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PECTIN METHYLESTERASE INHIBITOR6 Promotes
Arabidopsis Mucilage Release by Limiting Methylesterification
of Homogalacturonan in Seed Coat Epidermal Cells
CW
Susana Saez-Aguayo,a,b Marie-Christine Ralet,c Adeline Berger,a,b Lucy Botran,a,b David Ropartz,c
Annie Marion-Poll,a,b and Helen M. Northa,b,1
a Institut
National de la Recherche Agronomique, Institut Jean-Pierre Bourgin, Unité Mixte de Recherche 1318, Saclay Plant Sciences,
F-78026 Versailles, France
b AgroParisTech, Institut Jean-Pierre Bourgin, Unité Mixte de Recherche 1318, Saclay Plant Sciences, F-78026 Versailles, France
c Institut National de la Recherche Agronomique, Unité de Recherche 1268 Biopolymères Interactions Assemblages, F-44316 Nantes,
France
Imbibed seeds of the Arabidopsis thaliana accession Djarly are affected in mucilage release from seed coat epidermal cells.
The impaired locus was identified as a pectin methylesterase inhibitor gene, PECTIN METHYLESTERASE INHIBITOR6
(PMEI6), specifically expressed in seed coat epidermal cells at the time when mucilage polysaccharides are accumulated.
This spatio-temporal regulation appears to be modulated by GLABRA2 and LEUNIG HOMOLOG/MUCILAGE MODIFIED1, as
expression of PMEI6 is reduced in mutants of these transcription regulators. In pmei6, mucilage release was delayed and
outer cell walls of epidermal cells did not fragment. Pectin methylesterases (PMEs) demethylate homogalacturonan (HG), and
the majority of HG found in wild-type mucilage was in fact derived from outer cell wall fragments. This correlated with the
absence of methylesterified HG labeling in pmei6, whereas transgenic plants expressing the PMEI6 coding sequence under
the control of the 35S promoter had increased labeling of cell wall fragments. Activity tests on seeds from pmei6 and 35S:
PMEI6 transgenic plants showed that PMEI6 inhibits endogenous PME activities, in agreement with reduced overall
methylesterification of mucilage fractions and demucilaged seeds. Another regulator of PME activity in seed coat epidermal
cells, the subtilisin-like Ser protease SBT1.7, acts on different PMEs, as a pmei6 sbt1.7 mutant showed an additive phenotype.
INTRODUCTION
During seed coat formation, the developing seeds of myxospermous species, such as Arabidopsis thaliana, accumulate
polysaccharides in the apoplast of epidermal cells. When mature
seeds are imbibed, the rehydrated polysaccharides expand and
rupture the outer cell wall, and the released polysaccharides then
encapsulate the seed as viscous mucilage. The functional role of
this mucilage remains unclear, as Arabidopsis seeds without mucilage are viable and germinate under laboratory conditions. Certain reduced mucilage mutants have, however, been found to have
delayed germination or increased sensitivity to low water potential
compared with wild-type seeds (Penfield et al., 2001; Arsovski
et al., 2009a). Diverse physiological roles have been proposed; for
example, the adhesive properties of mucilage could glue seeds to
animals for dispersion, or to soil particles, thereby impeding predation by ants or retaining seeds in a favorable environment (Young
and Evans, 1973; García-Fayos et al., 2010; Engelbrecht and García-
1 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.plantcell.org) is: Helen M. North (helen.
[email protected]).
C
Some figures in this article are displayed in color online but in black and
white in the print edition.
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.112.106575
Fayos, 2012). Recent studies have shown that in the desert
shrub Artemisia sphaerocephala, seed mucilage can promote
seedling emergence and the growth of soil microflora and maintain
seed viability by enabling the repair of damaged seed DNA (Yang
et al., 2011; 2012a; 2012b).
The accumulation of mucilage polysaccharides in the apoplast
of seed coat epidermal cells is polarized and requires a high
degree of temporal organization (Young et al., 2008). The deposition of mucilage polysaccharides begins at the junction of the
outer tangential and radial cell walls and results in the formation of
a cytoplasmic column in the center of the epidermis cell. In parallel,
an increase in Golgi stacks and the accumulation of starch granules
is also observed. Following mucilage synthesis, a secondary cell
wall is synthesized and fills the cytoplasmic column, forming the
columella and leading to programmed cell death (Western, 2012).
At the end of differentiation, the polygonal epidermal cells have
central volcano-shaped columella that are connected to reinforced
radial cell walls and surrounded by dehydrated polysaccharides
under a primary cell wall.
The composition and structure of Arabidopsis seed mucilage
is one of the best characterized and it has been shown to be
composed of two layers, termed water-soluble (outer layer) and
adherent (inner layer) (Western et al., 2000; Macquet et al., 2007a).
Both layers are composed mostly of the pectin rhamnogalacturonan
I (RG I), a repeat of the disaccharide (→4)-a-D-GalA-(1→2)-a-L-Rha(1→) (Goto, 1985; Western et al., 2000, 2004; Penfield et al., 2001;
Usadel et al., 2004; Macquet et al., 2007a). In contrast with the
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water-soluble layer, the adherent layer is tightly attached to the
seed and the RG I contains a small number of arabinan and
galactan ramifications (Dean et al., 2007; Macquet et al., 2007a,
2007b; Arsovski et al., 2009b; Huang et al., 2011; Walker et al.,
2011). The pectin homogalacturonan (HG), a repeat of galacturonic
acid, is also present as a minor mucilage component (Willats et al.,
2001; Macquet et al., 2007a). In the adherent mucilage, its degree
of methylesterification (DM) varies, being higher in the outer
compared with the inner region of the adherent layer (Macquet
et al., 2007a). The adherent mucilage also contains cellulose,
which is required for mucilage structuration and adherence
(Harpaz-Saad et al., 2011; Mendu et al., 2011; Sullivan et al.,
2011). Seed mucilage has become a model system for the study
of polysaccharides as its constituents also form part of more
complex plant cell walls.
As seed mucilage is nonessential in laboratory conditions,
a number of mutants affected in mucilage production have been
identified. The defective genes have been characterized and
encode transcription regulators or polysaccharide metabolism
enzymes. Enzymes implicated in the synthesis of mucilage
pectin have been highlighted from the reduced mucilage phenotype of mutants. MUCILAGE MODIFIED4 (MUM4)/RHAMNOSE SYNTHASE2 synthesizes the UDP-rhamnose required for
the production of RG I (Usadel et al., 2004; Western et al., 2004;
Oka et al., 2007). GALACTURONSYL TRANSFERASE11 (GAUT11)
and GALACTURONSOSYLTRANSFERASE-LIKE5 (GATL5) are
potentially involved in the synthesis of pectin present in mucilage
(Caffall et al., 2009; Kong et al., 2011; Western, 2012). As gatl5
mutants present a reduction in both rhamnose and galacturonic
acid, while gaut11 only seems to be affected in galacturonic acid
content, this suggests different roles in RG I or HG synthesis,
respectively (Western, 2012). Recently, mutants defective in the
cellulose synthase catalytic subunit CELLULOSE SYNTHASE5
(CESA5/MUM3), the Leu-rich receptor kinase FEI2, and the
glycophosphatidylinositol-anchored fasciclin-like arabinogalactan
protein SALT OVERLY SENSITIVE5 (SOS5) were found to affect
the production of cellulose present in mucilage (Harpaz-Saad
et al., 2011; Mendu et al., 2011; Sullivan et al., 2011).
Most of the transcription regulators identified (APETALA2,
ENHANCER OF GLABRA3, GLABRA2 [GL2], MYB5, MYB61,
TRANSPARENT TESTA8, TRANSPARENT TESTA GLABRA1,
and TRANSPARENT TESTA GLABRA2) regulate seed coat differentiation and are required for normal epidermal cell morphology
and mucilage production (reviewed in Western 2012). By contrast,
mutation of the transcriptional corepressor LEUNIG HOMOLOG1
(LUH1)/MUM1 only affects mucilage extrusion (Bui et al., 2011;
Huang et al., 2011; Walker et al., 2011). Three downstream targets
of LUH1/MUM1 are enzymes that affect polysaccharide maturation; MUM2 is a b-D-galactosidase and BXL1 a bifunctional b-Dxylosidase/a-L-arabinofuranosidase that trim galactan or arabinan
side chains, respectively, from RG I, and the subtilisin-like Ser
protease SBT1.7 is implicated in the modulation of HG methylesterification (Dean et al., 2007; Macquet et al., 2007b; Rautengarten
et al., 2008; Arsovski et al., 2009b). These three enzymes appear to
alter both mucilage and cell wall mechanical properties important for
mucilage liberation (Rautengarten et al., 2008; Arsovski et al., 2009b;
Walker et al., 2011). Notably, HG is synthesized and secreted in
a highly methyl-esterified state, and the DM is determined after
secretion by the relative activity of pectin methylesterases (PME)
and their inhibition by proteinaceous PME inhibitors (PMEIs). It
has been hypothesized that SBT1.7 could regulate the DM of
HG through either the degradation of PME or activation of PMEI
by limited proteolysis (Rautengarten et al., 2008).
PMEIs were identified first by purification from kiwi fruit (Actinidia
chinensis) as small acidic proteins with four conserved Cys residues and an N-terminal signal sequence for extracellular targeting
whose cleavage is required for inhibitor function (Camardella et al.,
2000; Giovane et al., 2004). Subsequently, PMEI structure and
interactions in a 1:1 complex with PME were described for Arabidopsis PMEI1 (Hothorn et al., 2004). These studies showed that
mature PMEIs have two major structural features, an N-terminal
a-helix hairpin anchor that interacts with the PME C terminus
and a four-helix bundle that docks into the PME active site,
thereby blocking access to the substrate. It has been suggested
that PMEI inhibition is not generalized against all PMEs but that
different PME isoforms will have preferential PMEI partners (Wolf
et al., 2009). In Arabidopsis, the PECTIN METHYLESTERASE
INHIBITOR (PMEI) gene family is large, with 69 members, and
although five of these have been shown to act as inhibitors, either
through assays with recombinant protein or the modification of
PME activity in transgenic plants, to date, no PMEI has been directly linked to a precise physiological role or associated with
a loss-of-function effect (Wolf et al., 2003, 2012; Raiola et al., 2004;
Lionetti et al., 2007; Peaucelle et al., 2008; Pelletier et al., 2010).
The Arabidopsis accession Shahdara was previously shown
to be a natural mutant from central Asia affected in MUM2
(Macquet et al., 2007b). Analysis of further accessions from this
region has identified Djarly as a novel mutant affected in mucilage release (S. Saez-Aguayo A. Macquet, O. Loudet, and H.M.
North, unpublished results). The gene affected in Djarly has been
identified through map-based cloning as encoding a PMEI
(PMEI6). Genetic, cytological, and biochemical approaches have
been used to demonstrate that the expression of PMEI6 in
epidermal cells of the seed coat is necessary for the inhibition of
PME activity on methylesterified HG in mucilage and the outer
primary cell wall of these cells.
RESULTS
The Djarly Accession Is Affected in PMEI6
Seeds of new accessions obtained from central Asia (http://
www.inra.fr/vast/collections.htm) were imbibed in the pectin dye
ruthenium red, and the Djarly accession, from Kyrgyzstan, was
found not to release mucilage (cf. Figures 1B and 1A). The gene
affecting mucilage release in Djarly was identified using mapbased cloning; the affected locus was localized between 19.520
and 19.585 Mb on chromosome 2. Homozygous mutants were
obtained for 18 insertion lines in 15 of the 22 annotated genes
(see Supplemental Table 1 online) and their seeds analyzed for
mucilage release. Only one mutant had seeds with a phenotype
equivalent to Djarly, and this had an insertion in the gene
At2g47670, predicted to encode a PMEI that we named PMEI6
(Figures 1C and 1G). Two additional insertion mutant alleles,
pmei6-2 and pmei6-3, were obtained, and neither released mucilage when they were imbibed with ruthenium red (Figures 1D, 1E,
PMEI6 Activity Promotes Mucilage Release
and 1G). To test for allelism, pmei6-1 was crossed to Djarly, pmei6-2,
and pmei6-3, and in all cases, F1 and F2 seed coats did not release
mucilage (Figure 1F; data not shown); the Djarly mutant allele was
termed pmei6-4. Furthermore, seeds from the backcrossed pmei6-1
mutant still exhibited the phenotype. The release of mucilage from
F1 seed coats of F2 seeds used for mapping or from the backcross
indicated that pmei6-1 and pmei6-4 mutant alleles were recessive and maternally inherited.
To identify the nature of the mutant allele in the Djarly accession, the At2g47670 gene was sequenced and three nucleotide
polymorphisms were identified compared with Columbia-0 (Col0). The first substituted Ser-110 for Arg, the second at Leu-159
was a synonymous mutation, and the third caused a frameshift
mutation due to a 1-bp insertion 581 bp after the ATG codon
that changed amino acids 194 to 205 followed by the introduction of a stop codon (Figure 1G). PMEIs inhibit PMEs
through the formation of a reversible 1:1 complex (D’Avino et al.,
2003; Hothorn, et al., 2004). Structural studies have shown that
PMEIs have two distinct functional domains: an a-helical hairpin
structure that stabilizes the PME:PMEI complex and a C-terminal
bundle domain that interacts directly with the PME active site,
thereby blocking substrate access (Hothorn et al., 2004). Modeling of the effects of the Djarly mutations on PMEI tertiary
structure indicated that the bundle domain would be modified, in
particular the a7 helix predicted to interact with the PME binding
cleft, which would explain the lack of PMEI6 function (see
Supplemental Figure 1 online) (Di Matteo et al., 2005).
Molecular complementation of the pmei6-1 mutant phenotype
was also performed; the mutant was transformed with a construct
containing the PMEI6 coding sequence (CDS) downstream of the
cauliflower mosaic virus 35S promoter, and 16 independent
transformants were obtained. When seeds were sown on agarose
plates, mucilage release was restored in at least 60% of seeds
from all 16 transformants compared with <22% in the pmei6-1
mutant (see Supplemental Figure 2A online).
The expression of PMEI6 was analyzed in the pmei6-1 mutant
by RT-PCR on RNA extracts from developing seeds using PMEI6specific primers situated either side of the pmei6-1 insertion at the
59 and 39 extremities of PMEI6 (Figure 1G). Amplification of the
control gene EF1a4 was similar in the wild type and pmei6-1
(Figure 1H). By contrast, no amplification was obtained using
primers on either side of the pmei6-1 T-DNA insertion site and
encompassing the entire CDS, indicating that no full-length
transcript was recreated by splicing out the T-DNA insertion and
that the pmei6-1 mutant is a knockout. Reduced fragment amplification was observed in the mutant for the PMEI6 59 region,
compared with the wild type, and only a faint fragment was observed for the PMEI6 39 region that could result from a low level of
transcript reinitiation from within the T-DNA insertion (Figure 1H).
Mucilage Release Is Delayed in pmei6
Unlike the wild type, pmei6 mutant seeds did not release mucilage within the first few minutes of imbibition in ruthenium red.
Nevertheless, it was noted that mucilage was released from
pmei6 seeds when imbibition time was increased, indicating that
mucilage release was delayed (Figures 2A to 2D). In addition to
this delay, the outer primary cell wall did not break up and leave
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Figure 1. PMEI6 Is Required for the Correct Release of Seed Mucilage.
(A) to (F) Seeds imbibed directly in the pectin stain ruthenium red. Wild-type
seeds (A) release mucilage in contrast with the Djarly accession (pmei6-4) (B)
and three pmei6 mutant alleles ([C] to [E]) that do not release mucilage. Genetic complementation is not observed in the F1 seed coat of F2 seeds from
a cross between pmei6-1 and pmei6-4 (F). WT, the wild type. Bars = 500 µm.
(G) Schematic representation of the structure of PMEI6 as annotated by
The Arabidopsis Information Resource (http://www.Arabidopsis.org/). The
sites and orientation of insertion lines in pmei6-1, pmei6-2, and pmei6-3
are indicated (LB, left border) and the position and nature of pmei6-4
mutations are shown. Numbers indicate the position of different features in
bp. Colored bars denote the positions of the primers used in (H).
(H) Effect of the pmei6-1 mutation on PMEI6 expression. RT-PCR
analysis was performed with PMEI6-specific primers. Purple, green, and
orange labels indicate primers amplifying the CDS (PMEI6 CDS) and the
region 59 (PMEI6 59) or 39 (PMEI6 39) of the DNA insertion, respectively. A
control amplification was performed with primers for EF1a4.
fragments attached to the top of columella; instead, it was observed attached to the hilum, as a large sheet of cell wall (Figure
2D). To quantify this delay in mucilage release, soluble extracts
were measured for GalA contents after imbibition of seeds for
different lengths of time in water (Figure 2E). The mucilage release
rate for mutant seeds was less than half that of the wild type: Vmax
[wild type] = 0.17 mg21$min21 compared with Vmax[pmei6-1] = 0.07
mg21$min21 and Vmax[pmei6-2] = 0.06 mg21$min21. Furthermore,
the total amount of GalA released from pmei6-1 and pmei6-2
seeds never reached wild-type levels, even after 24 h of imbibition (Figure 2E). Ruthenium red staining intensity and the
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Figure 2. Seed Mucilage Release Is Delayed in pmei6 Mutants.
(A) to (D) Observation of seed mucilage released from wild-type ([A] and
[C]) and pmei6-1 ([B] and [D]) seeds by ruthenium red staining. Seeds
were stained directly ([A] and [B]) or after imbibition for 1 h in water ([C]
and [D]). OW, outer wall. Bars = 200 µm.
(E) Rate of mucilage release in water, measured as the amount of released GalA sugars. Error bars represent SE values (n = 4). Vmax, rates of
mucilage release; WT, the wild type.
thickness of the adherent mucilage appeared the same in the
wild type and pmei6-1 after imbibition in either water or the
cation chelator EDTA (Figures 2C and 2D; see Supplemental
Figure 3 online).
Expression of PMEI6 in Seeds Is Specific to the Epidermal
Cells of the Seed Coat
In accordance with the delayed mucilage release observed for
pmei6 seeds, available in silico expression data indicated highest
expression levels in whole siliques or seeds and in particular the
seed coat (Le et al., 2010; http://seedgenenetwork.net/arabidopsis);
expression observed in other tissues, such as stems and
flowers buds, was weak in comparison (Schmid et al., 2005). To
confirm these data and to determine in which silique tissues
PMEI6 is expressed, quantitative RT-PCR (qRT-PCR) was
performed on dissected seeds, silique envelopes, and whole
siliques at developmental stages when mucilage polysaccharides
are produced and deposited (8, 10, 12, and 14 d after pollination
[DAP]). PMEI6 expression was highest in developing seeds at 8
DAP, corresponding to seeds with torpedo stage embryos (see
Supplemental Figure 4A online). A diminution of transcript accumulation was observed at later stages, in agreement with in silico
transcriptome data.
To obtain more detailed information about the spatial expression of PMEI6 within the seed, b-glucuronidase (GUS) reporter gene expression was observed in the pmei6-3 mutant.
This mutant was generated using an enhancer trap Ds element
that contains the GUS reporter gene (Sundaresan et al., 1995).
In pmei6-3, the GUS gene is correctly orientated for control by
the endogenous PMEI6 promoter. In addition, wild-type transformants containing a translational fusion with GFP were examined and in situ hybridization performed using a PMEI6-specific
probe. The PMEI6-GFP (for green fluorescent protein) produced
was not functional, since the phenotypes of the pmei6-1 mutant
were not restored when transformed with the same construct. For
reporter gene analyses, cell walls were counterstained with propidium iodide to facilitate visualization. Analyses were performed
on the same four seed developmental stages as for qRT-PCR.
Expression of PMEI6 transcripts and protein was observed to be
specific to the epidermal cells of the seed coat. At 10 DAP, expression delimited the cytoplasm found at the base of the epidermal cells and in a column containing accumulated starch
granules (see Supplemental Figures 4B, 4D, 4F, 4H, 4J, and 4L
online). As seed coat differentiation progresses, secondary cell
wall material accumulated to form the columella and reinforce
radial cell walls. The column of cytoplasm was thus reduced at 14
DAP, and PMEI6 expression was observed in this restricted cytoplasm (see Supplemental Figures 4C, 4E, 4G, 4I, 4K, and 4M
online).
Seed Coat Structure and Function Are Unaffected
by PMEI6 Mutation
The structure of mutant seed coat epidermal cells was examined
in more detail by scanning electron microscopy on mature dry
seeds. The epidermal cells of both wild-type and pmei6-1 mutant
seeds were equivalent in size and morphology with a characteristic polygonal form and a central columella (see Supplemental
Figures 5A to 5D online). No difference in the outer and radial cell
walls was observed that could explain the delay in mucilage release and the nonfragmentation of the outer cell wall in pmei6.
Developing seed coat epidermal cells from the pmei6-1 mutant
were also compared with the wild type in seed sections staged
from 8 to 14 DAP (see Supplemental Figures 5E to 5L online). Cell
structure and the timing of differentiation looked the same; in
particular, outer and radial cell walls and mucilage accumulation
appeared unchanged.
PMEI6 Activity Promotes Mucilage Release
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PMEI6 Mutation Modifies HG Methylesterification
in Adherent Mucilage
The adherent mucilage layer of wild-type Col-0 has previously
been shown to be composed of two domains that both contain
HG as a minor component. The DM of this HG is variable, being
moderate in the inner domain and high in the outer domain
(Macquet et al., 2007a; Figures 3A and 3B). To determine
whether PMEI6 influences this DM, whole-mount immunolabeling was performed on mature dry seeds of wild-type, pmei6-1,
and PMEI6 overexpressor lines (PMEI6ox) (wild-type plants
transformed with the same 35S:PMEI6 construct used to complement the pmei6-1 mutant that show higher steady state levels
of PMEI6 transcripts in developing seeds; see Supplemental
Figure 6A online). Seeds were costained for b-glycans present in
the epidermal cell columella and cell walls, as well as cellulose in
the mucilage, using the fluorescent probe Calcofluor (Willats et al.,
2001; Macquet et al., 2007a). In the pmei6-1 mutant, neither
moderately nor highly methyl-esterified HGs were detectable with
JIM5 and JIM7 antibodies, respectively (Figures 3C and 3D). By
contrast, labeling with both JIM antibodies was increased in
PMEI6ox (Figures 3E and 3F) compared with the wild type (Figures 3A and 3B). Similar increases in labeling were also observed
for pmei6-1 seeds complemented with the same construct (see
Supplemental Figures 2E to 2H online). Furthermore, Calcofluor
labeling in the pmei6-1 mutant showed naked columella, with no
fragments from outer cell wall breakage attached to their tops
(Figures 3C and 3D).
PMEI6 Inhibits PME Activity in Seeds
To determine if PMEI6 acts as an inhibitor of PME activity found
in seeds, assays were performed using total protein extracts
from mature dry seeds of the wild type, pmei6, and PMEI6ox;
PME activities were normalized to the average wild-type activity
(Figure 4A). Activity levels were higher in pmei6-1 mutant seeds,
with a 14% increase in global PME activity. In addition, PME
activity was decreased in seeds of overexpressor lines by 40%
compared with the wild type. Similarly, activity levels were reduced in three pmei6-1 transformants complemented with the
same 35S:PMEI6 construct (see Supplemental Figure 2B online).
PME activity was also determined for proteins released with
soluble mucilage from imbibed seeds, which correspond to
proteins present in the apoplasmic mucilage pocket of seed
coat epidermal cells (Figure 4B). The diameters of activity halos
and their staining intensity showed the same tendencies as for
whole seeds, with higher and lower PME activities in pmei6-1
and PMEI6ox-21, respectively, compared with the wild type.
Mucilage Composition and Partitioning Are Modified
in the pmei6 Mutants
Water-soluble mucilage, adherent mucilage, and demucilaged
seeds were obtained from the wild type, pmei6-1, and pmei6-2
mutants using a sequential extraction procedure (see Supplemental
Figure 7 online) and analyzed for their sugar and methanol contents. As the major component of Arabidopsis mucilage is RG I, the
majority of sugars present in the soluble and adherent mucilage
Figure 3. Labeling of Methylesterified HG in Adherent Mucilage Released from Seeds of the Wild type, pmei6-1, and a PMEI6 Overexpressor Line.
Confocal microscopy optical sections of adherent mucilage (AM) released from mature imbibed seeds. Composite image of double labeling
of b-glycans with calcofluor (pink) and partially or highly methylesterified
HG, respectively, with JIM5 ([A], [C], and [E]) or JIM7 ([B], [D], and [F])
antibodies (green). (A) and (B) show the wild type; (C) and (D) show the
pmei6-1 mutant; and (E) and (F) show the overexpressor line PMEI6ox21. C, columella. Laser gains used were identical for images obtained for
the same antibody. Bars = 25 mm.
from the wild type were Rha and GalA in a molar ratio close to 1,
in agreement with previous studies (Table 1) (Macquet et al.,
2007a, 2007b; Huang et al., 2011; Sullivan et al., 2011; Walker
et al., 2011). In pmei6-1 and pmei6-2 soluble mucilage, a reduction in total sugars of up to 50% was observed, which was
mainly due to a reduction in Rha and GalA (Table 1). By contrast,
adherent mucilage total sugars increased by over 25% in
pmei6-1 and pmei6-2, again due to modified Rha and GalA
contents (Table 1). The pmei6 mutants thus showed a redistribution of water-soluble to adherent mucilage; in the
wild type, water-soluble mucilage represented ;63% of total
mucilage, whereas in pmei6 mutants, this was reduced to
<50% (Table 1). Furthermore, an overall reduction in mucilage
total sugars was measured in mutant extracts, as previously
observed in mucilage release assays, and this was mainly due
to a reduction in GalA (Table 1; Figure 2E). By contrast, GalA
contents increased slightly in demucilaged seed extracts
(Table 1).
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Figure 4. PMEI6 Modulates PME Activity in Seeds.
(A) PME activities in total protein extracts from mature dry seeds of wildtype (WT), pmei6-1, and three overexpressor lines (PMEI6ox). Activities
were normalized to average wild-type activity (=1). Error bars represent SE
values (n = 3). Mann-Whitney U test with the wild type, ***P < 0.1%.
Similar results were obtained in two experiments with seeds from independent cultures.
(B) In-gel PME assay of protein extracted from soluble mucilage fraction
on imbibition of mature seeds. The level of pectin demethylesterification
was visualized by staining with ruthenium red (0.025%, w/v).
[See online article for color version of this figure.]
To determine whether the overall degree of methylesterification was modified in pmei6 mutants, a methanol release
assay was conducted by alkali treatment of extracts. Methanol
release was reduced by more than 95, 48, and 13.5% in pmei6
water-soluble mucilage, adherent mucilage, and demucilaged
seeds, respectively (Table 1), corresponding to a total decrease
of more than 24% compared with the wild type. Previous mass
spectrometry (MS) analysis of soluble mucilage digested by rhamnogalacturonan hydrolase identified a series of RG I oligomers that
did not appear to be methylated (Ralet et al., 2010). The methanol
measured in wild-type soluble and adherent mucilage is therefore
most likely derived from methylesterified HG domains. In effect,
immunolabeling experiments have detected small amounts of
methylesterified HG in wild-type adherent mucilage (Figure 3;
Macquet et al., 2007a).
To determine whether the reduction in GalA contents measured corresponded to GalA derived from RG I or HG, analyses
were performed on fractions obtained after precipitation of HG
pectin domains with ethanol (see Supplemental Figure 7 online).
GalA derived from HG showed a dramatic decrease in the mutants compared with the wild type for both water-soluble and
adherent mucilage extracts of >88 and 90%, respectively (Table 2).
By comparing to the values obtained for total GalA contents
(Tables 1 and 2), values for RG I–related GalA were calculated
(Table 2) and showed that in pmei6 mutants most of the GalA
redistributed from soluble to adherent mucilage was RGI-derived
GalA, while nearly all the HG-derived GalA was in demucilaged
seeds.
The occurrence of methylesterified HG domains in soluble
mucilage was assessed by MS analysis of pectin lyase digests.
Pectin lyase degrades highly methylesterified HG via a b-elimination
mechanism, producing unsaturated methylesterified GalA oligomers
that can be detected and identified by MS (Ralet et al., 2012).
The MS spectrum of the pectin lyase digest of wild-type soluble
mucilage was characterized by the presence of peaks attributed
to unsaturated methylesterified GalA oligomers with a degree of
polymerization of 3 to 7. The ions corresponding to dp6 and dp7
oligomers were visible in a zoom of the wild-type mass spectrum
region corresponding to a mass-to-charge ratio (m/z) 1100 to
1400 (Figure 5A). Unsaturated GalA oligomers of dp6 with 4 to 6
methyl groups (m/z 1135, 1149, and 1163) and unsaturated GalA
oligomers of dp7 with 5 or 6 methyl groups (m/z 1325 and 1339)
were observed. In addition, single sodiation (+22) of the carboxylic
functions of predominant ions was detected at m/z 1157, 1171,
1347, and 1361, and potassium adducts (m/z 1165 and 1179) and
single sodiation of potassium adducts (m/z 1187 and 1377) were
observed. By contrast, none of these peaks could be detected in
the mass spectrum of the pectin lyase digest of pmei6-1 soluble
mucilage (Figure 5B).
Taken together, the results obtained from MS and biochemical analyses reveal the presence of highly methylesterified
HG domains in wild-type mucilage that are absent from pmei6-1
mucilage.
LUH/MUM1 and GL2 Regulate PMEI6 Expression
Previous studies implicated LUH/MUM1 as a positive regulator
of MUM2, BXL1, and SBT1.7 expression and GL2 in the induction of MUM4 expression (Western et al., 2004; Bui et al.,
2011; Huang et al., 2011; Walker et al., 2011). To assess whether
PMEI6 is regulated by these transcription factors, transcript
abundance was analyzed by qRT-PCR in developing seeds of
the wild type, luh/mum1, and gl2. Steady state PMEI6 transcript
levels were markedly reduced in both gl2 and luh/mum1 relative
to the wild type (Figure 6A; see Supplemental Figure 6B online).
These results indicate that LUH/MUM1 and GL2 are required for
normal PMEI6 expression in the seed coat. To confirm this
analysis, PME activity assays were performed on protein extracts from mature dry seeds of luh/mum1 and gl2. Figure 6B
shows that the PME activities of gl2 and luh/mum1 were both
altered compared with the wild type, confirming that these
transcription factors influence PME activity. Nevertheless, while
activity levels in gl2 were higher than those of the wild type, as
observed for the pmei6-1 mutant, in luh/mum1, activity levels
decreased.
PMEI6 and SBT1-7 Regulate PME Activity through
Independent Pathways
A previously identified mutant, sbt1.7, does not release mucilage
and has similar phenotypes to those observed for pmei6 mutants: higher PME activity in seeds, reduced methylesterification
of mucilage, nonfragmentation of the outer cell wall, and its
shedding in large pieces attached to the chalazal end of the seed
(Rautengarten et al., 2008). It was hypothesized that the SBT1.7
subtilisin-like Ser protease could activate PMEI by removing the
PMEI6 Activity Promotes Mucilage Release
7 of 16
Table 1. Composition of Sequentially Extracted Mucilage and Demucilaged Seeds from the Wild Type, pmei6-1, and pmei6-2
The Wild Type
pmei6-1
pmei6-2
mg$g21 seed
Water-soluble mucilage polysaccharides
GalA
Rha
Fuc
Ara
Xyl
Man
Gal
Glc
Total sugars (SM)
SM CH3OH (µmol$g21)
Adherent mucilage polysaccharides
GalA
Rha
Fuc
Ara
Xyl
Man
Gal
Glc
Total sugars (AM)
AM CH3OH (µmol$g21)
Total mucilage polysaccharides
Total mucilage sugars
Total mucilage GalA
Total mucilage Rha
Total mucilage CH3OH (µmol$g21)
Demucilaged seed polysaccharides
GalA
Rha
Fuc
Ara
Xyl
Man
Gal
Glc
Total sugars (DS)
DS CH3OH (µmol$g21)
11.29
9.1
0.04
0.21
0.64
0.19
0.36
(0.33)
(0.14)
(0.04)
(0.02)
(0.03)
(0.06)
(0.05)
–
7.54 (0.29)
6.35 (0.53)
5.46
4.45
0.05
0.12
0.45
0.20
0.22
–
0.18
0.5
0.16
0.34
(0.02)
(0.04)
(0.01)
(0.08)
–
(0.89)
(0.91)
(0.05)
(0.01)
(0.08)
(0.08)
(0.03)
21.83 (1.85)
3.5 (0.19)
15.08 (1.25)
0.07 (0.02)
–
10.63 (0.88)
0.13 (0.05)
6.17 (0.37)
3.73 (0.23)
7.87 (0.72)
5.61 (0.22)
7.85 (0.31)
5.45 (0.27)
–
–
0.45
0.1
0.29
2.28
(0.19)
(0.1)
(0.04)
(0.12)
–
–
0.49
0.07
0.36
2.19
(0.20)
(0.07)
(0.08)
(0.08)
–
16.59 (1.19)
0.24 (0.02)
34.84
17.46
12.83
4.12
31.67
15.41
11.96
0.30
36.03 (0.32)
7.11 (0.51)
2 (0.17)
31.72 (0.65)
11.55 (0.45)
5.99 (0.4)
22.51 (1.38)
54.96 (0.2)
171.87 (6.49)
25.9 (1.44)
0.33 (0.01)
2.35 (0.05)
–
13.01 (0.89)
0.62 (0.08)
(4.42)
(2.57)
(2.69)
(1.45)
0.42 (0.12)
–
(0.76)
(0.17)
(0.37)
(0.09)
37.99 (0.75)
7.65 (0.39)
–
30.79 (1.12)
10.92 (0)
6.12 (0.35)
22.01 (0.37)
50.4 (1.02)
165.87 (5.95)
24.07 (2.28)
16.4 (1.18)
0.32 (0.06)
27.03 (2.89)
13.31 (1.2)
9.9 (0.5)
0.45 (0.1)
39.1
8.35
1.4
28.85
10.9
7.55
22.35
51.5
170
20.57
(0.2)
(0.45)
(1.4)
(0.25)
(0.5)
(1.05)
(0.75)
(0.2)
(6.19)
(0.45)
Values are means of four independently extracted samples from two biological repeats (6SD). AM, adherent mucilage; CH3OH, methanol; DS,
demucilaged seed; SM, water-soluble mucilage; –, not detected. Details of sequential extraction method are given in Supplemental Figure 7 online.
predicted inhibitory N-terminal signal sequence or inactivate
PME by proteolysis. To determine whether SBT1.7 and PMEI6
could act through regulating the same PME activity, double
mutants were obtained from a cross between pmei6-1 and
sbt1.7-1. On direct imbibition of seeds in ruthenium red, the
seeds of pmei6-1 sbt1.7-1 did not release mucilage, as observed for seeds of the parental genotypes (Figures 7A to 7D).
Furthermore, when seeds were imbibed for 1 h in either water or
EDTA, mucilage was released from all genotypes and staining of
adherent mucilage was similar in width and intensity (Figures 7E
to 7L). In the same way as for the parent genotypes, the outer
cell wall did not fragment in the double mutant and remained
attached to the hilum (Figures 7H and 7L). By contrast, while
PME activities measured on dry seeds of the parental genotypes
exhibited similar increases in PME activity compared with the
wild type, activities in pmei6-1 sbt1.7-1 extracts were higher and
corresponded to the addition of the increases observed for the
parents (Figure 7M), indicating that they act on different PME
activities.
DISCUSSION
The Djarly Accession Is a Naturally Occurring Mutant
of a PMEI Expressed in Seed Coat Epidermal Cells
A screen exploiting variability between Arabidopsis accessions
was used to identify a new accession, Djarly, with altered seed
mucilage release (Figure 1B). The locus affecting mucilage release
in Djarly was identified through a map-based cloning approach as
At2g47670, predicted to encode a PMEI, named here as PMEI6.
Mutations identified in PMEI6 in the Djarly accession are likely to
modify protein structure such that interaction of PMEI6 with the
8 of 16
The Plant Cell
Table 2. GalA Distribution between Rhamnogalacturonan and HG in Sequentially Extracted Mucilage and Demucilaged Seeds from the Wild Type,
pmei6-1, and pmei6-2
Total GalA
SM
AM
DS
Total
HG GalA
SM
AM
DS
Total
RG I GalA
SM
AM
DS
Total
The Wild Type
mg$g21 seeds
pmei6-1
mg$g21 seeds
pmei6-2
mg$g21 seeds
11.29
6.17
36.03
53.49
(0.33)
(0.37)
(0.32)
(0.60)
7.54
7.87
37.99
53.4
(0.29)
(0.72)
(0.75)
(0.51)
5.46
7.85
39.09
52.376
(0.89)
(0.3)
(0.21)
(0.99)
0.633
0.213
27.48
28.32
(0)
(0)
(0.28)
(0.28)
0.078
0.017
28.77
28.87
(0)
(0)
(0.32)
(0.32)
0.053
0.022
29.02
29.10
(0)
(0)
(0.33)
(0.33)
10.66
5.96
8.56
25.18
(0.56)
(0.37)
(0.61)
(0.32)
7.46
7.85
9.22
24.54
(0.48)
(0.72)
(0.42)
(0.18)
5.41
7.83
10.07
23.28
(1.51)
(0.30)
(0.44)
(0.66)
Values for HG, GalA, soluble mucilage, and adherent mucilage are means of two independently extracted samples from two biological repeats (6SD).
Values for total GalA are from Table 1. Other values were derived by calculations based on measured values. AM, adherent mucilage; DS, demucilaged
seeds; SM, soluble mucilage. Details of sequential extraction procedure are given in Supplemental Figure 7 online.
active site of target PMEs would be affected, thereby leaving the
site free for pectin binding (see Supplemental Figure 1 online).
The role of PMEI6 in mucilage release was confirmed by the
observation of the same mucilage defect in three independent
allelic mutants and molecular complementation of pmei6-1 phenotypes with the PMEI6 CDS (Figures 1C to 1F; see Supplemental
Figure 2 online).
The expression of PMEI6 in seeds was analyzed by qRT-PCR,
in situ hybridization, and the expression of reporter genes from
the PMEI6 promoter. Expression of both transcripts and protein
for PMEI6 was specific to the epidermal cells of the seed coat
where mucilage polysaccharides accumulate (see Supplemental
Figures 4B to 4O online). Maximal expression was observed in
seeds with embryos at the linear cotyledon stage (8 to 10 DAP,
depending on culture conditions) (see Supplemental Figure 4
online). Therefore, the spatio-temporal expression patterns were
in agreement with PMEI6 participating in the release of mucilage
polysaccharides on imbibition.
had increased activity in pmei6 mutants and reduced activity in
PMEI6ox transformants, this confirms the expression of PMEI6
in epidermal cells of the seed coat and suggests that it is secreted to the apoplasm (Figure 4B).
PMEI6 Modulates the Degree of HG Methylesterification
by Regulating PME Activity
A comparison of PME activity in protein extracts from mature dry
seeds of a pmei6 mutant and transgenic plants expressing the
PMEI6 CDS under the control of the 35S promoter showed that
activity levels were modulated with respect to the presence or
absence of PMEI6 expression (Figure 4; see Supplemental Figure
2B online). Mucilage release was restored in pmei6 transformants,
confirming that the decreased activity observed in transformants
was due to increased inhibition of PME activity by PMEI6 expression.
In addition, PME activity was detected in protein extracts obtained
from imbibed seeds; these extracts correspond to proteins present in
the apoplasm of seed coat epidermal cells. As these extracts also
Figure 5. Matrix-Assisted Laser-Desorption Ionization Time-of-Flight
Mass Spectra (Positive Ion Mode) of Soluble Mucilage Digested with
Pectin Lyase.
Hydrolysates from wild-type (WT) (A) and pmei6-1 (B) soluble mucilage.
u, unsaturated; numbers in bold indicate degree of polymerization; exponent number indicates the degree of methylesterification.
[See online article for color version of this figure.]
PMEI6 Activity Promotes Mucilage Release
9 of 16
PMEI6 and SBT1.7 Regulate Different PME Targets
In the pmei6 mutant, mucilage release was not completely
blocked, as observed for luh/mum1, mum2, and mum4 mutants,
but delayed (Figure 2E; Western et al., 2001). When mucilage was
released, it was found that this delay was not due to reduced
mucilage expansion and only a minor decrease was observed in
total mucilage amount (Figure 2D, Table 1; see Supplemental
Figure 3B online). Strikingly, on imbibition, the outer cell wall of the
seed coat epidermal cells did not rupture into fragments as in the
wild type but was observed as a sheet attached to the hilum of
pmei6 seeds, as has been reported for sbt1.7 mutants imbibed in
EDTA (Figures 7F, 7G, 7J, and 7K; Rautengarten et al., 2008).
In addition, imbibition of sbt1.7 seeds for 1 h in water resulted in
a similar extrusion of mucilage from most seeds, as observed in
pmei6 (Figure 7G). SBT1.7 was implicated in the regulation of
pectin methylesterification, as mutants had higher seed PME activities and the overall degree of methylesterification was reduced
in soluble mucilage and seeds after EDTA extraction (Figure 7M;
Rautengarten et al., 2008). Although similar increases in PME activity were observed in pmei6 and sbt1.7 (Figure 7M; Rautengarten
et al., 2008), the pmei6-1 sbt1.7-1 mutant showed an additive
phenotype for PME activity in seed extracts, demonstrating that
PMEI6 and SBT1.7 inhibit the activity of different PMEs (Figure
7M). Nevertheless, the similarity of mutant phenotypes implies that
these PMEs act, at least in part, on the same targets.
PMEI6 Modulates HG Methylesterification in the Outer Cell
Wall of Seed Coat Epidermal Cells
Figure 6. Expression of PMEI6 and PME Activity in Mutant Seeds for the
Transcription Regulators GL2 and LUH/MUM1.
(A) Comparison of PMEI6 seed expression in the wild type (WT), gl2, and
luh/mum1. qRT-PCR analysis was used to determine the PMEI6 transcript levels in developing seeds at 10 DAP, corresponding to linear
cotyledon stage embryos. Steady state mRNA levels are represented as
a percentage of the constitutive EF1a4 gene (%EF). Error bars represent
SE values (n = 4). Mann-Whitney U test with the wild type, ***P < 0.1%.
(B) PME activities in total protein extracts from mature dry seeds of the
wild type, luh/mum1, and gl2. Activities were normalized to average wildtype activity (=1). Error bars represent SE values (n = 4). Mann-Whitney U
test with the wild type, ***P < 0.1%. Similar results were obtained in two
experiments with seeds from independent cultures.
In measurements of sugars in sequential extracts from mature
dry seeds, the amounts of GalA derived from HG were reduced
in pmei6 mucilage with a corresponding increase in GalA in HG
from demucilaged seeds (Tables 1 and 2). Furthermore, none of
the methylesterified HG oligomers detected in wild-type soluble
mucilage were present in pmei6-1 (Figure 5). A possible source
for the HG that is present in wild-type seed mucilage, but associated with pmei6 demucilaged seeds, is the sheet of outer
cell wall that remains attached to the hilum. In accordance, when
methylesterified HG in adherent mucilage was labeled with JIM5
and JIM7 antibodies, no immunofluorescence was observed in
the mucilage of the pmei6-1 mutant, although labeling of the cell
wall fragment attached to the hilum was observed (see Supplemental
Figure 2D online). By contrast, increased fluorescence was seen for
complemented mutants and PMEI6ox transgenic plants, compared
with the wild type, but fluorescence was not present in the region of adherent mucilage to the sides of the columella (Figure 3;
see Supplemental Figures 2E to 2H online). In certain complemented
mutants, outer cell wall rupture sometimes produced large cell wall
fragments, and generally no immunofluorescence was observed in
the mucilage beneath these fragments, while the cell wall fragments
themselves showed strong labeling (see Supplemental Figure 2H
online). This confirms that the majority of the methylesterified HG
labeled in mucilage is in fact derived from fragments of the outer
cell wall, either through direct labeling of fragments or labeling of
HG that has diffused from fragments. The increased labeling
observed in transgenic plants expressing the PMEI6 CDS (Figure
3; see Supplemental Figures 2E to 2H online) would therefore be
10 of 16
The Plant Cell
Figure 7. pmei6-1 and sbt1.7 Have Similar and Additive Phenotypes.
(A) to (L) Ruthenium red staining of mucilage released from the seed coats of the wild type (WT) ([A], [E], and [I]), pmei6-1 ([B], [F], and [J]), sbt1.7-1
([C], [G], and [K]), and pmei6-1 sbt1.7-1 ([D], [H], and [L]). Seeds were stained directly ([A]) to [D]) or after imbibition for 1 h in water ([E] to [H]) or EDTA
([I] to [L]). Bars = 200 µm.
(M) PME activities in total protein extracts from mature dry seeds of the wild type, pmei6-1, sbt1.7-1, and pmei6-1 sbt1.7-1. Activities were normalized
to average wild-type activity (=1). Mean values are indicated in bars. Error bars indicate SE (n = 4). Mann-Whitney U test with the wild type, ***P < 0.1%.
Similar results were obtained in three experiments with seeds from independent cultures.
due mainly to increased methylesterification of HG in the outer
cell wall.
Differences observed in the localization of JIM5 and JIM7 labeling indicate that HG observed in the outer region of the adherent
mucilage is more highly methylesterified than that in mucilage
closer to the seed (Figures 3A and 3B; Macquet et al., 2007a). The
increased number of negatively charged carboxyl groups on
partially methylesterified HG would allow interactions through
Ca2+ cross-links, and this could slow its diffusion compared with
highly methylesterified HG. Although most HG-derived GalA
would appear to be associated with cell wall fragments in the wild
type, a small amount was still detected in the mucilage of pmei6
(Table 2). This is in agreement with the detection of patches of HG
epitopes within mucilage polysaccharides accumulated in the
apoplast of developing seed sections from the wild type (Walker
et al., 2011).
RG I Partitioning into Adherent and Soluble Layers Is
Affected by HG Methylesterification
Sugar contents determined for water-soluble and adherent mucilage extracts revealed that the major component of seed mucilage,
RG I, had a modified distribution from soluble to adherent mucilage
in pmei6 mutants (Tables 1 and 2). As PMEI6 modulates PME
activity, this indicates that the DM of the small amount of HG domains present in mucilage plays a significant role in RG I solubility.
PMEI6 Activity Promotes Mucilage Release
11 of 16
Figure 8. Model Integrating PMEI6 Regulation and Action on PME Activity in Seed Coat Epidermal Cells.
The transcription regulators TTG1, EGL3/TT8, and MYB5/TT2 form a complex that regulates GL2 and TTG2. GL2 is required for MUM4 and PMEI6
expression. In addition, two independent pathways act via the regulators MYB61 and LUH/MUM1. The latter is required for the expression of MUM2,
BXL1, SBT1.7, and PMEI6. PME activity is negatively regulated by both PMEI6 and SBT1.7 through independent PMEs. MUM1 is also predicted to
modulate PMEs. Arrows with dotted lines indicate links identified in this study. Boxes denote enzymes and ovals transcription regulators.
The conformation of RG I must also be regulated by HG within
mucilage, as imbibition of wild-type seeds in Ca2+ chelators
causes increased adherent mucilage expansion compared with imbibition in water (Figures 7E and 7I; Willats et al., 2001; Rautengarten
et al., 2008; Arsovski et al., 2009b; Harpaz-Saad et al., 2011).
Modifications in mucilage partitioning between the two layers of
mucilage has previously been reported for mum2, cesa5, fei2,
and sos5, with more adherent mucilage in the former and less in
the latter three (Macquet et al., 2007b; Mendu et al., 2011;
Harpaz-Saad et al., 2011; Sullivan et al., 2011). A similar redistribution probably occurs in sbt1.7, as the amount of soluble
mucilage extracted by EDTA was 50% less than that of the wild
type, while mum5 had 50% more soluble mucilage (Macquet
et al., 2007a; Rautengarten et al., 2008). It is interesting to note
that the three mutants with increased proportions of adherent
mucilage are affected in proteins required for pectin modifications; MUM2 trims RG I galactan side chains, while PMEI6
and SBT1.7 modify HG methylesterification (Table 1; Dean et al.,
2007; Macquet et al., 2007b; Rautengarten et al., 2008). Nevertheless, defective arabinan side chain trimming in an Arabidopsis bxl1 mutant did not affect partitioning (Arsovski et al.,
2009b). This suggests that while the major factor determining
mucilage adherence to the seed is cellulose, the amount of
pectin associated with the cellulose network is determined by
relatively minor modifications of certain pectin properties.
Modulation of Mucilage Release by HG Methylesterification
The question remains as to how reduced methylesterification of
HG in mucilage and the outer cell wall delays mucilage release
and prevents cell wall fragmentation. In the wild type, cell wall
rupture occurs at the junction between the outer and radial cell
wall and leaves fragments attached to the top of columella. In
the mutant, although outer cell walls detach from radial cell
walls, they remain attached to adjoining outer cell walls and
detach from the top of columella, implying that links between the
former are reinforced and to the latter weakened. While thicker
radial cell walls have been observed in luh/mum1 and mum2,
defective mucilage release in these mutants has not been associated with this modification (Walker et al., 2011). Furthermore, defects in CESA2, CESA5, and CESA9 also affect radial
cell wall thickness, and mucilage release is not affected in the
corresponding mutants (Stork et al., 2010; Mendu et al., 2011).
In effect, a comparison of pmei6 seed coat epidermal cell morphology and development did not reveal any difference with the
wild type (see Supplemental Figure 5 online). Plants overexpressing
PMEI6 showed no obvious difference in seed coat epidermal cell
size compared with the wild type (Figure 3), in contrast with 35S:
PMEI5-overexpressing lines (Müller et al., 2013). This suggests
that the PMEs inhibited by PMEI5 intervene in different seed
coat epidermal cell processes to the PME partners of PMEI6.
Demethylesterified HG can form Ca2+ cross-links, which reinforce pectin structure and rigidify cell walls. The reduced
methylesterification observed in pmei6 would therefore most
likely reinforce links between adjoining outer cell walls. The outer
cell wall fragments connected to the top of the columella were
detached in cesa5, fei2, and sos5 mutants treated with Ca2+
chelators (Harpaz-Saad et al., 2011), indicating that fixation occurs through cellulose and Ca2+ cross-links. It is possible that
even if Ca2+ cross-links are more abundant in pmei6, detachment
12 of 16
The Plant Cell
of the outer cell walls from the top of the columella occurs because
increased resistance to expanding mucilage polysaccharides at the
intersection between outer and radial cell walls allows pressure
from the swelling mucilage to build up for longer at the junction
between the outer cell wall and columella. Mucilage expansion
could also be reduced by an increased number of Ca2+ crosslinks between HGs, and together with decreased amounts of soluble mucilage in mutant seeds (Table 1), this would lessen pressure
on cell walls. Therefore, it seems likely that modifications in both
mucilage and cell wall properties contribute to a delay in pmei6
mucilage release.
Transcriptional Regulation of HG Methylesterification in
Seed Coat Epidermal Cells
The transcription regulators GL2 and LUH/MUM1 appear to
regulate PMEI6 expression, as the transcript steady state levels
in gl2 and luh/mum1 were strongly reduced (Figure 6A; see
Supplemental Figure 6B online). Furthermore, PME activity was
also modified in seed protein extracts from both mutants (Figure
6B). A similar increase in activity was observed in gl2 and pmei6
extracts (Figure 4A; see Supplemental Figure 2B online), in accordance with PMEI6 expression being regulated by GL2 (Figure
8). LUH/MUM1 has been implicated in a regulatory pathway for
pectin modification, with MUM2, BXL1, and SBT1.7 as downstream targets and PMEI6 can now be integrated into this
pathway (Figure 8; Walker et al., 2011). Nevertheless, in the
pmei6-1 sbt1.7-1 mutant, PME activity levels were even higher
than in pmei6-1 or sbt1.7-1 alone, and the decreased activity
observed in luh/mum1 extracts is therefore unexpected. Moreover, increased labeling of methylesterified HG was observed in
both mucilage and outer cell walls of the luh/mum1 mutant,
which led Walker et al. (2011) to propose that LUH/MUM1 promotes PME expression during seed coat maturation. Therefore, it
is possible that LUH/MUM1 also regulates the expression of
genes coding the PME targets of PMEI6 and SBT1.7 and acts as
a coordinator of pectin maturation during seed coat epidermal cell
development. In effect, transcriptome analyses have found that
both PME and PMEI genes are high confidence targets of the
BRASSINAZOLE-RESISTANT1 transcription factor (Sun et al.,
2010), indicating that in other cell types they share common
regulators of expression. Alternatively, reduced PME activity could
occur in luh/mum1 by feedback regulation above a threshold DM
of HG. A feedback mechanism has recently been described that
mediates the level of HG methylesterification in the cell wall and
induces PME expression through the brassinosteroid signaling
pathway (Wolf et al., 2012).
The identification of PMEI6 as a PMEI that modulates mucilage release by regulating the DM of HG present in the outer cell
wall and mucilage of seed coat epidermal cells is an important
step toward understanding pectin function. In Arabidopsis,
PMEI-related proteins are encoded by a large multigene family
with 69 members, with only PMEI6 associated with a loss-offunction phenotype. Future studies will be required to identify
the PME protein target(s) of PMEI6 and SBT1.7; in silico data
and promoter:GUS constructs have shown that several of the 66
members of the PME gene family are expressed in seed tissues
and seven PMEs appear to be strongly expressed in the seed
coat (Louvet et al., 2006; Le et al., 2010). It will also be important
to determine the precise nature of their HG substrates and to
elucidate how PME activity is fine-tuned in seed coat epidermal
cells. Finally, it will be interesting to ascertain whether the modulation of mucilage release through pectin modifications conveys
a physiological advantage to Arabidopsis accessions.
METHODS
Plant Materials and Growth Conditions
The nine accessions corresponding to the Arabidopsis thaliana population
Djarly were collected from their natural habitat, and Dja-1 was used in
this study (http://www.inra.fr/internet/Produits/vast/collections.htm). The
pmei6-1 (SM_3.19557), pmei6-2 (GK-790B12), luh-3 (SALK_107245C),
gl2-6 (SM_3.16350), and sbt1.7-1 (GK_140B02) (Col-0 accession) mutants were obtained from the Nottingham Arabidopsis Stock Centre
(http://Arabidopsis.info) (Tissier et al., 1999; Alonso et al., 2003; Rosso
et al., 2003). The enhancer trap line ET6082 (Landsberg erecta accession)
pmei6-3 was obtained from the Martienssen lab at the Cold Spring Harbor
Laboratory (http://genetrap.cshl.edu). Homozygous lines were identified
by PCR using the primers indicated in Supplemental Table 2 online with
genomic DNA extracts; an amplicon was only obtained for the insertion
border and not with primers flanking the insertion.
Seed production was performed in either a growth chamber (photoperiod of 16 h light at 21°C, 8 h dark at 18°C, 65% relative humidity, and
170 µmol m-2s21) or a glasshouse (18 to 28°C) with a minimum photoperiod of 13 h assured by supplementary lighting. Plants were grown in
compost (Tref substrates) and watered with Plan-Prod nutritive solution
(Fertil). Three successive backcrosses to wild-type Col-0 were performed
on the mutant pmei6-1. All phenotypic analyses were performed using
these backcrossed seeds. Reciprocal crosses were performed between
homozygous pmei6-1 and pmei6-2, pmei6-3, and Djarly. As the seed coat
is a maternal tissue, mucilage phenotypes of F1 and F2 seed coats were
determined on F2 and F3 seed, respectively. In all comparative analyses,
seeds used were from mutant and wild-type plants that had been simultaneously cultivated and harvested. Developing seeds and silique
envelopes were obtained by tagging flowers on the day of pollination and
dissecting siliques at the specified number of days afterwards. For inclusions, whole siliques were harvested.
Expression Analysis
Total RNA was extracted from developing seeds using the RNeasy plant
mini kit (Qiagen) and used as a template (1 µg) for first-strand cDNA
synthesis with an oligo(dT) (22-mer) primer and RevertAid H Minus RT
(Fermentas) according to the manufacturer’s instructions. For amplification of PCR products from single-stranded cDNA from the wild type and
pmei6-1 (Figures 1G and 1H), the following exon primers were used:
PMEI6 CDS, forward primer 59-GTCCCAATTTCTTAAAAAGTTTGGATTCCTCCAATAAAGAACATGACTTC-39 and reverse primer 59-CATAGCGTAGATTACAAGCCATCAAAAGCAAGCTTCTTGAGGAGAGCCAG-39;
PMEI6 59, reverse primer 59-GCAAGAGCCTCAGCACCCGT-39; PMEI6 39,
forward primer 59-GAGTAGAACACGGACGGTCA-39; EF1a4 primers were
as described by North et al. (2007). Quantitative real-time PCR reactions
were performed as previously described using primers that had been tested
for their efficiency rates and sensitivity on dilution series of cDNAs (Plessis
et al., 2011). PMEI6 and seed reference gene At4g12590 (Dekkers et al.,
2012) specific primers were as follows: PMEI6, forward primer, 59-GGCAGATAAGCGATCTCGCCAC-39; PMEI6, reverse primer 59-AGCCAGAGCATTGCTGCATAGTC-39; At4g12590, forward primer 59-TGGCATTGACTTGAGCACTGTCG-39; and At4g12590, reverse primer 59-TCGAGGTAGTGCCCATTCGTGCT-39. EF1a4 primers were as described by Plessis et al. (2011).
PMEI6 Activity Promotes Mucilage Release
In situ hybridization was performed on paraffin-embedded developing
seed sections as described by Sullivan et al. (2011). PMEI6-specific
probes were PCR amplified from single-stranded cDNA reverse transcribed from wild-type RNA extracted from developing siliques using
forward primer 59-CGTCCCAATTTCTTAAAAGTTTG-39 and reverse primer
59-GTAATACGACTCACTATAGCCCACTTGCTGGTGTTGTT-39 for antisense probe and forward primer 59-GTAATACGACTCACTATACGTCCAATTTCTTAAAAGTTTG-39 and reverse primer 59-GCCCACTTGCTGGTGTTGTT-39 for sense probe.
Cloning and Plant Transformation
DNA fragments containing 2 kb of promoter region and the PMEI6 CDS or
the PMEI6 CDS alone were amplified from wild-type Col-0 genomic DNA
by PCR using the following primers containing attB1 and attB2 recombination sequences: pPMEI6:PMEI6, forward primer 59-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCTTCCCACGCCATGCGACGAC-39;
and pPMEI6:PMEI6, reverse primer 59-GGGGACCACTTTGTACAAGAAAGCTGGGTCCAAGCCATCAAAAGCAAGCT-39; PMEI6 CDS, forward
primer 59-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGACTTCGTCTTCTTCTTCTCC-39; and PMEI6 CDS, reverse primer 59-GGGGACCACTTTGTACAAGAAAGCTGGGTTACAAGCCATCAAAAGCAAGCT-39.
(Note: nucleotides in bold type were added so that PMEI6 codons would
be in phase with GFP codons; underlined nucleotides correspond to the
start codon.) The resulting PCR products were then recombined into the
pDONR207 vector with BP clonase (Invitrogen) according to manufacturer’s instructions and transformed into Escherichia coli strain DH10B.
The PMEI6 gene fragments were sequenced to confirm that no PCRinduced errors were present and then recombined into the binary vector
pGWB2 or pGWB4, which allow the expression of the PMEI6 CDS under the
control of the cauliflower mosaic virus 35S promoter, or the production of
a PMEI6:GFP translational fusion, respectively (Nakagawa et al., 2007). The
resulting binary vectors were transformed into Agrobacterium tumefaciens
C58C1pMP90 by electroporation prior to stable transformation of wild-type
or pmei6-1 plants using the floral dip method of Clough and Bent (1998).
Cytochemical Staining and Immunolabeling Procedures
Mucilage released from mature dry seeds was either stained directly with
500 µg mL21 ruthenium red or after imbibition in water or 50 mM EDTA, pH
8.0, for 1 h. After EDTA treatment, seeds were rinsed with water prior to
staining. Ruthenium red staining was observed with a light microscope
(Axioplan 2; Zeiss). Developing seed sections were obtained from staged
siliques that had been paraffin embedded and stained with toluidine blue
O as described previously (Macquet et al., 2007b; Sullivan et al., 2011).
Immunolabeling was performed with two primary monoclonal antibodies, JIM5, which binds low-ester HG, and JIM7, which binds highester HG (Knox et al., 1990; Plant Probes), as previously described with
double labeling performed with Calcofluor (Macquet et al., 2007a; Sullivan
et al., 2011). Observation with either a Leica TCS-SP2-AOBS spectral
confocal laser scanning microscope (Leica Microsystems) or a Zeiss
LSM710 confocal microscope using a 405-nm diode laser line to excite
Calcofluor or a 488-nm argon laser line to excite Alexa Fluor 488. Fluorescence emission was detected between 412 and 490 nm or 504 and 579
nm for Calcofluor and Alexa Fluor 488, respectively. For comparison of
signal intensity within a given experiment, laser gain values were fixed.
Scanning Electron Microscopy and Reporter Gene analyses
Dry seeds were mounted onto a Peltier cooling stage with adhesive discs
(Deben) and observed with a SH-1500 tabletop scanning electron microscope (Hirox). Histochemical detection of GUS activity was performed
on developing seeds staged as described above using 0.5 or 1 mM potassium
13 of 16
ferricyanide/potassium ferrocyanide (Jefferson et al., 1987). Seeds were
then stained using a modified pseudo-Schiff propidium iodide protocol and
observed as previously described (Truernit et al., 2008). Developing seeds
were dissected from siliques of transgenic plants containing the PMEI6
promoter:PMEI6:GFP construct and stained with 1 mg mL21 propidium
iodide for 10 min, before rinsing twice with PBS. GFP and propidium iodide
fluorescence were observed as described previously (Sullivan et al., 2011).
DNA Sequencing and Genetic Analyses
Genomic DNA was extracted from flower buds of Col-0 and Djarly accessions as described by Doyle and Doyle (1990). PMEI6 DNA fragments
were then amplified by PCR, gel purified using the Wizard SV gel and PCR
cleanup system according to the manufacturer’s instructions (Promega),
and sequenced (GenoScreen). For mapping, crosses were performed
between Col-0 and Djarly accessions, and DNA was extracted from flower
buds of F2 progeny in a 96-tube format as described by Simon et al.
(2008). An approximate genome position was determined based on recombination percentages for 90 F2 progeny genotyped with simple sequence length polymorphism markers. The mapping interval was then
reduced using recombinants identified among an additional 6344 F2
progeny. Mucilage release phenotypes for F2 seed coats were determined
by ruthenium red staining of F3 seed lots.
Prediction of PMEI6 Protein Structure
Structural predictions for PMEI6 were obtained with the ESyPred3D
program (Lambert et al., 2002) using the homology modeling approach
and the kiwi PMEI as template (Di Matteo et al., 2005; Protein Data Bank ID
code 1XG2). Protein Data Bank files were analyzed with Rasmol 2.7.5.2
software (http://rasmol.org/).
Determination of PME Activity
Total protein extracts were obtained by grinding 200 mg of dry seeds in
400 mL of extraction buffer (1 M NaCl, 12.5 mM citric acid, and 50 mM
Na2HPO4, pH 6.5). The resulting homogenate was shaken for 1 h at 4°C
and then centrifuged at 20,000g for 15 min, and the supernatant was
retained. Protein concentrations were determined according to Bradford
(1976), and equal quantities of protein (>8 µg) in the same volume (20 µL)
were loaded into 6-mm-diameter wells in gels prepared with 0.1% (w/v) of
$85% esterified citrus fruit pectin (Sigma-Aldrich), 1% (w/v) agarose, 12.5
mM citric acid, and 50 mM Na2HPO4, pH 6.5. After incubation overnight at
28°C, plates were stained with 500 µg mL21 ruthenium red for 45 min and
destained with water for >3 h. Measurements of stained areas were performed with ImageJ 1.34S software (Freeware, National Institute of Health).
Extraction and Analysis of Monosaccharides from Mucilage and
Demucilaged Seeds
For determination of monosaccharide contents from soluble and adherent
mucilage and demucilaged seeds, a sequential extraction procedure was
used (see Supplemental Figure 7 online). Soluble and adherent mucilage
extracts were obtained as described previously (Sullivan et al., 2011).
Demucilaged seeds were ground and then washed with 70% ethanol (10
min at 20°C), hexane (1 h at 40°C), and acetone (20 min 20°C) and dried
prior to analyses. Material lost during this delipidation process was determined and taken into account in calculations to obtain values for whole
seeds as indicated in Tables 1 and 2. Rates of mucilage release were
determined using 20 mg of dry seeds that were imbibed by mixing in 1 mL
of distilled water for the indicated times (1, 2, 4, 6, 16, and 24 h) at 20°C.
After imbibition, seeds were centrifuged (3000g, 5 min) and supernatants
removed, filtered, and stabilized by treating for 5 min at 100°C. Extracts
were conserved at 4°C prior to acid and neutral sugar analyses.
14 of 16
The Plant Cell
For the recovery of HG domains, soluble and adherent mucilage
fractions were obtained as above from 2 g of seeds. Soluble mucilage was
then subjected to digestion with 1 nkat of rhamnogalacturonan hydrolase
(Swiss-Prot Q00018) provided by Novozymes after adjusting with 100 mM
sodium acetate, pH 4.5, to a final volume of 50 mM; samples were incubated for 16 h at 40°C. Enzyme hydrolysates were concentrated, dialyzed to remove all traces of salts, and then dried. Samples were
suspended in 2 mL of water and then HG domains precipitated by adding
an equal volume of ethanol and incubating for 1 h at 4°C. Precipitated HG
was recovered after centrifugation (3000g, 15 min), and pellets were
rinsed twice with 3 mL of 48% (v/v) ethanol for 30 min at 4°C. To remove all
traces of ethanol, the resulting pellet, corresponding to HG, was resuspended twice in 1 mL of water and dried.
Uronic acid (as GalA) was determined by the automated m-hydroxybiphenyl
and orcinol methods, respectively (Thibault, 1979). Individual neutral
sugars were analyzed as their alditol acetate derivatives (Blakeney et al.,
1983) by gas–liquid chromatography after hydrolysis with 2 M trifluoroacetic acid at 121°C for 2.5 h for soluble fractions and prehydrolysis (26 N
H2SO4, 1 h, 25°C) followed by hydrolysis (2 N H2SO4, 2 h, 100°C) for
insoluble fractions (demucilaged seeds).
The degree of methylesterification of mucilage and demucilaged seeds
was measured from methanol released by alkaline deesterification of
extracts with 0.2 M NaOH for 1 h at 4°C. After neutralization of extracts
with 0.2 M HCl, released methanol was measured as described previously
(Ralet et al., 2012).
MS Analysis
Soluble mucilage was obtained from 100 mg of seeds by imbibition in 1
mL of 50 mM Na-acetate, pH 5.0, and shaking for 3 h at 20°C. Supernatants with soluble mucilage were recovered by centrifugation (3000g, 5
min) and then filtered and incubated with pectin lyase for 16 h (1 nkat). For
MS, all chemical reagents used were HPLC grade. N-N-dimethylaniline/
2,5-dihydroxybenzoic acid matrix solution was prepared by dissolving
100 mg of 2,5-dihydroxybenzoic acid (Sigma-Aldrich) in 1 mL of water/
acetonitrile/N,N-dimethylaniline (Fisher Scientific) (49:49:2). One microliter of sample was directly mixed with 1 mL of matrix solution on a polished steel target plate. Acquisition was performed on an Autoflex III
matrix-assisted laser-desorption ionization tandem time of flight mass
spectrometer (Bruker Daltonics) equipped with a Smartbeam laser (355 nm)
in positive ion mode with a reflector. Laser power was adapted for each
sample. Mass spectra were automatically processed by FlexAnalysis
software (Bruker Daltonics).
Supplemental Figure 5. Differentiation of Seed Coat Epidermal Cells
in the pmei6-1 Mutant.
Supplemental Figure 6. Expression of PMEI6 in Overexpressor Lines
or GL2 and LUH/MUM1 Mutant Seeds.
Supplemental Figure 7. Schematic Representation of Extraction
Procedure Used for Analyses Presented in Tables 1 and 2.
Supplemental Table 1. Genes in the Chromosome 2 Mapping Interval
for Which Homozygous T-DNA Insertion Mutants Were Obtained and
Examined for Mucilage Release.
Supplemental Table 2. Sequences of Gene-Specific Primers used for
Genotyping of Homozygous pmei6 Mutants.
ACKNOWLEDGMENTS
This work was supported by the Agence Nationale de la Recherche
program (Grant ANR-08-BLAN-0061) that included a doctoral fellowship
to S.S.-A. The Leica TCS-SP2-AOBS and Zeiss 710 confocal microscopes in the Institut Jean-Pierre Bourgin Plateforme de Cytologie et
Imagerie Végétale were cofinanced by grants from Institut National de la
Recherche Agronomique and Ile-de-France region or the Conseil Général
des Yvelines, respectively. We thank Marie-Jeanne Crépeau and Tracy
Plainchamp for technical aid and Marc Galland for providing At4g12590
primers. We also thank Bertrand Dubreucq, Olivier Leprince, Gregory
Mouille, Jérôme Pelloux, and Sebastian Wolf for stimulating scientific
exchanges and technical advice and Michaël Anjuère, Fabrice Petitpas,
Hervé Ferry, and Christine Sallé for assistance with plant culture.
AUTHOR CONTRIBUTIONS
A.M.-P., H.M.N., S.S.-A., and M.-C.R. conceived the research. S.S.-A.
performed experiments. M.-C.R., D.R., and S.S.-A. performed biochemical measurements. L.B., H.M.N., and S.S.-A. performed genetic analyses. A.B., H.M.N., and S.S.-A. performed cytological analyses and in situ
hybridization. H.M.N., S.S.-A., and M.-C.R. analyzed data. H.M.N. and
S.S.-A. performed activity assays and wrote the article.
Received October 25, 2012; revised December 20, 2012; accepted
January 3, 2013; published January 29, 2013.
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome
Initiative or GenBank/EMBL databases under the following accession
numbers: EF1a4, At5g60390; GL2, At1g79840; LUH/MUM1, At2g32700;
PMEI6, At2g47670; SBT1.7, At5g67360; and seed reference gene,
At4g12590.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Predicted Three-Dimensional Structure for
PMEI6 Wild-Type and Mutant Proteins.
Supplemental Figure 2. Complementation of pmei6-1 Mucilage
Phenotypes by Transformation with a 35S Promoter:PMEI6 Construct.
Supplemental Figure 3. Ruthenium Red Staining of Adherent Mucilage Released from Seeds Imbibed in a Cation Chelator.
Supplemental Figure 4. Expression of PMEI6 in Developing Seeds.
REFERENCES
Alonso, J.M., et al. (2003). Genome-wide insertional mutagenesis of
Arabidopsis thaliana. Science 301: 653–657.
Arsovski, A.A., Popma, T.M., Haughn, G.W., Carpita, N.C., McCann, M.C.,
and Western, T.L. (2009b). AtBXL1 encodes a bifunctional b-D-xylosidase/a-L-arabinofuranosidase required for pectic arabinan modification in
Arabidopsis mucilage secretory cells. Plant Physiol. 150: 1219–1234.
Arsovski, A.A., Villota, M.M., Rowland, O., Subramaniam, R., and
Western, T.L. (2009a). MUM ENHANCERS are important for seed
coat mucilage production and mucilage secretory cell differentiation
in Arabidopsis thaliana. J. Exp. Bot. 60: 2601–2612.
Blakeney, A.B., Harris, P.J., Henry, R.J., and Stone, B.A. (1983). A
simple and rapid preparation of alditol acetates for monosaccharide
analysis. Carbohydr. Res. 113: 291–299.
Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of
protein-dye binding. Anal. Biochem. 72: 248–254.
PMEI6 Activity Promotes Mucilage Release
Bui, M., Lim, N., Sijacic, P., and Liu, Z. (2011). LEUNIG_HOMOLOG
and LEUNIG regulate seed mucilage extrusion in Arabidopsis. J.
Integr. Plant Biol. 53: 399–408.
Caffall, K.H., Pattathil, S., Phillips, S.E., Hahn, M.G., and Mohnen,
D. (2009). Arabidopsis thaliana T-DNA mutants implicate GAUT
genes in the biosynthesis of pectin and xylan in cell walls and seed
testa. Mol. Plant 2: 1000–1014.
Camardella, L., Carratore, V., Ciardiello, M.A., Servillo, L.,
Balestrieri, C., and Giovane, A. (2000). Kiwi protein inhibitor of
pectin methylesterase amino-acid sequence and structural importance of two disulfide bridges. Eur. J. Biochem. 267: 4561–4565.
Clough, S.J., and Bent, A.F. (1998). Floral dip: A simplified method for
Agrobacterium-mediated transformation of Arabidopsis thaliana.
Plant J. 16: 735–743.
D’Avino, R., Camardella, L., Christensen, T.M., Giovane, A., and
Servillo, L. (2003). Tomato pectin methylesterase: Modeling, fluorescence, and inhibitor interaction studies-comparison with the
bacterial (Erwinia chrysanthemi) enzyme. Proteins 53: 830–839.
Dean, G.H., Zheng, H., Tewari, J., Huang, J., Young, D.S., Hwang, Y.T.,
Western, T.L., Carpita, N.C., McCann, M.C., Mansfield, S.D., and
Haughn, G.W. (2007). The Arabidopsis MUM2 gene encodes a bgalactosidase required for the production of seed coat mucilage with
correct hydration properties. Plant Cell 19: 4007–4021.
Dekkers, B.J.W., Willems, L., Bassel, G.W., van Bolderen-Veldkamp,
R.P., Ligterink, W., Hilhorst, H.W.M., and Bentsink, L. (2012). Identification of reference genes for RT-qPCR expression analysis in Arabidopsis and tomato seeds. Plant Cell Physiol. 53: 28–37.
Di Matteo, A., Giovane, A., Raiola, A., Camardella, L., Bonivento,
D., De Lorenzo, G., Cervone, F., Bellincampi, D., and Tsernoglou, D.
(2005). Structural basis for the interaction between pectin
methylesterase and a specific inhibitor protein. Plant Cell 17:
849–858.
Doyle, J.J., and Doyle, J.L. (1990). Isolation of plant DNA from fresh
tissues. Focus 12: 13–15.
Engelbrecht, M., and García-Fayos, P. (2012). Mucilage secretion by
seeds doubles the chance to escape removal by ants. Plant Ecol.
213: 1167–1175.
García-Fayos, P., Bochet, E., and Cerdà, A. (2010). Seed removal
susceptibility through soil erosion shapes vegetation composition.
Plant Soil 334: 289–297.
Giovane, A., Servillo, L., Balestrieri, C., Raiola, A., D’Avino, R.,
Tamburrini, M., Ciardiello, M.A., and Camardella, L. (2004). Pectin
methylesterase inhibitor. Biochim. Biophys. Acta 1696: 245–252.
Goto, N. (1985). A mucilage polysaccharide secreted from testa of
Arabidopsis thaliana. Arabidopsis Inf. Serv. 22: 143–145.
Harpaz-Saad, S., McFarlane, H.E., Xu, S., Divi, U.K., Forward, B.,
Western, T.L., and Kieber, J.J. (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.
Hothorn, M., Wolf, S., Aloy, P., Greiner, S., and Scheffzek, K.
(2004). Structural insights into the target specificity of plant invertase and pectin methylesterase inhibitory proteins. Plant Cell 16:
3437–3447.
Huang, J., DeBowles, D., Esfandiari, E., Dean, G., Carpita, N.C.,
and Haughn, G.W. (2011). The Arabidopsis transcription factor
LUH/MUM1 is required for extrusion of seed coat mucilage. Plant
Physiol. 156: 491–502.
Jefferson, R.A., Kavanagh, T.A., and Bevan, M.W. (1987). GUS fusions: Beta-glucuronidase as a sensitive and versatile gene fusion
marker in higher plants. EMBO J. 6: 3901–3907.
Kong, Y., Zhou, G., Yin, Y., Xu, Y., Pattathil, S., and Hahn, M.G.
(2011). Molecular analysis of a family of Arabidopsis genes related
to galacturonosyltransferases. Plant Physiol. 155: 1791–1805.
15 of 16
Knox, J.P., Linstead, P.J., King, J., Cooper, C., and Roberts, K.
(1990). Pectin esterification is spatially regulated both within cell
walls and between developing tissues or root apices. Planta 181:
512–521.
Lambert, C., Léonard, N., De Bolle, X., and Depiereux, E. (2002).
ESyPred3D: Prediction of proteins 3D structures. Bioinformatics 18:
1250–1256.
Le, B.H., et al. (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.
Lionetti, V., Raiola, A., Camardella, L., Giovane, A., Obel, N., Pauly, M.,
Favaron, F., Cervone, F., and Bellincampi, D. (2007). Overexpression
of pectin methylesterase inhibitors in Arabidopsis restricts fungal infection by Botrytis cinerea. Plant Physiol. 143: 1871–1880.
Louvet, R., Cavel, E., Gutierrez, L., Guénin, S., Roger, D., Gillet, F.,
Guerineau, F., and Pelloux, J. (2006). Comprehensive expression
profiling of the pectin methylesterase gene family during silique
development in Arabidopsis thaliana. Planta 224: 782–791.
Macquet, A., Ralet, M.-C., Kronenberger, J., Marion-Poll, A., and
North, H.M. (2007a). In situ, chemical and macromolecular study of
the composition of Arabidopsis thaliana seed coat mucilage. Plant
Cell Physiol. 48: 984–999.
Macquet, A., Ralet, M.-C., Loudet, O., Kronenberger, J., Mouille,
G., Marion-Poll, A., and North, H.M. (2007b). A naturally occurring
mutation in an Arabidopsis accession affects a b-D-galactosidase
that increases the hydrophilic potential of rhamnogalacturonan I in
seed mucilage. Plant Cell 19: 3990–4006.
Mendu, V., Griffiths, J.S., Persson, S., Stork, J., Downie, A.B.,
Voiniciuc, C., Haughn, G.W., and DeBolt, S. (2011). Subfunctionalization of cellulose synthases in seed coat epidermal cells
mediates secondary radial wall synthesis and mucilage attachment.
Plant Physiol. 157: 441–453.
Müller, K., Levesque-Tremblay, G., Bartels, S., Weitbrecht, K.,
Wormit, A., Usadel, B., Haughn, G., and Kermode, A.R. (2013).
Demethylesterification of cell wall pectins in Arabidopsis thaliana
plays a role in seed germination. Plant Physiol. 161: 305–316.
Nakagawa, T., Kurose, T., Hino, T., Tanaka, K., Kawamukai, M.,
Niwa, Y., Toyooka, K., Matsuoka, K., Jinbo, T., and Kimura, T.
(2007). Development of series of gateway binary vectors, pGWBs,
for realizing efficient construction of fusion genes for plant transformation. J. Biosci. Bioeng. 104: 34–41.
North, H.M., De Almeida, A., Boutin, J.-P., Frey, A., To, A., Botran,
L., Sotta, B., and Marion-Poll, A. (2007). The Arabidopsis ABAdeficient mutant aba4 demonstrates that the major route for stressinduced ABA accumulation is via neoxanthin isomers. Plant J. 50:
810–824.
Oka, T., Nemoto, T., and Jigami, Y. (2007). Functional analysis of
Arabidopsis thaliana RHM2/MUM4, a multidomain protein involved
in UDP-D-glucose to UDP-L-rhamnose conversion. J. Biol. Chem.
282: 5389–5403.
Peaucelle, A., Louvet, R., Johansen, J.N., Höfte, H., Laufs, P.,
Pelloux, J., and Mouille, G. (2008). Arabidopsis phyllotaxis is
controlled by the methyl-esterification status of cell-wall pectins.
Curr. Biol. 18: 1943–1948.
Pelletier, S., et al. (2010). A role for pectin de-methylesterification in
a developmentally regulated growth acceleration in dark-grown
Arabidopsis hypocotyls. New Phytol. 188: 726–739.
Penfield, S., Meissner, R.C., Shoue, D.A., Carpita, N.C., and Bevan,
M.W. (2001). MYB61 is required for mucilage deposition and extrusion in the Arabidopsis seed coat. Plant Cell 13: 2777–2791.
Plessis, A., et al. (2011). New ABA-hypersensitive Arabidopsis mutants are affected in loci mediating responses to water deficit and
Dickeya dadantii infection. PLoS ONE 6: e20243.
16 of 16
The Plant Cell
Raiola, A., Camardella, L., Giovane, A., Mattei, B., De Lorenzo, G.,
Cervone, F., and Bellincampi, D. (2004). Two Arabidopsis thaliana
genes encode functional pectin methylesterase inhibitors. FEBS
Lett. 557: 199–203.
Ralet, M.-C., Tranquet, O., Poulain, D., Moïse, A., and Guillon, F.
(2010). Monoclonal antibodies to rhamnogalacturonan I backbone.
Planta 231: 1373–1383.
Ralet, M.-C., Williams, M.A.K., Tanhatan-Nasseri, A., Ropartz, D.,
Quéméner, B., and Bonnin, E. (2012). Innovative enzymatic approach to resolve homogalacturonans based on their methylesterification pattern. Biomacromolecules 13: 1615–1624.
Rautengarten, C., Usadel, B., Neumetzler, L., Hartmann, J., Büssis, D.,
and Altmann, T. (2008). A subtilisin-like serine protease essential for
mucilage release from Arabidopsis seed coats. Plant J. 54: 466–480.
Rosso, M.G., Li, Y., Strizhov, N., Reiss, B., Dekker, K., and
Weisshaar, B. (2003). An Arabidopsis thaliana T-DNA mutagenized
population (GABI-Kat) for flanking sequence tag-based reverse
genetics. Plant Mol. Biol. 53: 247–259.
Schmid, M., Davison, T.S., Henz, S.R., Pape, U.J., Demar, M.,
Vingron, M., Schölkopf, B., Weigel, D., and Lohmann, J.U. (2005).
A gene expression map of Arabidopsis thaliana development. Nat.
Genet. 37: 501–506.
Simon, M., Loudet, O., Durand, S., Bérard, A., Brunel, D., Sennesal,
F.-X., Durand-Tardif, M., Pelletier, G., and Camilleri, C. (2008).
Quantitative trait loci mapping in five new large recombinant inbred line
populations of Arabidopsis thaliana genotyped with consensus singlenucleotide polymorphism markers. Genetics 178: 2253–2264.
Stork, J., Harris, D., Griffiths, J., Williams, B., Beisson, F., LiBeisson, Y., Mendu, V., Haughn, G., and Debolt, S. (2010). CELLULOSE SYNTHASE9 serves a nonredundant role in secondary cell
wall synthesis in Arabidopsis epidermal testa cells. Plant Physiol.
153: 580–589.
Sullivan, S., Ralet, M.-C., Berger, A., Diatloff, E., Bischoff, V., Gonneau,
M., Marion-Poll, A., and North, H.M. (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.
Sun, Y., et al. (2010). Integration of brassinosteroid signal transduction with the transcription network for plant growth regulation in
Arabidopsis. Dev. Cell 19: 765–777.
Sundaresan, V., Springer, P., Volpe, T., Haward, S., Jones, J.D.G.,
Dean, C., Ma, H., and Martienssen, R. (1995). Patterns of gene
action in plant development revealed by enhancer trap and gene
trap transposable elements. Genes Dev. 9: 1797–1810.
Thibault, J.-F. (1979). Automatisation du dosage des substances
pectiques par la méthode au métahydroxydiphényle. Lebensml.
-Wiss. Technol. 12: 247–251.
Tissier, A.F., Marillonnet, S., Klimyuk, V., Patel, K., Torres, M.A.,
Murphy, G., and Jones, J.D.G. (1999). Multiple independent defective suppressor-mutator transposon insertions in Arabidopsis: A
tool for functional genomics. Plant Cell 11: 1841–1852.
Truernit, E., Bauby, H., Dubreucq, B., Grandjean, O., Runions, J.,
Barthélémy, J., and Palauqui, J.-C. (2008). High-resolution wholemount imaging of three-dimensional tissue organization and gene
expression enables the study of Phloem development and structure
in Arabidopsis. Plant Cell 20: 1494–1503.
Usadel, B., Kuschinsky, A.M., Rosso, M.G., Eckermann, N., and
Pauly, M. (2004). RHM2 is involved in mucilage pectin synthesis
and is required for the development of the seed coat in Arabidopsis.
Plant Physiol. 134: 286–295.
Walker, M., Tehseen, M., Doblin, M.S., Pettolino, F.A., Wilson,
S.M., Bacic, A., and Golz, J.F. (2011). The transcriptional regulator
LEUNIG_HOMOLOG regulates mucilage release from the Arabidopsis testa. Plant Physiol. 156: 46–60.
Willats, W.G.T., McCartney, L., and Knox, J.P. (2001). In-situ analysis of pectic polysaccharides in seed mucilage and at the root
surface of Arabidopsis thaliana. Planta 213: 37–44.
Western, T.L. (2012). The sticky tale of seed coat mucilages: Production, genetics, and role in seed germination and dispersal. Seed
Sci. Res. 22: 1–25.
Western, T.L., Burn, J., Tan, W.L., Skinner, D.J., Martin-McCaffrey,
L., Moffatt, B.A., and Haughn, G.W. (2001). Isolation and characterization of mutants defective in seed coat mucilage secretory cell
development in Arabidopsis. Plant Physiol. 127: 998–1011.
Western, T.L., Skinner, D.J., and Haughn, G.W. (2000). Differentiation of mucilage secretory cells of the Arabidopsis seed coat. Plant
Physiol. 122: 345–356.
Western, T.L., Young, D.S., Dean, G.H., Tan, W.L., Samuels, A.L.,
and Haughn, G.W. (2004). MUCILAGE-MODIFIED4 encodes a putative pectin biosynthetic enzyme developmentally regulated by
APETALA2, TRANSPARENT TESTA GLABRA1, and GLABRA2 in
the Arabidopsis seed coat. Plant Physiol. 134: 296–306.
Wolf, S., Grsic-Rausch, S., Rausch, T., and Greiner, S. (2003).
Identification of pollen-expressed pectin methylesterase inhibitors
in Arabidopsis. FEBS Lett. 555: 551–555.
Wolf, S., Mouille, G., and Pelloux, J. (2009). Homogalacturonan
methyl-esterification and plant development. Mol. Plant 2: 851–860.
Wolf, S., Mravec, J., Greiner, S., Mouille, G., and Höfte, H. (2012).
Plant cell wall homeostasis is mediated by brassinosteroid feedback signaling. Curr. Biol. 22: 1732–1737.
Yang, X., Baskin, C.C., Baskin, J.M., Liu, G., and Huang, Z. (2012a).
Seed mucilage improves seedling emergence of a sand desert
shrub. PLoS ONE 7: e34597.
Yang, X., Baskin, C.C., Baskin, J.M., Zhang, W., and Huang, Z.
(2012b). Degradation of seed mucilage by soil microflora promotes
early seedling growth of a desert sand dune plant. Plant Cell Environ. 35: 872–883.
Yang, X., Zhang, W., Dong, M., Boubriak, I., and Huang, Z. (2011).
The achene mucilage hydrated in desert dew assists seed cells in
maintaining DNA integrity: adaptive strategy of desert plant Artemisia sphaerocephala. PLoS ONE 6: e24346.
Young, J.A., and Evans, R.A. (1973). Mucilaginous seed coats. Weed
Sci. 21: 52–54.
Young, R.E., McFarlane, H.E., Hahn, M.G., Western, T.L., Haughn,
G.W., and Samuels, A.L. (2008). Analysis of the Golgi apparatus in
Arabidopsis seed coat cells during polarized secretion of pectin-rich
mucilage. Plant Cell 20: 1623–1638.
PECTIN METHYLESTERASE INHIBITOR6 Promotes Arabidopsis Mucilage Release by
Limiting Methylesterification of Homogalacturonan in Seed Coat Epidermal Cells
Susana Saez-Aguayo, Marie-Christine Ralet, Adeline Berger, Lucy Botran, David Ropartz, Annie
Marion-Poll and Helen M. North
Plant Cell; originally published online January 29, 2013;
DOI 10.1105/tpc.112.106575
This information is current as of June 17, 2017
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