Identification and Characterization of Arabidopsis Seed
Coat Mucilage Proteins1[OPEN]
Allen Yi-Lun Tsai 2, Tadashi Kunieda 3, Jason Rogalski, Leonard J. Foster, Brian E. Ellis, and
George W. Haughn*
Department of Botany (A.Y.-L.T., T.K., G.W.H.), Michael Smith Laboratories (A.Y.-L.T., J.R., L.J.F., B.E.E.), and
Department of Biochemistry and Molecular Biology (L.J.F.), University of British Columbia, Vancouver, British
Columbia, Canada V6T 1Z4
ORCID ID: 0000-0001-8164-8826 (G.W.H.).
Plant cell wall proteins are important regulators of cell wall architecture and function. However, because cell wall proteins are
difficult to extract and analyze, they are generally poorly understood. Here, we describe the identification and characterization
of proteins integral to the Arabidopsis (Arabidopsis thaliana) seed coat mucilage, a specialized layer of the extracellular matrix
composed of plant cell wall carbohydrates that is used as a model for cell wall research. The proteins identified in mucilage
include those previously identified by genetic analysis, and several mucilage proteins are reduced in mucilage-deficient mutant
seeds, suggesting that these proteins are genuinely associated with the mucilage. Arabidopsis mucilage has both nonadherent
and adherent layers. Both layers have similar protein profiles except for proteins involved in lipid metabolism, which are present
exclusively in the adherent mucilage. The most abundant mucilage proteins include a family of proteins named TESTA
ABUNDANT1 (TBA1) to TBA3; a less abundant fourth homolog was named TBA-LIKE (TBAL). TBA and TBAL transcripts
and promoter activities were detected in developing seed coats, and their expression requires seed coat differentiation
regulators. TBA proteins are secreted to the mucilage pocket during differentiation. Although reverse genetics failed to
identify a function for TBAs/TBAL, the TBA promoters are highly expressed and cell type specific and so should be very
useful tools for targeting proteins to the seed coat epidermis. Altogether, these results highlight the mucilage proteome as a
model for cell walls in general, as it shares similarities with other cell wall proteomes while also containing mucilage-specific
features.
The plant cell wall plays key roles in structural support, cell-cell cohesion, and interaction of the cell with
the environment. It is a dynamic structure and can be
strengthened or loosened in response to environmental
or developmental cues (Fry, 2000; Passardi et al., 2004).
1
This work was supported by the National Sciences and Engineering Research Council (NSERC; Discovery Grants to B.E.E. and G.W.
H.), a British Columbia Proteomic Network Graduate/Postdoctoral
Training Grant (to A.Y.-L.T.), the NSERC Collaborative Research and
Training Experience Program Working on Walls (to A.Y.-L.T., G.W.H.,
and B.E.E.), and the Japan Society for the Promotion of Science (Postdoctoral Fellowship for Research Abroad to T.K.).
2
Present address: Graduate School of Science and Technology,
Kumamoto University, 2-39-1 Kurokami, Chuo-ku, Kumamoto 8608555, Japan.
3
Present address: Department of Biology, Faculty of Science and
Engineering, Konan University, 8-9-1 Okamoto, Higashinada-ku,
Kobe 658-8501, Japan.
* Address correspondence to [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is:
George W. Haughn ([email protected]).
A.Y.-L.T. designed the research; A.Y.-L.T., T.K., and J.R. performed the research; A.Y.-L.T., L.J.F., B.E.E., and G.W.H. analyzed
the data and wrote the article.
[OPEN]
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Plant cell walls typically contain cellulose and hemicellulose and may include pectin or lignin depending
on the type of wall. In addition to these carbohydrate
components, 5% to 10% of the cell wall biomass consists
of proteins (Cassab and Varner, 1988; Burton et al.,
2010). Despite being a relatively minor component in
terms of cell wall biomass, these proteins are critical
regulators of the cell wall architecture and, therefore, its
physical properties. For example, structural proteins
can cross-link various cell wall polysaccharides
(Showalter, 1993), while carbohydrate-active enzymes
modify polysaccharide structure.
Since cell wall proteins are generally difficult to extract and analyze, they remain a relatively poorly understood component of the cell wall. Several factors
complicate the analysis of cell wall proteins. First, they
often undergo extensive posttranslational modifications, such as Pro hydroxylation, glycosylation, and the
addition of GPI anchors (Jamet et al., 2008b; Albenne
et al., 2013). These modifications not only alter protein
mass, thereby complicating protein identification, but
they also can anchor the proteins in the apoplast by
covalent or noncovalent interactions (Kieliszewski and
Lamport, 1994; Spiro, 2002), which make cell wall
protein extraction and identification more challenging.
The extraction of cell wall proteins typically requires
harsh conditions (Lee et al., 2004; Jamet et al., 2008b)
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Tsai et al.
that often lead to protein degradation and contamination with cytoplasmic proteins, with a resulting decrease in the quality of proteomic data. In addition, the
cell wall resides in extracellular space and abuts the
perimeters of adjacent cells. Since a variety of cell types
with distinctive cell walls are found in most tissues and
organs, it is common that cell wall extracts typically
include carbohydrate and proteins derived from multiple cell types, and the relative contribution of specific
cell types is difficult to assess. Despite these problems,
several studies have characterized cell wall proteomes
from different tissue types in various plant species, including the model plant Arabidopsis (Arabidopsis thaliana; for review, see Albenne et al., 2013). Out of the
;5,000 Arabidopsis genes that encode a predicted signal peptide to allow a protein to enter the secretory
pathway, 1,000 to 2,000 are thought to be cell wall
proteins (Jamet et al., 2006). However, currently, most
published cell wall proteomes contain less than
100 proteins each and are contaminated by cytoplasmic
proteins to a variable extent, depending on the tissue
type and extraction techniques (Albenne et al., 2013).
This suggests that many cell wall proteins remain to be
discovered and characterized and emphasizes the need
for better models and more robust methodologies.
Arabidopsis seed coat mucilage is a specialized layer
of the extracellular matrix composed of cell wall carbohydrates arranged in a distinct structure (for review,
see Arsovski et al., 2010; Haughn and Western, 2012;
Western, 2012; North et al., 2014, Voiniciuc et al., 2015c)
that is used as a model to study cell wall structure and
function. It contains cellulose and hemicellulose
(Macquet et al., 2007a; Young et al., 2008; Harpaz-Saad
et al., 2011; Mendu et al., 2011; Sullivan et al., 2011;
Griffiths et al., 2014; Yu et al., 2014; Voiniciuc et al.,
2015a, 2015b; Hu et al., 2016a, 2016b) but is particularly
rich in pectin, with unsubstituted rhamnogalacturonan
I making up ;85% of the total mucilage carbohydrate
(Western et al., 2000, 2001; Willats et al., 2001; Dean
et al., 2007; Macquet et al., 2007a; Young et al., 2008).
Similar to cell walls, Arabidopsis seed coat mucilage
also contains proteins. Forward and reverse genetics
studies have identified several loci required for proper
mucilage synthesis, secretion, and extrusion (for review, see Haughn and Chaudhury, 2005; Arsovski
et al., 2010; Haughn and Western, 2012; Western, 2012;
North et al., 2014; Francoz et al., 2015). Several of these
gene products are believed to be secreted to the mucilage pocket or adjacent primary wall in the developing
seed coat. For example, the mucilage-modifying enzyme MUCILAGE MODIFIED2 (MUM2) is secreted to
the mucilage pocket during mucilage synthesis
(Western et al., 2001; Dean et al., 2007; Macquet et al.,
2007b). PEROXIDASE36 (PER36) has been shown to
localize to the radial and tangential primary cell wall
adjacent to the mucilage pocket (Kunieda et al., 2013).
Two other genes that encode proteins needed for normal
mucilage, SUBTILISIN-LIKE SERINE PROTEASE1.7
(SBT1.7; Rautengarten et al., 2008) and arabinofuranosidase b-XYLOSIDASE1 (BXL1; Arsovski et al., 2009),
contain signal peptides and modify mucilage carbohydrates. However, a thorough analysis of mucilage proteins has not been described.
The deposition of seed coat mucilage is known as
myxospermy and is common in angiosperms (Young
and Evans, 1973; Grubert, 1974). During differentiation,
Arabidopsis seed coat epidermal cells synthesize mucilage components and deposit them between the
plasma membrane and the primary wall at the junction
between the radial and tangential cell walls, forming a
ring-shaped mucilage pocket surrounding a volcanoshaped cytoplasmic column (Western et al., 2000;
Windsor et al., 2000). A cellulose-rich secondary cell
wall, the columella, is subsequently deposited beneath
the mucilage, gradually replacing the cytoplasm
(Western et al., 2000; Windsor et al., 2000). Upon exposure of mature seeds to water, the pectin-rich mucilage swells rapidly, ruptures the primary wall, and
extrudes to encapsulate the seed. The extruded Arabidopsis seed mucilage has at least two distinct layers,
nonadherent and adherent (Western et al., 2000;
Macquet et al., 2007a). The outermost layer (nonadherent layer) is amorphous in appearance, composed
primarily of pectin and, as its name suggests, easily
separated from the seed by gentle shaking. The layer of
mucilage adjacent to the seed coat has a distinct ray-like
structure, has cellulose and hemicellulose in addition to
pectin, and is strongly adherent to the seed surface.
Relative to cell wall preparations from most other tissue
types, seed coat mucilage can be easily extracted in large
amounts without contamination with cell wall material
from other cell types (Haughn and Chaudhury, 2005;
Haughn and Western, 2012; North et al., 2014). These
advantages suggest that seed coat mucilage can yield cell
wall proteomes that are potentially of higher quality
than cell wall proteomes derived from other tissues.
Here, we describe the extraction and proteomic analysis
of the mature Arabidopsis seed coat mucilage and discuss the protein profiles of the mucilage in comparison
with other cell wall proteomes. In addition, we characterize a family of unknown proteins that are particularly
abundant in seed coat mucilage and strongly expressed
in the developing seed coat.
RESULTS
Proteins Are a Component of Mucilage Extracted from
Arabidopsis Seeds
In order to identify and characterize proteins integral
to the seed coat mucilage, a protocol was developed to
extract seed coat mucilage for protein analyses (Fig. 1).
Upon hydration, Arabidopsis seed coat epidermal cells
extrude mucilage as two distinct layers: an outer nonadherent layer that detaches easily from the hydrated
seed and a dense halo-like adherent layer that is bound
tightly to the seed coat (Fig. 1A; Western et al., 2000).
We took advantage of the different physical properties
of these two layers to separate them by sequential extraction (Fig. 1B). Seeds imbibed in water were shaken
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Identification of Seed Coat Mucilage Proteins
Figure 1. Strategy to isolate and identify seed
coat mucilage proteins. A, Columbia-0 (Col-0)
seed coat mucilage stained with Ruthenium
Red. Double-headed arrows depict the two
mucilage layers. Bar = 100 mm. B, Schematic
depiction of the extraction and identification of
mucilage proteins. The nonadherent mucilage
and adherent mucilage were extracted sequentially. Proteins in each mucilage layer
were identified by mass spectrometry (MS) after
chemical deglycosylation and trypsin digestion. ddH2O, Distilled, deionized water.
gently to separate the nonadherent mucilage. The seeds
were then shaken at high speed for several hours to
remove the adherent mucilage (Fig. 1B). Ruthenium
Red and Calcofluor White staining showed that the
pectin and cellulosic components of seed coat mucilage
were almost completely removed by the sequential
extraction (Fig. 1B; Supplemental Fig. S1). The harvested adherent and nonadherent mucilage samples
were chemically deglycosylated, trypsin digested, and
analyzed by MS to identify mucilage-associated proteins in these samples (Fig. 1B; Supplemental Data Sets
S1 and S2). Of those proteins detected by this process,
only the ones identified in more than one biological
replicate with MASCOT scores greater than 40 (where a
score of 25 or greater corresponds to a 5% false discovery rate), and at least once with multiple peptides,
were considered for further analyses. Based on these
criteria, 30 proteins were considered to be robustly
identified from mucilage, all containing predicted signal peptides (Supplemental Data Set S3). Cruciferin A1
and cruciferin C were discarded from further analyses,
since they are not known to be secreted to the apoplast.
This leaves a total 28 potential mucilage proteins
identified (Table I; Supplemental Data Set S3). One
protein was found only in the nonadherent layer,
15 only in the adherent layer, and the remaining
12 proteins were found in both mucilage layers (Table I).
Mucilage Proteins Are Functionally Similar to Other Cell
Wall Proteins
When proteins found in each mucilage layer were
sorted by their predicted functions (Table I; Fig. 2A),
most fell within the various functional categories of cell
wall proteins as defined by Albenne et al. (2013). These
categories include carbohydrate-active enzymes, oxidoreductases, proteases, proteins involved in lipid
metabolism, arabinogalactan proteins, as well as miscellaneous proteins and proteins with unknown functions. The fact that seed coat mucilage-associated
proteins appear to be functionally similar to proteins
from other Arabidopsis cell wall proteomes reinforces
the concept that seed coat mucilage is a specialized type
of cell wall (Haughn and Western, 2012). On the other
hand, nearly half of the specific mucilage-associated
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Tsai et al.
Table I. Total proteins identified in mature Col-0 seed coat mucilage
Accession No.
ATG No.
Name
O04575
Q39168
O04573
Q9M8X3
At1g62080
At1g62000
At1g62060
At3g04170
Q9FK75
At5g45670
Q9FGY1
Q9LU14
At5g49360
At3g16370
O65351
At5g67360
Q9LV33
Q9SD46
Q94CH6
At3g18080
At3g50990
At1g75900
Q9FFN4
Q8L7S6
Q9SCV4
Q9SUS0
At5g63800
At1g65590
At2g28470
At4g23560
Q9M8X6
At3g04200
Q9LLR6
At5g59310
Q9LS40
At3g18490
Q8VY93
At4g26790
Q9FMK9
At5g63140
Q9LDB4
At3g08770
Q9LEY1
At5g08260
Q94BT2
At3g07390
Q9LZX4
At3g60900
Q9LHF1
P43297
At3g24480
At1g47128
Q9SVU5
At4g28780
Q66GR0
At5g06390
TESTA ABUNDANT (TBA3)
TESTA ABUNDANT (TBA1)
TESTA ABUNDANT (TBA2)
RmlC-like cupin superfamily
protein
GDSL motif esterase/
acyltransferase/lipase
b-XYLOSIDASE1 (BXL1)
GDSL motif esterase/
acyltransferase/lipase
SUBTILISIN-LIKE SERINE
PROTEASE1.7 (Sbt1.7, ARA12)
B-S GLUCOSIDASE44 (BGLU44)
PEROXIDASE36 (PER36)
GDSL motif esterase/
acyltransferase/lipase
MUCILAGE MODIFIED2 (MUM2)
b-HEXOSAMINIDASE3 (HEXO3)
b-GALACTOSIDASE8 (BGAL8)
GLYCOSYL HYDROLASE9B15
(GH9B15)
RmlC-like cupin superfamily
protein
LIPID TRANSFER PROTEIN4
(LTP4)
ASPARTIC PROTEASE IN GUARD
CELL1 (ASPG1)
GDSL motif esterase/
acyltransferase/lipase
PURPLE ACID PHOSPHATASE29
(PAP29)
LIPID TRANSFER PROTEIN6
(LTP6)
SERINE CARBOXYPEPTIDASELIKE35 (SCPL35)
AUXIN-INDUCED IN ROOT
CULTURES12 (AIR12)
FASCICLIN-LIKE
ARABINOGALACTAN
PROTEIN10 (FLA10)
Leu-rich repeat family protein
RESPONSIVE TO
DEHYDRATION21A (RD21a)
GDSL motif esterase/
acyltransferase/lipase
FASCICLIN-LIKE
ARABINOGALACTAN
PROTEIN17 (FLA17)
protein isoforms were unique to mucilage and not
identified in other cell wall proteomes to date (Fig. 2B),
including a family of unknown proteins (discussed
below), RmlC-like cupin superfamily proteins, and
GDSL lipases (Table I). Homologs of the RmlC-like
cupin superfamily proteins and GDSL lipases are
commonly found in other cell walls (Albenne et al.,
2013), suggesting that these proteins may represent
mucilage-specific isoforms.
In general, the adherent layer displays a richer and
more diverse protein profile compared with the nonadherent layer, including 15 proteins that are unique to
Location
Function
WallProtDB
Both
Both
Both
Adherent
Unknown
Unknown
Unknown
Oxidoreductase
Not
Not
Not
Not
Adherent
Lipid metabolism
Not detected
Adherent
Adherent
Carbohydrate-active enzyme
Lipid metabolism
Multiple tissues
Leaves
Both
Protease
Multiple tissues
Both
Both
Adherent
Carbohydrate-active enzyme
Oxidoreductase
Lipid metabolism
Multiple tissues
Hypocotyl
Not detected
Both
Both
Both
Adherent
Carbohydrate-active
Carbohydrate-active
Carbohydrate-active
Carbohydrate-active
Multiple tissues
Multiple tissues
Leaves
Not detected
Both
Oxidoreductase
Not detected
Adherent
Lipid metabolism
Not detected
Both
Protease
Multiple tissues
Adherent
Lipid metabolism
Not detected
Adherent
Miscellaneous
Roots
Adherent
Lipid metabolism
Roots
Both
Protease
Not detected
Adherent
Oxidoreductase
Multiple tissues
Adherent
Arabinogalactan protein
Cell culture
Adherent
Adherent
Structural
Protease
Multiple tissues
Multiple tissues
Adherent
Lipid metabolism
Not detected
Nonadherent
Arabinogalactan protein
Not detected
enzyme
enzyme
enzyme
enzyme
detected
detected
detected
detected
the adherent layer (Table I). Interestingly, this adherentspecific group includes a number of proteins involved in
lipid metabolism (Fig. 2A). Otherwise, the numbers of
proteins that belong to each predicted functional class are
more or less comparable between the two mucilage layers
(Fig. 2A). This suggests that the types of protein-mediated
biological processes associated with mucilage modification in the apoplast are comparable within the two layers.
Since the seeds from which mucilage was obtained in
these experiments had not been processed in any way
prior to hydration and mucilage extraction, the possibility remains that the proteins we identified originate
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Identification of Seed Coat Mucilage Proteins
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. Numbers
denote the number of proteins in each category,
while percentages denote the proportion of proteins that occupy each category.
from sources other than the mucilage. In order to address
this concern, the nonadherent mucilage protein profiles
obtained from Col-0 seeds were compared with protein
profiles obtained from the seed surface extracts from the
mucilage mutants mum2-10 and apetala 2-7 (ap2-7; Fig.
3A). mum2-10 seeds synthesize mucilage but do not extrude it when hydrated (Dean et al., 2007; Macquet et al.,
2007b), whereas ap2-7 seed coat epidermal cells fail to
differentiate and, therefore, do not synthesize mucilage
(Jofuku et al., 1994; Western et al., 2001; Dean et al.,
2011). Since mucilage can only be extracted from hydrated Col-0 seeds, proteins that are significantly overrepresented in Col-0 nonadherent mucilage compared
with mum2-10 and/or ap2-7 seed surface extracts would
be predicted to be derived from the extruded mucilage.
Several mucilage proteins identified were indeed found
at much higher levels in Col-0 compared with mum2-10
and ap2-7 (Fig. 3B; Supplemental Data Sets S4 and S5).
Overall, the recovery of mucilage-associated protein was
reduced by ;90% when mum2-10 seed was used and by
;99% when ap2-7 seed was used compared with Col-0
seed (Fig. 3B). These data support the hypothesis that
proteins identified in this study are derived from extruded mucilage of seed coat epidermal cells and not
from the primary wall.
The Identity of Many Mucilage Proteins Is Consistent with
a Role in Mucilage/Cell Wall Modification
The collection of enzymes identified by our proteomics analyses includes all the secreted enzymes
required for normal mucilage extrusion that have
been identified previously by genetic analysis:
MUM2 (At5g63800/Q9FFN4; Dean et al., 2007), BXL1
(At5g49360/Q9FGY1; Arsovski et al., 2009), PER36
(At3g50990/Q9SD46; Kunieda et al., 2013), and SBT1.7
(At5g67360/O65351; Rautengarten et al., 2008; Table I).
Their identification here thus validates the robustness
of the proteomic analysis.
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Tsai et al.
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. ddH 2 O, Distilled, deionized water. B, Relative
levels of mucilage proteins in Col-0
nonadherent mucilage, mum2-10
seed surface extract, and ap2-7 seed
surface extract. Values are normalized to Col-0. Averages 6 SD are
shown; n = 3. *, P , 0.01; and **,
P , 0.001.
In an attempt to determine the roles of other mucilage
proteins we identified, plant lines with T-DNA insertions
in the genes b-GLUCOSIDASE44 (At3g18080/Q9LV33),
b-HEXOSAMINIDASE3 (HEXO3; At1g65590/Q8L7S6),
ASPARTIC PROTEASE IN GUARD CELL1 (ASPG1;
At3g18490/Q9LS40), AUXIN INDUCIBLE IN ROOTS12
(At3g07390/Q94BT2), RESPONSIVE TO DEHYDRATION1a
(RD21a; At1g47128/P43297), and SERINE CARBOXYPEPTIDASE-LIKE35 (At5g08260/Q9LEY1) were characterized (Supplemental Fig. S2; Supplemental Table S1).
These genes were chosen because they do not appear to
have homologs that are also expressed in seed coat epidermal cells, thus decreasing the possibility of functional
redundancy obscuring mutant phenotypes. In each case,
the T-DNA insertion decreased or eliminated the
steady-state levels of transcript in homozygous lines
(Supplemental Fig. S2). Seeds of each insertional mutant
were imbibed in water, 0.05 M EDTA, 0.05 M CaCl2, or 0.5 M
Na2CO3, stained with Ruthenium Red, and examined for
seed mucilage abnormalities. EDTA is believed to loosen
mucilage by disrupting the homogalacturonan salt bridges
through Ca2+ chelation (Western et al., 2001; Rautengarten
et al., 2008; Saez-Aguayo et al., 2013; Voiniciuc et al., 2013).
Na2CO3 treatment also loosens mucilage, possibly by
cleaving cross-linking ester bonds between homogalacturonan polymers (Selvendran and Ryden, 1990; Fry,
2000; McCartney and Knox, 2002). In contrast, mucilage
extruded in a CaCl2 solution is more compact and stains
more intensely with Ruthenium Red than mucilage
extruded in water, presumably by enhancing Ca2+ salt
bridging between mucilage homogalacturonan molecules.
However, no clear mucilage defects were found in any
mucilage protein mutant lines under the conditions tested
(Supplemental Fig. S3), suggesting that if the corresponding gene products have a role in mucilage modification, the
mutant phenotype must be relatively subtle or conditional.
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Identification of Seed Coat Mucilage Proteins
TBA Proteins Were Found to Be Highly Abundant in Seed
Coat Mucilage
Among the mucilage proteins identified, three proteins of
the unknown protein family 0540 (UPF0540), At1g62000/
Q39168, At1g62060/O04573, and At1g62080/O04575, were
of particular interest because they were consistently identified as the most abundant proteins in almost all samples
(Table I; Supplemental Data Set S4). Consistent with these
protein data, the corresponding genes were found to be
expressed in the seed coat at very high levels (Supplemental
Fig. S4; Schmid et al., 2005; Winter et al., 2007; Le et al., 2010;
Dean et al., 2011). However, the proteins encoded by these
genes do not contain known functional domains other than
putative signal peptides, so no function has been ascribed to
them to date. Members of the UPF0540 protein family are
strongly conserved, as they share 79% amino acid sequence
identity and 81% similarity with one another (Fig. 4). Furthermore, the loci that encode these proteins are tightly
clustered on chromosome 1, suggesting that the gene family
may have expanded through tandem duplication events.
Due to the abundance of the UPF0540 proteins in the seed
coat, these genes were named TESTA ABUNDANT1
(TBA1; At1g62000/Q39168), TBA2 (At1g62060/O04573),
and TBA3 (At1g62080/O04575). Interestingly, a peptide from a fourth member of the UPF0540 family,
At1g62220/O04587, also was detected in adherent
mucilage (Supplemental Data Sets S1 and S3). However, At1g62220/O04587 was identified with only one
peptide with a score below the cutoff for statistical significance. Due to the strong similarities in amino acid
sequences and expression patterns between At1g62220/
O04587 and the TBA proteins, At1g62220/O04587 was
named TBA-LIKE (TBAL).
TBAs are small proteins (;150 amino acids) with
many conserved Ser and Thr residues predicted by
NetOGlyc to be O-glycosylated (Steentoft et al., 2013;
Fig. 4). These characteristics suggest that TBAs and
TBAL may function as structural proteins that interact
with various polysaccharides in seed coat mucilage.
TBA Proteins Are Synthesized in the Developing Seed
Coat Epidermis and Secreted to the Apoplast
Public microarray data suggest that TBA and TBAL
are expressed uniquely in the seed coat, and reverse
transcription (RT)-PCR results are consistent with this
pattern (Fig. 5). TBA and TBAL transcripts could only
be detected in siliques (Fig. 5A) and, more specifically,
in the 7- and 10-d post anthesis (DPA) seed coat (Fig.
5B). TBA2 expression levels are by far the highest and
peaked at 7 DPA (coinciding with mucilage synthesis;
Fig. 5B), whereas the expression levels of the remaining
genes were lower and peaked at 10 DPA (coinciding
with columella synthesis; Fig. 5B).
To verify the expression pattern of TBA and TBAL,
reporter assays were performed on tissues of plants
carrying chimeric genes encoding the GUS gene under
the control of the TBA and TBAL native promoters.
Consistent with RT-PCR data, GUS activity could be
detected in developing seeds at 7 and 10 DPA (Fig. 6B)
but not in seedlings, leaves, stems (Fig. 6A), and embryos (Fig. 6B). GUS under the control of TBA2p
appeared earlier compared with other promoters, although in general, all TBA and TBAL promoters were
active by 10 DPA (Fig. 6B). These data support the
hypothesis that all four promoters are active primarily
in the seed coat. The fact that the TBA promoter-GUS
patterns mirror the presence of TBA transcripts implies
that the expression of the TBA genes is largely regulated
by their upstream cis-regulatory elements.
Several transcription factors are known to regulate
the differentiation of seed coat epidermis and the synthesis of seed coat mucilage. Some of these master
regulators include NAC-REGULATED SEED MORPHOLOGY1 (NARS1), NARS2, MUM1, MYELOBLASTOSIS61 (MYB61) and TRANSPARENT TESTA
GLABRA1 (TTG1; Koornneef, 1981; Penfield et al.,
2001; Kunieda et al., 2008; Huang et al., 2011). Since the
TBA genes are expressed exclusively in the seed coat,
we asked whether they are under the control of these
transcription factors. RT-PCR results showed that TBA
and TBAL transcripts were absent in developing seeds
of both the nars1 nars2 double mutant and ttg1-1 (Fig. 7),
which suggests that NARS1/NARS2 and TTG1 are all
required for TBA and TBAL expression. Intriguingly,
TBA2 transcripts appear to be absent in the Landsberg
erecta ecotype, suggesting that there may be TBA expression variation among different natural accessions.
The subcellular localization of TBAs was characterized
using C-terminally tagged citrine-yellow fluorescent protein (cYFP) TBA translational fusion constructs driven by
Figure 4. Amino acid sequences of the TBA proteins. Amino acid sequence alignment is shown
for TBA1, TBA2, and TBA3. Dark gray highlights
amino acid residues that are identical, and light
gray highlights amino acid residues that are similar. Underlined residues denote signal peptides
predicted by SignalP (http://www.cbs.dtu.dk/
services/SignalP/). Boldface S and T residues are
predicted by NetOGlyc (http://www.cbs.dtu.dk/
services/NetOGlyc/) to be O-glycosylated.
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Tsai et al.
try to detect TBA-cYFP extracted from developing siliques. Interestingly, full-length TBA-cYFP proteins
were difficult to detect in the siliques of TBA-cYFP
transgenic plants by immunoblotting. The abundance
of TBA-cYFP was quite variable among different lines,
and TBA3-cYFP appears to be partially insoluble
(Supplemental Fig. S5). These results suggest that TBAcYFP proteins are likely unstable and may undergo
proteolysis or other posttranslational modification
within the mucilage pocket.
TBA Proteins Are Likely Functionally Redundant
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. CYTOSOLIC GLYCERALDEHYDE-3PHOSPHATE DEHYDROGENASE (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, and error bars denote SD. A second
biological replicate was processed with similar results.
their endogenous promoters. In agreement with the
expression data, cYFP signals were detected in the developing seed coat. No cYFP signal was observed in 4-DPA
seeds (Fig. 8). By 6 DPA, TBA1-cYFP and TBA2-cYFP could
be detected in the seed coat epidermal lateral cell walls and
the developing mucilage pockets. All three TBAs could be
detected in the mucilage pockets by 8 DPA (Fig. 8). By
10 DPA, the cYFP signal was absent from the mucilage
pocket but was observed in the developing columella (Fig.
8). This expression pattern coincides spatiotemporally with
the TBA transcript and promoter activity patterns (Figs. 5
and 6) and reinforces the characterization of TBA proteins
as mucilage proteins. Furthermore, cYFP fluorescence was
detected only in the outer epidermal layer of the seed coat
(Fig. 8), suggesting that TBA proteins may only be synthesized in mucilage-secretory cells.
Despite the fact that TBAs were initially identified in
mature mucilage, TBA-cYFP fluorescence was absent
from mucilage pockets by 10 DPA, which raised the
possibility that TBAs might be unstable proteins. To test
this idea, immunoblot experiments were performed to
In an attempt to determine the function of the TBA
proteins, we characterized tba loss-of-function mutants.
Since the three TBA proteins are highly conserved and
share a similar expression pattern, we anticipated that
they might be functionally redundant. Furthermore,
because all these genes are closely linked on the chromosome, the construction of double, triple, and quadruple tba mutants is relatively difficult. To overcome
these problems, an artificial microRNA (amiRNA)
driven by the UBIQUITIN EXTENSION PROTEIN1
promoter (UBQ1p) was designed to knock down all
three TBA homologs and TBAL simultaneously. When
TBA1 transcript levels in developing siliques were
quantified in 12 UBQ1p:TBA-amiRNA transgenic lines,
four lines showed significant down-regulation of TBA1,
but only line 5 showed significant down-regulation in
all three TBA genes and TBAL (Fig. 9A). However, none
of the UBQ1p:TBA-amiRNA lines showed any mucilage
defects when their seeds were imbibed in water, 0.05 M
EDTA, 0.05 M CaCl2, or 0.5 M Na2CO3 followed by Ruthenium Red staining (Fig. 9B). These results suggest
that either the TBA genes are functionally redundant,
even at low levels of expression, or that the amiRNA
knockdown lines possess mucilage phenotypes that are
not clearly discernible with Ruthenium Red staining.
DISCUSSION
Proteins Are an Integral Part of the Arabidopsis Seed
Coat Mucilage
Arabidopsis seed coat mucilage is a specialized layer
of the extracellular matrix composed of cell wall carbohydrates arranged in a distinct structure in the apoplast of seed coat epidermal cells. Because of its
accessibility and dispensability, mucilage has been
used as a genetic model for studying the structure and
function of the plant cell wall (Arsovski et al., 2009;
Haughn and Western, 2012). Forward genetic analysis
has enabled the identification of several proteins that
are secreted with mucilage and required for normal
mucilage structure. To more comprehensively define
the array of proteins involved in mucilage structure and
modification, we used proteomic analysis to examine
mucilage extruded by mature Arabidopsis seeds.
The mucilage extracted by extensive shaking yielded
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Identification of Seed Coat Mucilage Proteins
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. Bars = 500 mm. 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.
The 10-DPA seed coats and embryos were dissected and stained separately, as shown in the two columns at right. Bars = 500 mm
for siliques and 100 mm for dissected seed coats and embryos.
protein preparations that possessed a consistent array
of secreted polypeptides relatively free of intracellular
proteins. The 28 proteins identified in Col-0 seed coat
mucilage by this approach may not be very numerous,
but they represent the same classes of proteins found in
the cell wall proteomes of other tissue types, which
generally contain less than 100 proteins (Albenne et al.,
2013). This suggests that the mucilage protein extraction protocol is at least comparable with other cell wall
proteome studies in terms of protein recovery rate.
Furthermore, the mucilage proteins identified include
all proteins believed to be secreted to the mucilage
pocket that are not membrane anchored: MUM2,
SBT1.7, BXL1, and PER36 (Dean et al., 2007; Macquet
et al., 2007b; Rautengarten et al., 2008; Arsovski et al.,
2009; Kunieda et al., 2013). In addition, proteomic
analysis of mucilage extracts from the seeds of ap2
mutants that fail to differentiate a seed coat epidermis
showed decreases among the most abundant mucilage
proteins with the exception of PER36 (see below),
which, unlike the others, localizes to the primary cell
wall surrounding the mucilage. Therefore, we believe
that our method is sufficiently robust in characterizing
the mature mucilage proteome.
consistent with the idea that mucilage and cell walls
share many biosynthetic and functional processes. Seed
coat mucilage has the experimental advantage over
other types of cell walls that it is actively extruded and
can be extracted without tissue homogenization and
associated cytoplasmic contamination (Supplemental
Data Sets S1–S3). All of the mucilage proteins identified
contain predicted signal peptides, while proteins
without signal peptides detected in mucilage generally
scored poorly (Supplemental Data Set S3), suggesting
that this method indeed strongly favors apoplastic
proteins. This reenforces mucilage as a strong model in
which to study cell wall proteins, as it has markedly reduced cytoplasmic contamination compared with other
cell wall proteomes while retaining a comparable protein
recovery rate (Albenne et al., 2013). However, because the
extraction of mucilage requires its hydration-induced extrusion, the proteomic profile established for mature
mucilage may not include proteins that are normally
present only in early developmental stages. Characterizing the mucilage proteins from developing seeds will require other analytical strategies.
Seed Coat Mucilage Proteome Is a Specialized Cell
Wall Proteome
Seed Coat Mucilage Is a Suitable Model for Cell Wall
Protein Analyses
Most of the proteins of mature seed coat mucilage are
functionally similar to proteins found in primary cell
walls from other tissues (Albenne et al., 2013; Fig. 2),
Carbohydrate-active enzymes identified in the seed
mucilage proteome include two enzymes previously
detected by molecular genetic analyses. The mucilage
proteins MUM2 and BXL1 are required for the removal of
arabinogalactan side chains of cell wall polysaccharides
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Tsai et al.
Figure 7. TBA and TBAL expression requires NARS1, NARS2, and
TTG1. RT-PCR detection is shown for 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. Ler, Landsberg erecta.
and for proper mucilage extrusion (Dean et al., 2007;
Macquet et al., 2007b; Arsovski et al., 2009). Four other
carbohydrate-active enzymes not previously known to be
mucilage associated also were identified, including
HEXO3 (At1g65590/Q8L7S6). HEXO3 has been shown
to be involved in the removal of GlcNAc residues from
glycoproteins and in the formation of paucimannosidic
N-glycan (Gutternigg et al., 2007; Liebminger et al., 2011;
this study). HEXO3 localizes primarily to the plasma
membrane, although a minor fraction has been suggested
to be soluble in the apoplast, which would be consistent
with our findings (Liebminger et al., 2011). However, the
overall biological role of HEXO3 and its ortholog HEXO1
remains unknown, as no defects in growth and stress
responses were found in their respective mutants
(Liebminger et al., 2011).
Proteases are commonly found in cell walls, and four
were detected in the mucilage proteome. SBT1.7 has
been identified previously by its mutant mucilage
phenotype of defective extrusion and altered homogalacturonan methylation state. It has been suggested
that SBT1.7 participates in the removal of the inhibitor
domain from a pectin methylesterase (Rautengarten
et al., 2008). Two other proteases detected in mucilage
have been connected to roles in other tissues. Ectopic
expression of ASPG1 (At3g18490/Q9LS40) enhances
abscisic acid (ABA)-induced stomata closure, reactive
oxygen species production, and drought resistance (Yao
et al., 2012), and ASPG1 expression also is induced by
ABA (Yao et al., 2012). RD21a (At1g47128/P43297) is
up-regulated during drought stress, suggesting that it
also may be connected to ABA-regulated processes
(Koizumi et al., 1993). In addition, RD21a is known to
facilitate apoptosis, which the outer seed coat epidermal
cells eventually undergo at the end of seed development
(Lampl et al., 2013). However, no obvious mucilage defects were observed in either aspg1 or rd21a seeds.
Oxidoreductases can potentially modify cell wall
components either by regulating the production and
turnover of reactive oxygen species or by participating in
the oxidative modification of other cellular metabolites.
Mucilage proteins of this category include PER36, which
was shown previously to facilitate mucilage extrusion by
weakening the primary cell wall (Kunieda et al., 2013).
PER36 localizes to the radial and tangential primary
walls of the mucilage pockets but was not detected in the
mucilage itself (Kunieda et al., 2013). Therefore, it is
likely that PER36 is not part of the mucilage proteome
but, rather, a contaminant from the primary wall. Consistent with this, PER36 was the only protein found to be
more abundant in mum2-10 seed surface extracts compared with Col-0 (Fig. 3B). Primary cell wall proteins
such as PER36 would be expected to be overrepresented
in the proteome extracted from mum2-10 seeds, since
mucilage extrudes very poorly from this mutant.
Proteins involved in lipid metabolism also are common in cell wall proteomes (Albenne et al., 2013). These
proteins are likely involved in the synthesis and modification of cuticles deposited outside the cell wall. In
seed coat mucilage, proteins involved in lipid metabolism associate exclusively with the adherent layer,
making it the only obvious distinction between the
proteomes of the two mucilage layers (Fig. 2A). Since
the seed coat epidermis likely has a cuticle (Watanabe
et al., 2004; Panikashvili et al., 2009) and the primary
wall remains attached to the top of the columella embedded in the adherent layer, these proteins may be
involved in the synthesis/modification of a seed coat
cuticle. The fact that proteins involved in lipid metabolism are found only in the adherent layer suggests that
they are very strongly bound to the primary cell wall,
either covalently cross-linked with other cell wall polymers or perhaps anchored by hydrophobic interactions with the cuticle. In contrast, PER36 was observed
in the primary cell wall, but it was found in both adherent and nonadherent mucilage in our analysis,
suggesting that it is less strongly bound to the cell wall
than the proteins involved in lipid metabolism.
One difference between the mucilage and other cell wall
proteomes is the apparent lack of structural proteins in
mucilage. No extensins and Hyp-rich glycoproteins, typically major components of cell wall proteomes, were observed (Jamet et al., 2008a; Albenne et al., 2013), although
the Leu-rich repeat family protein At3g24480/Q9LHF1
might play a structural role. Furthermore, the TBA protein
structure (short, no identifiable protein domains, potential
glycosylation sites) and abundance (see below) are characteristics consistent with those of structural proteins, although we have no direct evidence supporting this
hypothesis. Arabinogalactan proteins also may function as structural proteins, as has been suggested for
ARABINOXYLAN PECTIN ARABINOGALACTAN
PROTEIN1 (Tan et al., 2013). Two fasciclin-like arabinogalactan proteins, FLA10 (At3g60900/Q9LZX4)
and FLA17 (At5g06390/Q66GR0), were identified in
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Identification of Seed Coat Mucilage Proteins
Figure 8. TBA proteins are secreted to
the seed coat epidermis apoplast.
Confocal microscopy images denote
the localization of cYFP-tagged TBA1,
TBA2, and TBA3 in developing seed
coats driven by their respective endogenous promoters. C, Columella; CC,
cytoplasmic column; M, mucilage
pockets. Bars = 10 mm.
mucilage, but their loss-of-function phenotypes did not
provide any insight into a possible structural role. Surprisingly, SALT OVERLY SENSITIVE5 (SOS5/FLA4;
At2g46550), the only FLA known to be required for
normal mucilage structure (Harpaz-Saad et al., 2011;
Griffiths et al., 2014), was not identified as a component
of the mature mucilage proteome. It may be possible that
SOS5 impacts mucilage structure indirectly, perhaps by
facilitating matrix polysaccharide biosynthesis in the
Golgi or acting as a GPI-anchored carrier of carbohydrates to the apoplast.
Despite the identification of numerous new mucilage
proteins, in addition to the four proteins (MUM2, BXL1,
PER36, and SBT1.7) previously known to regulate
mucilage extrusion and structure (Dean et al., 2007;
Macquet et al., 2007b; Rautengarten et al., 2008;
Arsovski et al., 2009; Kunieda et al., 2013), the biological
roles of the new proteins in mucilage remain unknown.
Analysis of loss-of-function mutants for many of the
genes encoding these proteins failed to identify mucilage extrusion and morphology defects. This may reflect functional redundancy. Alternatively, it may
indicate that these mucilage proteins play roles that do
not directly impact the extrusion, adherence, or structure of the mature mucilage and that, therefore, do not
generate a loss-of-function phenotype readily detectable by Ruthenium Red staining and light microscopy.
TBA Proteins Are Seed Coat-Specific Proteins with
Potential Roles in Seed Coat Differentiation
Among the mucilage proteins most frequently identified in this study are a family of three uncharacterized
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Tsai et al.
Figure 9. Down-regulation of TBA and TBAL does
not affect mucilage extrusion. A, Quantitative RTPCR analysis of the 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, and error bars denote SD. Asterisks denote transcript
levels significantly different from the wild type at
P , 0.05 (*) and P , 0.01 (**). B, Ruthenium Redstained seed coat mucilage from the four independent UBQ1p:TBA-amiRNA lines shown in A.
Seeds were imbibed in water, 0.05 M EDTA, 0.5 M
Na2CO3, or 0.05 M CaCl2 prior to staining. Bars =
100 mm.
proteins designated TBA, which are highly and specifically expressed in the seed coat epidermis during late
seed development (Figs. 5 and 6). The expression of
TBAs requires the seed coat differentiation master regulators NARS1, NARS2, and TTG1 (Fig. 7) and coincides temporally and spatially with mucilage synthesis
and secretion, while ectopically expressed TBAs were
shown to localize to the seed coat mucilage and the
columella (Fig. 8). Since TBAs are abundant and are
predicted to be heavily glycosylated while lacking
known functional domains, they may function as
structural proteins and cross-link other cell wall polysaccharides (Fig. 4). However, our attempts to use
amiRNA to knock down all of the TBA homologs simultaneously failed to produce a Ruthenium Redstained mucilage mutant phenotype (Fig. 9), so this
hypothesis remains to be validated. It is interesting that,
following the completion of mucilage synthesis during
columella formation, cYFP fluorescence in the mucilage
pocket abruptly disappears (Fig. 8; 10 DPA), leaving
fluorescence only in the columella. These observations
suggest that the TBA-cYFP proteins may be actively
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Identification of Seed Coat Mucilage Proteins
degraded, although we cannot rule out the possibility
that the fluorescence of the intact protein is being
quenched due to changes in the chemical environment
of the mucilage pocket.
The seed coat specificity and relative strength of the
TBA promoters make them valuable tools for seed coat
mucilage studies. Since the seed coat mucilage is not
essential to plant fitness under laboratory conditions, it
can tolerate genetic perturbation, which has made it a
powerful genetic model for cell wall analysis. The
specificity of the TBA promoters makes them suitable
tools to genetically manipulate the seed coat epidermis
in general and seed mucilage specifically. Another
Arabidopsis seed coat-specific promoter, from the
DIRIGENT PROTEIN1 gene (DP1), differs from expression of the TBA promoters in that DP1 is expressed
in both the epidermal and palisade cell layers and peaks
in expression during midseed development (Esfandiari
et al., 2013). Since the TBA promoters are active only in
the epidermis late in seed development, the TBA and
DP1 promoters complement each other by covering
different temporal and spatial domains.
In summary, a novel method was developed to extract and detect proteins integral to the Arabidopsis
seed coat mucilage. A total of 28 proteins were identified in mature seed coat mucilage, mostly with predicted functions consistent with a cell wall proteome.
The protein profiles are largely similar between the
adherent and nonadherent mucilage, with the exception of lipid metabolism proteins that occur exclusively
in the adherent layer mucilage. Three homologous,
previously undescribed proteins we named TBA were
highly abundant in seed coat mucilage. Although their
functions remain to be determined, their seed coat
epidermis-specific promoters should prove to be useful
tools for targeted gene expression.
MATERIALS AND METHODS
Plant Materials and Growth Conditions
Most Arabidopsis (Arabidopsis thaliana) plants used in this study were derived from the Col-0 ecotype, except for ttg1-1, which is derived from the
Landsberg erecta ecotype. Seeds were germinated on plates with Arabidopsis
medium (Haughn and Somerville, 1986) at 7% agar and transferred to soil
SunshineMix 4 (SunGro). Plants were grown with continuous fluorescent illumination of 80 to 140 mE m22 s21 at 20°C to 22°C. T-DNA insertion lines used in
this study were obtained from the ABRC and are listed in Supplemental Table
S1. T-DNA insertion lines were selected with a PCR-based assay using primers
listed in Supplemental Table S2.
Seed Coat Mucilage Extraction
Dry seeds (40 mg) were imbibed with 800 mL of double-distilled water in a
microcentrifuge tube. The seeds were gently shaken on a tabletop shaker for 1 h
at 120 rpm. Supernatants that contain the nonadherent mucilage were collected.
The seeds were washed once with 200 mL of double-distilled water, which was
pooled with the supernatant to form the nonadherent mucilage fraction. To
obtain the adherent mucilage, 800 mL of double-distilled water was added to
the seeds after extracting the nonadherent mucilage, and the seeds were secured
horizontally to a tabletop vortex and shaken at top speed for 3 h. The supernatants containing the adherent mucilage were collected. The seeds were
washed once with 200 mL of double-distilled water, which was pooled with the
supernatant as the adherent layer fraction. The mucilage samples were freeze
dried overnight and then chemically deglycosylated as described by Edge et al.
(1981). Briefly, 15 mL of anisole (Sigma-Aldrich) and 135 mL of trifluoromethanesulfonic acid (Sigma-Aldrich) were added to one freeze-dried
mucilage sample in a Reacti-vial. The samples were sealed and incubated at
4°C for 2 h. Four microliters of 0.2% Bromophenol Blue was added to each
sample, and 60% pyridine (Sigma-Aldrich) was added drop wise to each mucilage sample on ice until the solution turned light blue. The neutralized mucilage samples were dialyzed overnight in double-distilled water with dialysis
tubing pore sizes of 3,500 to 5,000 D and then freeze dried overnight.
MS
Mucilage protein samples were resuspended in SDS-PAGE sample buffer and
separated briefly on a 10% SDS-PAGE gel until all of the Mr markers just entered
the resolving gel. The proteins were stained with blue silver (Candiano et al.,
2004), and the entire lane was excised from the gel as one gel slice. Protein samples
were analyzed by tandem mass spectrometry (MS/MS) at the Centre for HighThroughput Biology Proteomics Core Facility at the University of British Columbia. In brief, samples were subjected to reduction/alkylation with DTT/
iodoacetamide followed by digestion with trypsin essentially as described by
Shevchenko et al. (1996). The resulting peptides were desalted and concentrated
with STAGE tips (Rappsilber et al., 2003) and analyzed by liquid chromatography-MS/MS on a linear-trapping quadrupole-Orbitrap mass spectrometer (LTQ
Orbitrap Velos) online coupled to an Agilent 1290 series HPLC device using a
nanospray ionization source (ThermoFisher Scientific) including a 2-cm-long
100-mm i.d. fused silica trap column, a 20-cm-long 50-mm i.d. fused silica fritted
analytical column, and a 20-mm i.d. fused silica gold-coated spray tip (6-mmdiameter opening, pulled on a P-2000 laser puller from Sutter Instruments,
coated on a Leica EM SCD005 Super Cool Sputtering Device). The trap column is
packed with 5-mm-diameter Aqua C-18 beads (Phenomenex; www.phenomenex.
com), while the analytical column is packed with 1.9 mm-diameter Reprosil-Pur
C-18-AQ beads (Dr. Maisch; www.dr-maisch.com). Standard 90-min gradients
were run from 10% to 32% buffer B (0.5% acetic acid and 80% acetonitrile) over
51 min, then from 32% to 40% in the next 5 min, then increased to 100% over a
2-min period, held at 100% for 2.5 min, and then dropped to 0% for another
20 min. The HPLC system included an Agilent 1290 series pump and autosampler
with thermostat. The thermostat temperature was set at 6°C. The sample was
loaded on the trap column at 5 mL min21, and the analysis was performed at
0.1 mL min21. The LTQ-Orbitrap was set to acquire a full-range scan at 60,000
resolution from 350 to 1,600 Th in the Orbitrap to simultaneously fragment the top
ten peptide ions by CID and the top five peptide ions by HCD (resolution, 7,500)
in each cycle in the LTQ (minimum intensity, 1,000 counts). Parent ions were then
excluded from MS/MS for the next 30 s. Singly charged ions were excluded, since
in electrospray ionization (ESI) mode, peptides usually carry multiple charges.
The Orbitrap was continuously recalibrated using lock-mass function. For mass
accuracy, the error of mass measurement is typically within 5 ppm and is not
allowed to exceed 10 ppm.
For quantitative analyses, Col-0 mucilage along with mum2-10 and ap2-7
seed surface extracts were prepared from 80 mg of seeds using the protocol
described above for nonadherent mucilage. All protein samples were reduced/
alkylated and digested as described above and then dimethylated with light,
medium, and heavy formaldehyde. Col-0 samples were labeled with light
formaldehyde for all replicates. mum2-10 samples were labeled with medium
formaldehyde for replicates 1 and 2 and with heavy formaldehyde for replicate
3. ap2-7 samples were labeled with heavy formaldehyde for replicates 1 and
2 and with medium formaldehyde for replicate 3. Samples from all three genotypes were pooled before the MS analysis. For replicates 1 and 2, samples were
analyzed using the LTQ Orbitrap Velo as described above. For replicate 3,
samples were analyzed using the Impact II quadrupole-time of flight mass
spectrometer (Bruker Daltonics) online coupled to an Easy nano LC 1000 HPLC
device (ThermoFisher Scientific) using a captive spray nanospray ionization
source (Bruker Daltonics) including a 2-cm-long 100-mm i.d. fused silica fritted
trap column and a 75-mm i.d. fused silica analytical column with an integrated
spray tip (6–8-mm-diameter opening pulled on a P-2000 laser puller from Sutter
Instruments). The trap column is packed with 5-mm Aqua C-18 beads
(Phenomenex; www.phenomenex.com), while the analytical column is packed
with 1.9-mm-diameter Reprosil-Pur C-18-AQ beads (Dr. Maisch; www.drmaisch.com). The analytical column was held at 50°C by an in-house constructed column heater. Samples were resuspended and loaded in buffer A
(0.1% aqueous formic acid). Standard 45-min gradients were run from 10% to
60% buffer B (0.1% formic acid and 80% acetonitrile) over 28 min, then increased
to 100% over 2 min, and held at 100% for 15 min. The liquid chromatograph
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Tsai et al.
thermostat temperature was set at 7°C. The sample was loaded on the trap
column at 850 Bar, and the analysis was performed at a flow rate of 0.25 mL
min21. The Impact II quadrupole-time of flight mass spectrometer was set to
acquire in a data-dependent auto-MS/MS mode with inactive focus fragmenting the 20 most abundant ions (one at the time at a rate of 18 Hz) after each
full-range scan from m/z 200 to 2,000 Th (at a rate of 5 Hz). The isolation window
for MS/MS was 2 to 3 Th depending on parent ion mass-to-charge ratio, and the
collision energy ranged from 23 to 65 eV depending on ion mass and charge.
Parent ions were then excluded from MS/MS for the next 0.4 min and reconsidered if their intensity increased more than 5 times. Singly charged ions were
excluded, since in ESI mode, peptides usually carry multiple charges. Strict
active exclusion was applied. For mass accuracy, the error of mass measurement is typically within 5 ppm and is not allowed to exceed 10 ppm. The nano
ESI source was operated at 1,700 V capillary voltage, 0.20 Bar nano buster
pressure, 3 L min21 drying gas, and 150°C drying temperature.
MS Data Analyses
For all qualitative analyses and replicates 1 and 2 of the quantitative analyses,
liquid chromatography-MS/MS data were processed with Proteome Discoverer version 1.2 (ThermoFisher Scientific) and then searched against the
Uniprot-Swissprot Arabidopsis database using the MASCOT algorithm version
2.4 (Perkins et al., 1999; http://www.matrixscience.com). The database contains 12,069 sequences; no contaminants were added in the search space. The
following parameters were applied: peptide mass accuracy, 10 ppm; fragment
mass accuracy, 0.6 D; trypsin enzyme specificity, fixed modifications, carbamidomethyl, variable modifications, Met oxidation; deamidated N, Q, and
N-acetyl peptides, ESI-TRAP fragment characteristics. Only those peptides with
IonScores exceeding the individually calculated 99% confidence limit (as opposed to the average limit for the whole experiment) were considered as accurately identified. Proteome Discoverer parameters were as follows: event
detector, mass precision of 4 ppm (corresponds to extracted ion chromatograms
at 612 ppm max error); S/N threshold, 1; quantitation method, ratio calculation; replace missing quantitation values with minimum intensity, yes; use
single peak quantitation channels, yes; protein quantification, use all peptides,
yes. In order for a protein to be considered a true mucilage protein in qualitative
analysis, it must be identified in at least two out of the three biological replicates
with MASCOT protein scores .40 (a score of $25 corresponds to a false discovery rate of #5%), and identified in at least one out of the three biological
replicates with two or more unique peptides.
For replicate 3 of the quantitative analysis, data analysis was performed using
MaxQuant 1.5.3.30 (Cox and Mann, 2008) with the Arabidopsis protein sequence database plus common contaminants. The search was performed using
the following parameters: peptide mass accuracy, 10 ppm; fragment mass accuracy, 0.05 D; trypsin enzyme specificity, fixed modifications, carbamidomethyl, variable modifications, Met oxidation; and N-acetyl proteins. Only
those peptides exceeding the individually calculated 99% confidence limit (as
opposed to the average limit for the whole experiment) were considered as
accurately identified. Relative protein levels from mum2-10 and ap2-7 seed
surface extracts were normalized to Col-0 nonadherent mucilage. Proteins that
could be detected and quantified in all three replicates were analyzed. A twotailed Student’s t test was used to determine the statistical significance of relative protein level differences between mum2-10, ap2-7, and Col-0.
Generation of Transgenic Plants
The TBAp:GUS constructs were generated using the pBI101 vector, with the
promoter fragments amplified from Col-0 genomic DNA. The TBAp:TBA-cYFP
translational fusion constructs were assembled in the citrine-pCambia1300 vector
as described by Debono et al. (2009), with the promoter and coding region
fragments amplified from Col-0 genomic DNA. The TBA amiRNA constructs
were designed and built as described by Schwab et al. (2006) using the UBQ1pCambia1300 vector as described by Ambrose et al. (2011). Primers used for
these constructs are listed in Supplemental Table S3. The TBA and TBAL promoters were defined as the DNA sequence extending upstream of the TBA start
codon to the next annotated gene, not including pseudogenes, to capture as
much of the promoter region as possible without introducing another gene.
TBA1p is ;0.5 kb, TBA2p is ;1.3 kb, TBA3p is ;1.8 kb, and TBALp is ;1.4 kb
long. Col-0 Arabidopsis plants were transformed using the floral dip method
(Clough and Bent, 1998); at least 20 independent transgenic lines were selected
for each construct. Results from at least three representative lines are shown.
Microscopy
For seed coat mucilage staining with Ruthenium Red, ;20 dry seeds were
imbibed in 1 mL of distilled, deionized water, 0.05 M EDTA, 0.05 M CaCl2, or 0.5
M Na2CO3 for 1 h and washed twice with double-distilled water. The seeds were
then stained with 0.01% (w/v) Ruthenium Red (Sigma-Aldrich) for 1 h and
washed once with double-distilled water. Seeds were imaged with a DFC450 C
camera (Leica) on an Axioskop 2 upright light microscope (Carl Zeiss).
Histochemical GUS assays were performed essentially as described by
Esfandiari et al. (2013). Tissue samples were vacuum infiltrated with GUS
staining solution (0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 20 mM Na2EDTA, and 0.1% [v/v] Triton X-100, supplemented with 1 mg
mL21 5-bromo-4-chloro-3-indolyl-b-D-glucuronide [Gold BioTechnology] in
100 mM phosphate buffer [pH 7]), incubated at 37°C for 16 h, and then washed
several times with 75% ethanol. Tissues were imaged with a DP72 camera
(Olympus) mounted on an SZX10 stereomicroscope (Olympus).
All confocal images were acquired from an Ultraview VoX Spinning Disk
Confocal System (PerkinElmer).
For cellulose staining, seeds after mucilage extraction were stained with 0.1%
(w/v) Calcofluor White for 5 min and then washed twice with double-distilled
water. The seeds were inspected under UV light with the Axioskop 2 microscope.
Immunoblot Analyses
Developing siliques were ground in liquid nitrogen and added to extraction
buffer containing 50 mM Tris-HCl (pH 8), 150 mM NaCl, 1 mM EDTA, 10% (v/v)
glycerol, 1% Triton X-100, 1 mM PMSF, and protease inhibitor cocktail (Roche).
The homogenates were centrifuged at 15,000 rpm for 10 min at 4°C, and the supernatant was collected. Protein concentrations were determined by Bradford
assay (Bio-Rad), and 100-mg protein samples were electrophoretically resolved by
12% SDS-PAGE. cYFP fusion proteins were detected using a mouse anti-GFP
polyclonal antibody (Roche) and horseradish peroxidase-conjugated goat antimouse polyclonal antibody (Santa Cruz Biotechnology). ECL Prime westernblotting detection reagent (GE Healthcare) was used for target detection.
Expression Analyses
Accession Numbers
RNA was extracted from various plant tissues using Trizol reagent (Life
Technologies), except that siliques were processed with the RNAqueous Total RNA
Isolation Kit (Ambion) while developing seed coats and embryos were processed
with the RNAqueous-Micro Total RNA Isolation Kit (Ambion) according to the
manufacturer’s instructions. cDNA synthesis was carried out using SuperScript II
reverse transcriptase (Life Technologies) according to the manufacturer’s instructions. Quantitative PCR (qPCR) was performed using iQ SYBR Green Supermix
(Bio-Rad) and the primers listed in Supplemental Table S3. The qPCRs were
assayed with the iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad).
GAPC transcripts were used as an internal control. A two-tailed Student’s t test
was used to determine the statistical significance of differences in TBA and TBAL
expression levels between the amiRNA lines and the wild type.
For gene expression analysis of TBAs in transcription factor mutants, RNA
extraction and cDNA synthesis were performed as described previously
(Kunieda et al., 2013). PCR was performed using Mango-Taq polymerase
(Bioline) and the same primers that were used for qPCR.
Sequence data from this article can be found in the GenBank/EMBL data
libraries under the following accession numbers: At1g65590/Q8L7S6, HEXO3;
At5g63800/Q9FFN4, MUM2; At5g49360/Q9FGY1, BXL1; At3g50990/
Q9SD46, PER36; At5g67360/O65351, SBT1.7; At3g18080/Q9LV33, BGLU44;
At1g65590/Q8L7S6, HEXO3; At3g18490/Q9LS40, ASPG1; At3g07390/
Q94BT2, AIR12; At1g47128/P43297, RD21a; At5g08260/Q9LEY1, SCPL35;
At1g62000/Q39168, TBA1; At1g62060/O04573, TBA2; At1g62080/O04575,
TBA3; At5g63140/Q9FMK9, PAP29; At5g06390/Q66GR0, FLA17; At3g60900/
Q9LZX4, FLA10; At2g28470/Q9SCV4, BGAL8, At4g23560/Q9SUS0, GH9B15;
At5g59310/Q9LLR6, LTP4; At3g08770/Q9LDB4, LTP6; At3g04170/Q9M8X3,
RmlC-like cupin superfamily protein; At5g45670/Q9FK75, GDSL motif esterase/acyltransferase/lipase; At3g16370/Q9LU14, GDSL motif esterase/acyltransferase/lipase; At1g75900/Q94CH6, GDSL motif esterase/acyltransferase/
lipase; At4g26790/Q8VY93, GDSL motif esterase/acyltransferase/lipase;
At4g28780/Q9SVU5, GDSL motif esterase/acyltransferase/lipase; At3g24480/
Q9LHF1, Leu-rich repeat family protein.
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Identification of Seed Coat Mucilage Proteins
Supplemental Data
The following supplemental materials are available.
Supplemental Figure S1. Mucilage extraction removes pectin and cellulose
from the seed coat.
Supplemental Figure S2. Expression (RT-PCR) of genes mutated by
T-DNA insertion.
Supplemental Figure S3. Imbibed seeds from plants homozygous for a
T-DNA insertion in genes encoding proteins found in seed mucilage.
Supplemental Figure S4. TBA and TBAL expression patterns in various
plant tissues.
Supplemental Figure S5. Detection TBA-cYFP by immunoblotting.
Supplemental Table S1. T-DNA insertion lines used in this study.
Supplemental Table S2. Primers used for mucilage protein T-DNA line
analyses in this study.
Supplemental Table S3. Primers used for TBA and TBAL constructs in this
study.
Supplemental Data Set S1. Col-0 adherent mucilage protein MS data.
Supplemental Data Set S2. Col-0 nonadherent mucilage protein MS data.
Supplemental Data Set S3. Summary of Col-0 mucilage protein identification.
Supplemental Data Set S4. mum2-10 and ap2-7 mucilage protein quantification MS data relative to Col-0.
Supplemental Data Set S5. Summary of mum2-10 and ap2-7 mucilage protein quantification relative to Col-0.
ACKNOWLEDGMENTS
We thank Suzanne Perry, Jamie Hackworth, and Jenny Hyung-Mee Moon of
the University of British Columbia Centre for High-Throughput Biology
Proteomics Core Facility for technical assistance, data analyses, and equipment
use; the University of British Columbia Bioimaging Facility for technical
assistance and equipment use; and Ikuko Hara-Nishimura and Dr. Tomoo
Shimada (Department of Botany, Kyoto University) for providing the nars1
nars2 mutant.
Received October 20, 2016; accepted December 15, 2016; published December
21, 2016.
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