Role in Structuring the
1058
Adherent Mucilage of Arabidopsis Seeds. Plant Physiol 156: 1725-1739
1059
1060
Tan L, Eberhard S, Pattathil S, Warder C, Glushka J, Yuan C, Hao Z, Zhu X, Avci U,
1061
Miller JS, Baldwin D, Pham C, Orlando R, Darvill A, Hahn MG, Kieliszewski MJ, Mohnen
1062
D (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan covalently
1063
linked to an arabinogalactan protein. Plant Cell 25: 270-287
1064
1065
Voiniciuc C, Yang B, Schmidt MH, Günl M, Usadel B (2015a) Starting to gel: how
1066
Arabidopsis seed coat epidermal cells produce specialized secondary cell walls. Int J Mol Sci
1067
16:3452-3473
1068
51
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
1069
Voiniciuc C, Schmidt MH, Berger A, Yang B, Ebert B, Scheller HV, North HM, Usadel B,
1070
Günl M (2015b) MUCILAGE-RELATED10 produces galactoglucomannan that maintains
1071
pectin and cellulose architecture in Arabidopsis seed mucilage. Plant Physiol 169: 403-420
1072
1073
Voiniciuc C, Guenl M, Schmidt MH, Usadel B (2015c) Highly branched xylan made by
1074
IRX14 and MUCI21 links mucilage to Arabidopsis seeds. Plant Physiol 169: 2481-2495
1075
1076
Voiniciuc C, Dean GH, Griffiths JS, Kirchsteiger K, Hwang YT, Gillett A, Dow G, Western
1077
TL, Estelle M, Haughn GW (2013) FLYING SAUCER1 is a transmembrane RING E3
1078
ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed
1079
mucilage. Plant Cell 25: 944-959
1080
1081
Watanabe M, Tanaka H, Watanabe D, Machida C, Machida Y (2004) The ACR4 receptor-
1082
like kinase is required for surface formation of epidermis-related tissues in Arabidopsis thaliana.
1083
Plant J 39: 298-308
1084
1085
Western TL (2012) The sticky tale of seed coat mucilages: production, genetics and role in seed
1086
germination and dispersal. Seed Sci Res 22: 1-25
1087
1088
Western TL, Burn J, Tan WL, Skinner DJ, Martin-McCaffrey L, Moffatt BA, Haughn
1089
GW (2001) Isolation and characterization of mutants defective in seed coat mucilage secretory
1090
cell development in Arabidopsis. Plant Physiol 127: 998-1011
1091
52
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
1092
Western TL, Skinner DJ, Haughn GW (2000) Differentiation of mucilage secretory cells of
1093
the Arabidopsis seed coat. Plant Physiol 122: 345-356
1094
1095
Willats WG, McCartney L, Knox JP (2001) In situ analysis of pectic polysaccharides in seed
1096
mucilage and at the root surface of Arabidopsis thaliana. Planta 213: 37-44
1097
1098
Windsor JB, Symonds VV, Mendenhall J, Lloyd AM (2000) Arabidopsis seed coat
1099
development: morphological differentiation of the outer integument. Plant J 22: 483-493
1100
1101
Winter D, Vinegar B, Nahal H, Ammar R, Wilson GV, Provart NJ (2007) An "Electronic
1102
Fluorescent Pictograph" browser for exploring and analyzing large-scale biological data sets.
1103
PLoS One 2: e718
1104
1105
Yao X, Xiong W, Ye T, Wu Y (2012) Overexpression of the aspartic protease ASPG1 gene
1106
confers drought avoidance in Arabidopsis. J Exp Bot 63: 2579-2593
1107
1108
Young RE, McFarlane HE, Hahn MG, Western TL, Haughn GW, Samuels AL (2008)
1109
Analysis of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of
1110
pectin-rich mucilage. Plant Cell 20: 1623-1638
1111
1112
Young JA, Evans RA (1973) Mucilaginous seed coats. Weed Sci 21: 52-54
1113
53
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
1114
Yu L, Shi D, Li J, Kong Y, Yu Y, Chai G, Hu R, Wang J, Hahn MG, Zhou G (2014)
1115
CELLULOSE SYNTHASE-LIKE A2, a glucomannan synthase, is involved in maintaining
1116
adherent mucilage structure in Arabidopsis seed. Plant Physiol 164: 1842-1856
54
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
A
Non-adherent
mucilage
Adherent
mucilage
B
Col-0
Gentle
rotation in
ddH2O
Col-0
Non-adherent
mucilage
Prolonged
vortexing in
ddH2O
Col-0
Adherent
mucilage
Chemical de-glycosylation
Identification by mass-spectrometry
Non-adherent
mucilage proteome
13 proteins
Adherent
mucilage proteome
27 proteins
Figure 1. Strategy to isolate and identify seed coat mucilage proteins
A) Col-0 seed coat mucilage stained with Ruthenium Red. Double-headed arrows depict the two mucilage layers. Bar = 100 µm.
B) Schematic depiction of the extraction and identification of mucilage proteins. The non-adherent mucilage and adherent
mucilage were extracted sequentially. Proteins in each mucilage layer were identified by mass spectrometry after chemical deglycosylation and trypsin digest.
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
8
Number of proteins
A
7
Adherent
6
Non-adherent
5
4
3
2
1
0
B
Previously
unidentified
in other wall
proteomes
13 (46.43%)
Multiple
tissues
9 (32.14%)
Leaves
2 (7.14%)
Roots
2
(4.14%)
Suspension culture
1 (3.57%)
Hypocotyl
1 (3.57%)
Figure 2. Mucilage proteins are functionally similar to other cell wall proteins
A) Numbers of seed coat mucilage proteins from each mucilage layer sorted by the cell wall protein functional categories.
B) Proportions of mucilage proteins previously identified in cell walls from other tissue types as documented by WallProtDB.
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Numbers denote the number of proteins Copyright
in each category,
while the
percentages
denote the
proportion
of proteins that occupy
© 2016 American
Society
of Plant Biologists.
All rights
reserved.
each category.
A
Gentle
rotation in
ddH2O
Col-0
Col-0
Col-0 non-adherent
mucilage
Quantification of proteins
mum2-10 seed
surface extract
mum2-10
ap2-7 seed
surface extract
mum2-10
ap2-7
Gentle
rotation in
ddH2O
Gentle
rotation in
ddH2O
B
2
1.8
Relative protein level
ap2-7
Col-0
1.6
mum2-10
1.4
ap2-7
*
1.2
1
0.8
0.6
0.4
0.2
0
**
**
**
**
**
**
**
TESTA ABUNDANT TESTA ABUNDANT TESTA ABUNDANT
1 (TBA1)
2 (TBA2)
3 (TBA3)
**
Subtilisin-like
proteinase 1.7
(Sbt1.7, ARA12)
**
PEROXIDASE 36
(PER36)
Figure 3. Proteins identified are genuinely associated with mucilage
A) Schematic depiction of the mucilage protein quantification in mum2-10 and ap2-7 seed surface extracts relative to Col-0 nonadherent mucilage.
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
B) Relative levels of mucilage proteins in Col-0
non-adherent
mucilage,
mum2-10
seed surface
extract
and ap2-7 seed surface
Copyright
© 2016 American
Society
of Plant Biologists.
All rights
reserved.
extract. Values are normalized to Col-0. Averages ± SD are shown, N= 3, * P<0.01, ** P<0.001.
1
50
TBA1 MNATKFVVLLVIGILCAIVTARQVKDLSTETKLGASLPKTTTKGIGAQLS
TBA2 MNATKFVVLLVIGILCAIVTARQVEEVSKETKLGTSLPKSTNKGIGAQLS
TBA3 MNATKFLVLLVIGVLCAIVTARQVKDLSTETKLGASLPKTTTKGIGAQLS
51
100
TBA1 ATGTTYSTSSVVSYANGFNNPKGPGANSFESANTFTSGQVTAKGRKARVS
TBA2 AAGLTYSGSSVSSSASAFNNPKGPGASASESGYTSTIGQVIAKGRKARVS
TBA3 ATGSTFSSSSVVSYANGFNNPKGPGANAFESGSTFTSGQVTAKGRKARVS
101
150
TBA1 STSASAAEGDAAAAVTRKAAAARANGKVASASRVKGSSEKKKG--KGKKD
TBA2 SASASAATGEAAAAVTRKAAAARAKGKVASASRVKGSSEKKKKDHKGKKD
TBA3 SASASTATGEAAAAVTRKAAAARAKGKVASASRVKGSSEKKKKDRKGKKD
Figure 4. Amino acid sequences of the TBA proteins
Amino acid sequence alignments of TBA1, TBA2 and TBA3. Dark grey highlights amino acid residues that are identical, and light
grey highlights amino acid residues that are similar. Underlined residues denote signal peptides predicted by SignalP
(http://www.cbs.dtu.dk/services/SignalP/). Bolded S and T residues are predicted by NetOGlyc
(http://www.cbs.dtu.dk/services/NetOGlyc/) to be O-glycosylated.
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
A
TBA1
TBA2
TBA3
TBAL
GAPC
25
Relative expression
B
TBA1
20
TBA2
TBA3
15
TBAL
10
5
0
Silique
valves
4 DPA
seeds
7 DPA
7 DPA
10 DPA
10 DPA
seed coats embryos seed coats embryos
Figure 5. TBA and TBAL transcripts are found predominantly in the developing seed coat
A) RT-PCR detection of TBA1, TBA2, TBA3 and TBAL transcripts in seedlings, roots, rosette and cauline leaves, stem, inflorescence
and siliques. GAPC transcripts are shown as cDNA loading controls.
B) Quantitative RT-PCR results showing the relative expression levels of TBA1, TBA2, TBA3 and TBAL in empty silique valves, 4
DPA seeds, 7 DPA seed coats, 7 DPA embryos, 10 DPA seed coats and 10 DPA embryos. Expression levels were relative to GAPC
transcript levels. N = 3, error bars denote SD. A second biological replicate was processed with similar results.
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Siliques
B
Seed coat
Embryo
4 DPA
A
Seedling
Rosette leaf
Stem
Inflorescence
TBA1p:
7 DPA
GUS
10 DPA
TBA1p:
GUS
TBA2p:
GUS
4 DPA
TBA2p:
7 DPA
GUS
10 DPA
4 DPA
TBA3p:
GUS
TBA3p:
7 DPA
GUS
10 DPA
TBALp:
GUS
4 DPA
TBALp:
7 DPA
GUS
10 DPA
Col-0
4 DPA
Col-0
7 DPA
10 DPA
Figure 6. TBA and TBAL promoters are active exclusively in the seed coat
A) TBA1p:GUS, TBA2p:GUS, TBA3p:GUS, TBALp:GUS and Col-0 seedlings, rosette leaves, stems, and inflorescences stained for GUS activities. Bar = 500 µm.
B) TBA1p:GUS, TBA2p:GUS, TBA3p:GUS, TBALp:GUS and Col-0 siliques and developing seeds at 4, 7 and 10 DPA stained for GUS activities. 10 DPA seed coats and embryos
were dissected and stained separately as shown on the two columns on the right. Bar = 500 µm for siliques, and 100 µm for dissected seed coats and embryos.
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
TBA1
TBA2
TBA3
TBAL
GAPC
Figure 7. TBA and TBAL expression requires NARS1, NARS2 and TTG1
RT-PCR detection of TBA and TBAL transcripts in 7 DPA seeds of nars1 nars2, mum1-1, myb61-1, ttg1-1 and their respective
ecotype backgrounds. GAPC transcripts are shown as cDNA loading controls.
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Top-down
CC
M
Cross-section
M
C
M
M
CC
Top-down
TBA1p:
TBA1-cYFP
M
M
C
M
M
TBA2p:
TBA2-cYFP
Cross-section
M
CC
C
M
M
CC
Top-down
C
M
M
CC M
C
Cross-section
TBA3p:
TBA3-cYFP
M
M
C
CC
Late heart
~4 DPA
Bent cotyledon
~6 DPA
Walking stick
~8 DPA
Mature embryo
~10 DPA
Figure 8. TBA proteins are secreted to theDownloaded
seed coat epidermis
from on Juneapoplast
18, 2017 - Published by www.plantphysiol.org
© 2016
American Society
of Plant
Allin
rights
reserved.seed coats driven by
Confocal microscopy images denoting theCopyright
localization
of cYFP-tagged
TBA1,
TBA2Biologists.
and TBA3
developing
their respective endogenous promoters. M: mucilage pockets, CC: cytoplasmic column, and C: columella. Bar = 10 µm.
1.8
A
**
1.6
*
TBA1
TBA2
Relative expression
normalized to Col-0
1.4
TBA3
1.2
TBAL
1
**
0.8
**
0.6
*
0.4
0.2
0
B
**
**
** **
**
amiRNA-5
amiRNA-6
**
ddH2O
0.05M
EDTA
**
ami-RNA-8
**
amiRNA-10
0.5M
Na2CO3
Col-0
0.05M
CaCl2
Col-0
TBAamiRNA
#5
TBAamiRNA
#6
TBAamiRNA
#8
TBAamiRNA
#10
Figure 9. Down-regulation of TBA and TBAL does not affect mucilage extrusion
A) qRT-PCR analysis of expression of TBA and TBAL in 10 DPA siliques from four independent UBQ1p:TBA-amiRNA lines and Col-0.
All expression levels were relative to GAPC, and were then normalized to Col-0 expression levels. N = 3, error bars denote SD. *
denotes transcript level significantly different from WT where P<0.05, ** denotes transcript level significantly different from WT
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
where P<0.01.
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
B) Ruthenium Red-stained seed coat mucilage from the 4 independent UBQ1p:TBA-amiRNA lines shown in (A). Seeds were
imbibed in water, 0.05 M EDTA, 0.5 M Na CO , or 0.05 M CaCl prior to staining. Bar = 100 µm.
Parsed Citations
Albenne C, Canut H, Jamet E (2013) Plant cell wall proteomics: the leadership of Arabidopsis thaliana. Front Plant Sci 4: 111
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Ambrose C, Allard JF, Cytrynbaum EN, Wasteneys GO (2011) A CLASP-modulated cell edge barrier mechanism drives cell-wide
cortical microtubule organization in Arabidopsis. Nat Commun 2: 430
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Arsovski AA, Haughn GW, Western TL (2010) Seed coat mucilage cells of Arabidopsis thaliana as a model for plant cell wall
research. Plant Signal Behav 5: 796-801
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Arsovski AA, Popma TM, Haughn GW, Carpita NC, McCann MC, Western TL (2009) AtBXL1 encodes a bifunctional beta-Dxylosidase/alpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells. Plant
Physiol 150: 1219-1234
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Burton RA, Gidley MJ, Fincher GB (2010) Heterogeneity in the chemistry, structure and function of plant cell walls. Nat Chem Biol
6: 724-732
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Candiano G, Bruschi M, Musante L, Santucci L, Ghiggeri GM, Carnemolla B, Orecchia P, Zardi L, Righetti PG (2004) Blue silver: a
very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 25: 132713-132733
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Cassab GI, Varner JE (1988) Cell wall proteins. Annu Rev Plant Physiol Plant Mol Biol 39: 321-353
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant
J 16: 735-743
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Cox, J., Mann, M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and
proteome-wide quantitation. Nature Biotechnol 26: 1367-1372
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Dean G, Cao Y, Xiang D, Provart NJ, Ramsay L, Ahad A, White R, Selvaraj G, Datla R, Haughn G (2011) Analysis of gene expression
patterns during seed coat development in Arabidopsis. Mol Plant 4: 1074-1091
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Dean GH, Zheng H, Tewari J, Huang J, Young DS, Hwang YT, Western TL, Carpita NC, McCann MC, Mansfield SD, Haughn GW
(2007) The Arabidopsis MUM2 gene encodes a beta-galactosidase required for the production of seed coat mucilage with correct
hydration properties. Plant Cell 19: 4007-4021
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Debono A, Yeats TH, Rose JK, Bird D, Jetter R, Kunst L, Samuels L (2009) Arabidopsis LTPG is a glycosylphosphatidylinositolanchored lipid transfer protein required for export of lipids to the plant surface. Plant Cell 21: 1230-1238
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Edge AS, Faltynek CR, Hof L, Reichert LE Jr, Weber P (1981) Deglycosylation of glycoproteins by trifluoromethanesulfonic acid.
Anal Biochem 118: 131-137
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Esfandiari E, Jin Z, Abdeen A, Griffiths JS, Western TL, Haughn GW (2013) Identification and analysis of an outer seed coat-specific
promoter from Arabidopsis thaliana. Plant Mol Biol 81: 93-104
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Francoz E, Ranocha P, Burlat V, Dunand C (2015) Arabidopsis seed mucilage secretory cells: regulation and dynamics. Trends
Plant Sci 20: 515-524
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Fry SC (2000) The Growing Plant Cell Wall: Chemical and Metabolic Analysis. (Caldwell, NJ: The Blackburn Press)
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Griffiths JS, Tsai AY, Xue H, Voiniciuc C, Sola K, Seifert GJ, Mansfield SD, Haughn GW (2014) SALT-OVERLY SENSITIVE5
mediates Arabidopsis seed coat mucilage adherence and organization through pectins. Plant Physiol 165: 991-1004
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Grubert M (1974) Studies on the distribution of myxospermy among seeds and fruits of Angiospermae and its ecological
importance. Acta Biol Venez 8: 315-551
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Gutternigg M, Kretschmer-Lubich D, Paschinger K, Rendic D, Hader J, Geier P, Ranftl R, Jantsch V, Lochnit G, Wilson IB (2007)
Biosynthesis of truncated N-linked oligosaccharides results from non-orthologous hexosaminidase-mediated mechanisms in
nematodes, plants, and insects. J Biol Chem 282: 27825-277840
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Harpaz-Saad S, McFarlane HE, Xu S, Divi UK, Forward B, Western TL, Kieber JJ (2011) Cellulose synthesis via the FEI2 RLK/SOS5
pathway and cellulose synthase 5 is required for the structure of seed coat mucilage in Arabidopsis. Plant J 68: 941-953
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Haughn GW, Western TL (2012) Arabidopsis seed coat mucilage is a specialized cell wall that can be used as a model for genetic
analysis of plant cell wall structure and function. Front Plant Sci 3: 64
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Haughn G, Chaudhury A (2005) Genetic analysis of seed coat development in Arabidopsis. Trends Plant Sci 10: 472-477
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Haughn GW, Somerville C (1986) Sulfonylurea-resistant mutants of Arabidopsis thaliana. Mol Gen Genet 204: 430-434
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Huang J, DeBowles D, Esfandiari E, Dean G, Carpita NC, Haughn GW (2011) The Arabidopsis transcription factor LUH/MUM1 is
required for extrusion of seed coat mucilage. Plant Physiol 156: 491-502
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Hu R, Li J, Wang X, Zhao X, Wang Z, Yang X, Tang Q, He G, Zhou G, Kong Y (2016a) Xylan synthesized by Irregular Xylem 14 (IRX14)
maintains the structure of seed coat mucilage in Arabidopsis. J Exp Bot 67: 1243-1257
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Hu R, Li J, Yang X, Zhao X, Wang X, Tang Q, He G, Zhou G, Kong Y (2016b). Irregular xylem 7 (IRX7) is required for anchoring seed
coat mucilage in Arabidopsis. Plant Mol Biol 92: 25-38
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Jamet E, Albenne C, Boudart G, Irshad M, Canut H, Pont-Lezica R (2008a) Recent advances in plant cell wall proteomics.
Proteomics 8: 893-908
Pubmed: Author and Title
CrossRef: Author and Title
Downloaded
Google Scholar: Author Only Title Only Author
and Title from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Jamet E, Boudart G, Borderies G, Charmont S, Lafitte C, Rossignol M, Canut H, Pont-Lezica R (2008b) Isolation of plant cell wall
proteins. Methods Mol Biol 425: 187-201
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Jamet E, Canut H, Boudart G, Pont-Lezica RF (2006) Cell wall proteins: a new insight through proteomics. Trends Plant Sci 11: 3339
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Jofuku KD, den Boer BG, Van Montagu M, Okamuro JK (1994) Control of Arabidopsis flower and seed development by the
homeotic gene APETALA2. Plant Cell 6: 1211-1225
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kieliszewski MJ, Lamport DTA (1994) Extensin: repetitive motifs, functional sites, post-translational codes, and phylogeny. Plant J
5: 157-172
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Koizumi M, Yamaguchi-Shinozaki K, Tsuji H, Shinozaki K (1993) Structure and expression of two genes that encode distinct
drought-inducible cysteine proteinases in Arabidopsis thaliana. Gene 129: 175-182
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Koornneef M (1981) The complex syndrome of ttg mutants. Arabid Inf Serv 18: 45-51
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kunieda T, Shimada T, Kondo M, Nishimura M, Nishitani K, Hara-Nishimura I (2013) Spatiotemporal secretion of PEROXIDASE36 is
required for seed coat mucilage extrusion in Arabidopsis. Plant Cell 25: 1355-1367
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Kunieda T, Mitsuda N, Ohme-Takagi M, Takeda S, Aida M, Tasaka M, Kondo M, Nishimura M, Hara-Nishimura I (2008) NAC family
proteins NARS1/NAC2 and NARS2/NAM in the outer integument regulate embryogenesis in Arabidopsis. Plant Cell 20: 2631-2642
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Lampl N, Alkan N, Davydov O, Fluhr R (2013) Set-point control of RD21 protease activity by AtSerpin1 controls cell death in
Arabidopsis. Plant J 74: 498-510
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Le BH, Cheng C, Bui AQ, Wagmaister JA, Henry KF, Pelletier J, Kwong L, Belmonte M, Kirkbride R, Horvath S, Drews GN, Fischer
RL, Okamuro JK, Harada JJ, Goldberg RB (2010) Global analysis of gene activity during Arabidopsis seed development and
identification of seed-specific transcription factors. Proc Natl Acad Sci USA 107: 8063-8070
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Lee SJ, Saravanan RS, Damasceno CM, Yamane H, Kim BD, Rose JK (2004) Digging deeper into the plant cell wall proteome. Plant
Physiol Biochem 42: 979-988
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Liebminger E, Veit C, Pabst M, Batoux M, Zipfel C, Altmann F, Mach L, Strasser R (2011) Beta-N-acetylhexosaminidases HEXO1 and
HEXO3 are responsible for the formation of paucimannosidic N-glycans in Arabidopsis thaliana. J Biol Chem 286: 10793-10802
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Macquet A, Ralet MC, Loudet O, Kronenberger J, Mouille G, Marion-Poll A, North HM (2007a) A naturally occurring mutation in an
Arabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed
mucilage. Plant Cell 19: 3990-4006
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
from A,
onNorth
June 18,
- Published
www.plantphysiol.org
Macquet A, Ralet MC, KronenbergerDownloaded
J, Marion-Poll
HM2017
(2007b)
In-situ,bychemical
and macromolecular study of the
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
composition of Arabidopsis thaliana seed coat mucilage. Plant Cell Physiol 48: 984-999
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
McCartney L, Knox JP (2002) Regulation of pectic polysaccharide domains in relation to cell development and cell properties in
the pea testa. J Exp Bot 53: 707-713
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Mendu V, Griffiths J, Persson S, Stork J, Downie B, Vioniciuc C, Haughn G, Debolt S (2011) Subfunctionalization of cellulose
synthases in seed coat epidermal cells mediate secondary radial wall synthesis and mucilage attachment. Plant Physiol 157: 441453
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
North HM, Berger A, Saez-Aguayo S, Ralet MC (2014) Understanding polysaccharide production and properties using seed coat
mutants: future perspectives for the exploitation of natural variants. Ann Bot 114: 1251-1263
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Panikashvili D, Shi JX, Schreiber L, Aharoni A (2009) The Arabidopsis DCR encoding a soluble BAHD acyltransferase is required
for cutin polyester formation and seed hydration properties. Plant Physiol 151: 1773-1789
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Passardi F, Penel C, Dunand C (2004) Performing the paradoxical: how plant peroxidases modify the cell wall. Trends Plant Sci 9:
534-540
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Penfield S, Meissner RC, Shoue DA, Carpita NC, Bevan MW (2001) MYB61 is required for mucilage deposition and extrusion in the
Arabidopsis seed coat. Plant Cell 13: 2777-2791
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Perkins DN, Pappin DJ, Creasy DM, Cottrell JS (1999) Probability-based protein identification by searching sequence databases
using mass spectrometry data. Electrophoresis 20: 3551-3567
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Rappsilber J, Ishihama Y, Mann M (2003) Stop and go extraction tips for matrix-assisted laser desorption/ionization,
nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal Chem 75: 663-670
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Rautengarten C, Usadel B, Neumetzler L, Hartmann J, Büssis D, Altmann T (2008) A subtilisin-like serine protease essential for
mucilage release from Arabidopsis seed coats. Plant J 54: 466-480
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Saez-Aguayo S, Ralet MC, Berger A, Botran L, Ropartz D, Marion-Poll A, North HM (2013) PECTIN METHYLESTERASE INHIBITOR6
promotes Arabidopsis mucilage release by limiting methylesterification of homogalacturonan in seed coat epidermal cells. Plant
Cell 25: 308-323
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Schmid M, Davison TS, Henz SR, Pape UJ, Demar M, Vingron M, Schölkopf B, Weigel D, Lohmann JU (2005) A gene expression
map of Arabidopsis thaliana development. Nat Genet 37: 501-506
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D (2006) Highly specific gene silencing by artificial microRNAs in
Arabidopsis. Plant Cell 18: 1121-1133
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Selvendran RR, Ryden P (1990) Isolation and analysis of plant cell walls. In: Methods in Plant Biochemistry, Vol. 2: Carbohydrates,
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
P.M. Dey and J.B. Harbourne, eds (San Diego, CA: Academic Press), 549-575
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.
Anal Chem 68: 850-858
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Showalter AM (1993) Structure and function of plant cell wall proteins. Plant Cell 5: 9-23
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Spiro RG (2002) Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds.
Glycobiology 12: 43R-56R
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Steentoft C, Vakhrushev SY, Joshi HJ, Kong Y, Vester-Christensen MB, Schjoldager KT, Lavrsen K, Dabelsteen S, Pedersen NB,
Marcos-Silva L, Gupta R, Bennett EP, Mandel U, Brunak S, Wandall HH, Levery SB, Clausen H (2013) Precision mapping of the
human O-GalNAc glycoproteome through SimpleCell technology. EMBO J 32:1478-1488
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Sullivan S, Ralet M-C, Berger A, Diatloff E, Bischoff V, Gonneau M, Marion-Poll A, North HM (2011) CESA5 is Required for the
Synthesis of Cellulose with a Role in Structuring the Adherent Mucilage of Arabidopsis Seeds. Plant Physiol 156: 1725-1739
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Tan L, Eberhard S, Pattathil S, Warder C, Glushka J, Yuan C, Hao Z, Zhu X, Avci U, Miller JS, Baldwin D, Pham C, Orlando R, Darvill
A, Hahn MG, Kieliszewski MJ, Mohnen D (2013) An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan
covalently linked to an arabinogalactan protein. Plant Cell 25: 270-287
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Voiniciuc C, Yang B, Schmidt MH, Günl M, Usadel B (2015a) Starting to gel: how Arabidopsis seed coat epidermal cells produce
specialized secondary cell walls. Int J Mol Sci 16:3452-3473
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Voiniciuc C, Schmidt MH, Berger A, Yang B, Ebert B, Scheller HV, North HM, Usadel B, Günl M (2015b) MUCILAGE-RELATED10
produces galactoglucomannan that maintains pectin and cellulose architecture in Arabidopsis seed mucilage. Plant Physiol 169:
403-420
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Voiniciuc C, Guenl M, Schmidt MH, Usadel B (2015c) Highly branched xylan made by IRX14 and MUCI21 links mucilage to
Arabidopsis seeds. Plant Physiol 169: 2481-2495
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Voiniciuc C, Dean GH, Griffiths JS, Kirchsteiger K, Hwang YT, Gillett A, Dow G, Western TL, Estelle M, Haughn GW (2013) FLYING
SAUCER1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis
seed mucilage. Plant Cell 25: 944-959
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Watanabe M, Tanaka H, Watanabe D, Machida C, Machida Y (2004) The ACR4 receptor-like kinase is required for surface formation
of epidermis-related tissues in Arabidopsis thaliana. Plant J 39: 298-308
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Western TL (2012) The sticky tale of seed coat mucilages: production, genetics and role in seed germination and dispersal. Seed
Sci Res 22: 1-25
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Western TL, Burn J, Tan WL, Skinner DJ, Martin-McCaffrey L, Moffatt BA, Haughn GW (2001) Isolation and characterization of
mutants defective in seed coat mucilage secretory cell development in Arabidopsis. Plant Physiol 127: 998-1011
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Western TL, Skinner DJ, Haughn GW (2000) Differentiation of mucilage secretory cells of the Arabidopsis seed coat. Plant Physiol
122: 345-356
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Willats WG, McCartney L, Knox JP (2001) In situ analysis of pectic polysaccharides in seed mucilage and at the root surface of
Arabidopsis thaliana. Planta 213: 37-44
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Windsor JB, Symonds VV, Mendenhall J, Lloyd AM (2000) Arabidopsis seed coat development: morphological differentiation of the
outer integument. Plant J 22: 483-493
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Winter D, Vinegar B, Nahal H, Ammar R, Wilson GV, Provart NJ (2007) An "Electronic Fluorescent Pictograph" browser for
exploring and analyzing large-scale biological data sets. PLoS One 2: e718
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Yao X, Xiong W, Ye T, Wu Y (2012) Overexpression of the aspartic protease ASPG1 gene confers drought avoidance in
Arabidopsis. J Exp Bot 63: 2579-2593
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Young RE, McFarlane HE, Hahn MG, Western TL, Haughn GW, Samuels AL (2008) Analysis of the Golgi apparatus in Arabidopsis
seed coat cells during polarized secretion of pectin-rich mucilage. Plant Cell 20: 1623-1638
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Young JA, Evans RA (1973) Mucilaginous seed coats. Weed Sci 21: 52-54
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Yu L, Shi D, Li J, Kong Y, Yu Y, Chai G, Hu R, Wang J, Hahn MG, Zhou G (2014) CELLULOSE SYNTHASE-LIKE A2, a glucomannan
synthase, is involved in maintaining adherent mucilage structure in Arabidopsis seed. Plant Physiol 164: 1842-1856
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.