Mannans and endo-β-mannanases (MAN) in Brachypodium distachyon: expression profiling and possible role of the BdMAN genes during coleorhiza-limited seed germination
Journal of Experimental Botany
Mannans and endo-β-mannanases (MAN) in Brachypodium distachyon: expression profiling and possible role of the BdMAN genes during coleorhiza-limited seed germination
Virginia González-Calle 0
Cristina Barrero-Sicilia 0
Pilar Carbonero 0
Raquel Iglesias-Fernández 0
0 Centro de Biotecnología y Genómica de Plantas (UPM-INIA), and ETSI Agrónomos, Campus de Montegancedo, Universidad Politécnica de Madrid, Pozuelo de Alarcón , 28223-Madrid , Spain
Immunolocalization of mannans in the seeds of Brachypodium distachyon reveals the presence of these polysaccharides in the root embryo and in the coleorhiza in the early stages of germination (12 h), decreasing thereafter to the point of being hardly detected at 27 h. Concurrently, the activity of endo-β-mannanases (MANs; EC 22.214.171.124) that catalyse the hydrolysis of β-1,4 bonds in mannan polymers, increases as germination progresses. The MAN gene family is represented by six members in the Brachypodium genome, and their expression has been explored in different organs and especially in germinating seeds. Transcripts of BdMAN2, BdMAN4 and BdMAN6 accumulate in embryos, with a maximum at 24-30 h, and are detected in the coleorhiza and in the root by in situ hybridization analyses, before root protrusion (germination sensu stricto). BdMAN4 is not only present in the embryo root and coleorhiza, but is abundant in the de-embryonated (endosperm) imbibed seeds, while BdMAN2 and BdMAN6 are faintly expressed in endosperm during post-germination (36-42 h). BdMAN4 and BdMAN6 transcripts are detected in the aleurone layer. These data indicate that BdMAN2, BdMAN4 and BdMAN6 are important for germination sensu stricto and that BdMAN4 and BdMAN6 may also influence reserve mobilization. Whether the coleorhiza in monocots and the micropylar endosperm in eudicots have similar functions, is discussed.
BdMAN gene family; Brachypodium distachyon; coleorhiza; endo-β-mannanases; germination; MAN gene expression; mannan immunolocalization; mRNA in situ hybridization
Poaceae grains (caryopses) include the seed proper, formed by
a triploid endosperm and a diploid embryo, surrounded by
the maternal tissues of the seed coat (testa) and the pericarp.
The coleorhiza is a non-vascularized multicellular embryonic
tissue, covering the seminal roots of Poaceae seeds. The
coleorhiza has been thought to have a role in protecting the
emerging root (Sargent and Osborne, 1980) and, more recently, it
has been also associated with the regulation of dormancy,
since abscisic acid (ABA) sensitivity is reduced in this tissue
during germination of non-dormant barley seeds and the gene
encoding the HvABA8´OH-1 enzyme, that is critical for ABA
degradation, is expressed in the coleorhiza. During
germination, both in barley and in Brachypodium seeds, the coleorhiza
is the first structure that protrudes after the pericarp and testa
rupture (coleorhiza emergence), followed by the coleorhiza
rupture that allows root emergence (root emergence), and
indicates the end of germination sensu stricto (Millar et al.,
2006; Barrero et al., 2009, 2012; Gao and Ayele, 2014).
The germination process can be separated into
germination sensu stricto and subsequent reserve mobilization
(post-germination) and has been more deeply investigated
in eudicotyledonous than in monocotyledonous seeds. In
© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.
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Arabidopsis thaliana, Sisymbrium officinale, Lepidium
sativum and Nicotiana tabacum, the germination sensu stricto
occurs in two different steps; first, the testa ruptures and,
afterwards, the micropylar endosperm breakage takes
place, allowing the radicle to emerge (Leubner-Metzger
and Meins, 2000; Nonogaki et al., 2000; Müller et al., 2006;
Iglesias-Fernández and Matilla, 2010; Iglesias-Fernández
et al., 2011a, b; Weitbrecht et al., 2011; Nonogaki, 2014).
Addition of ABA to the imbibition medium specifically
blocks endosperm weakening and prevents its rupture
(Müller et al., 2006; Piskurewicz et al., 2009; Carrillo-Barral
et al., 2014). It is assumed that testa rupture is influenced by
the driving force of the imbibed elongating radicle and that
the endosperm rupture is mainly produced by the
weakening of the endosperm cell walls (CWs) by enzymes,
specifically those localized to the micropylar endosperm, such as
endo-β-1,4-mannanases (MANs), endo-ß-1,3-glucanases,
expansins, xyloglucan-transglycosylases/hydrolases (XTHs)
and pectin-methylesterases (Leubner-Metzger, 2005;
Nonogaki et al., 2007; Iglesias-Fernández et al., 2011a, b;
Endo et al., 2012; Martínez-Andújar et al., 2012;
RodríguezGacio et al., 2012; Scheler et al., 2014).
Since the endosperm CWs of several eudicot seeds are
rich in mannans (Lee et al., 2012), the MAN activity and
the expression of MAN genes upon seed germination have
been further characterized and their transcriptional
regulation studied. In A. thaliana, four MAN genes (AtMAN2,
AtMAN5, AtMAN6 and AtMAN7) are expressed in
germinating seeds and their transcripts are restricted to the
micropylar endosperm and to the radicle, disappearing as soon
as the radicle emerges. Moreover, knock-out mutants in the
AtMAN5, AtMAN6 and AtMAN7 genes, as well as, in the
AtbZIP44 gene encoding an important activating
transcription factor of AtMAN7, have a significantly retarded
germination as compared to that of wild-type seeds, indicating a
role for these MAN genes and their regulators during
germination sensu stricto (Iglesias-Fernández et al., 2011a, b, 2013;
Rodríguez-Gacio et al., 2012; Yan et al., 2014).
Brachypodium distachyon is being considered a model
species for the genetics and molecular genomics of cereals, due
in part to its small sequenced genome (~355 Mbp), short life
cycle, self-fertility, diploidy and its close phylogenetic
relationship with important crop plants of the tribe Triticeae within
the Poaceae family, such as wheat and barley (International
Brachypodium Initiative, 2010; Mochida and Shinozaki,
2013; Girin et al., 2014). In Poaceae seeds, the
β-1,3-1,4glucans are abundant in the endosperm cell walls (Burton
and Fincher, 2009; Guillon et al., 2011) and genes encoding
hydrolytic enzymes involved in their degradation, such as
endo-β-1,3-glucanases and endo-β-1,3-1,4-glucanases, have
been associated with rice post-germination events (2–4 days
of imbibition; Akiyama et al., 2004) and with the elongation
of barley coleoptiles (Takeda et al., 2010). Although mannan
content is lower than glucan content in Brachypodium seeds
(Rancour et al., 2012), the function of mannans and MANs
may be relevant in its germinating seeds.
In this work, mannan polysaccharides were
immunolocalized to the root and the coleorhiza of germinating seeds early
in imbibition, decreased thereafter at later stages, and the
enzymatic activity of endo-β-mannanases increased as
germination progressed. The MAN gene family of B. distachyon
was annotated and the expression of its six members explored
in vegetative and reproductive organs. Interestingly, genes
BdMAN2, BdMAN4 and BdMAN6 were clearly induced
upon seed germination and mRNA in situ hybridization
analyses demonstrated that these transcripts were found in
the coleorhiza and the root during germination sensu stricto.
BdMAN4 and BdMAN6 were also expressed in the aleurone
layer, and may also be involved in post-germinative reserve
Materials and Methods
Biological material, growth conditions and germination assays
The diploid inbred Brachypodium distachyon strain Bd21 (kindly
provided by Prof. Garvin from the University of Minnesota, USA;
International Brachypodium Initiative, 2010) was used in this work.
Seeds were surface-sterilized with 1% NaOCl for 10 min and washed
in sterile water, before germinating on Petri dishes, containing two
filter papers (Whatman 3) moistened with 8ml of sterile water, at
22ºC in the dark for 2 d. They were then transferred to pots in the
greenhouse under long-day conditions (16h/8h, light/darkness; light
intensity 155 µmol photons m−2 s−1) for sampling roots (6-week-old
plants), young and old leaves (6- and 12-week-old plants) and spikes.
For the germination experiments that lasted up to 42 h, seeds were
incubated in the dark at 22ºC, in Petri dishes with moistened filter
papers, using triplicate lots of 25 non-stratified after-ripened seeds
(stored at 22ºC and 30% relative humidity in the dark for 3 months).
Seed samples were separated into embryo and endosperm
(deembryonated seeds) at 0, 12, 24, 30, 36 and 42 h of imbibition, and
used for RNA quantification and for protein extraction to determine
MAN enzymatic activity.
Endo-β-1,4-mannanase (MAN) activity assays
Seed samples, obtained as described above, were homogenized
in 100 mM sodium acetate buffer (pH 4.5) containing 1 M NaCl
and 0.5% ascorbic acid, at 4ºC for 2 h in an orbital shaker (VWR
International Eurolab, Barcelona, Spain). The homogenates were
centrifuged at 15,000 × g for 45 min, and 80 μl of this supernatant
was mixed with 150 μl of 0.25% mannan (1,4-β-D-mannan from
carob; Megazyme International Ireland Ltd., Wicklow, Ireland).
Incubation was at 30ºC for different periods of time and the
enzymatic activity was determined by the increase in reducing sugar
production per mg of protein, as determined by the 4-OH-Benzoic Acid
Hydrazide (PAH-BAH; Sigma-Aldrich) method (Lever, 1977). The
MAN from Aspergillus niger (Megazyme) was used to establish the
control curve (one unit of MAN activity defined as the amount of
enzyme that releases 1 nmol of reducing sugar per minute under
the experimental conditions). Protein concentration was
determined with the Bradford reagent (Bio-Rad Laboratories, Munich,
Germany) using bovine serum albumin (BSA) as a standard.
Bioinformatic tools: BdMANs identification and phylogenetic
The deduced protein sequences of the six MAN genes were
obtained from the B. distachyon genome using the TBLASTN tool
at the Phytozome v8.0 Database (Goodstein et al., 2012; www.
phytozome.net), using the eight OsMAN proteins from the Oryza
sativa genome as query sequences (Yuan et al., 2007). The Interpro
Program (PFAM database; Bateman et al., 2002; http://pfam.sanger.
ac.uk) was used to confirm the presence of the MAN conserved
domain (glycosyl-hydrolase family 5). The complete amino acid
sequences deduced from B. distachyon, O. sativa and Arabidopsis
thaliana MAN genes were aligned by means of the CLUSTAL W
program (Thompson et al., 1994) and utilized to construct a
phylogenetic dendrogram, using the neighbor-joining algorithm, a
bootstrap analysis with 1,000 replicates, complete deletion and the Jones
Taylor Thornton matrix, as settings. The MEME program software
version 4.0 (Tamura et al., 2007) was used to identify conserved
motifs within the deduced MAN proteins and to validate the
phylogenetic tree (Table 1). Default parameters were used with the
following exceptions: the maximum number of motifs to find was set
to 22 and the minimum width was set to eight amino-acid residues
(Bailey et al., 2009; http://meme.sdsc.edu/meme4_6_0/intro.htlm).
A single capital letter represents a single residue relative frequency,
if this is greater than 50% than twice that of the second most
frequent residue in the same position. If no single residue matches
these criteria, a pair of residues, represented by capital letters in
brackets, is given if the sum of their relative frequencies exceeds
75%. If none of these characteristics are satisfied, a lowercase letter
is given when the relative frequency of a residue is greater than 40%,
if not, x is set.
The major biochemical parameters of the deduced MAN proteins
from B. distachyon and O. sativa are listed in Supplementary Table
S1. Both isoeletric point (pI) and molecular weight (MW) were
predicted using the Compute pI/MW tool (Gasteiger et al., 2005; http://
www.expasy.ch/tools/pi_tool.html) and the putative signal peptide
cleavage site and sub-cellular localization were deduced by the
SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP) and TargetP
1.1 tools (http://www.cbs.dtu.dk/services/TargetP/), respectively
(Emanuelsson et al., 2007).
Real time quantitative PCR (RT-qPCR) analyses
Total RNA was purified from roots (6-week-old plants), young
and old leaves (6- and 12–week-old plants) and spikes by the
phenol/chloroform method (Lagrimini et al., 1987). For the isolation
of RNA from seeds at different stages of development (0–10 d
after pollination: dap) and at different time points of germination
(12, 24, 30, 36 and 42 h), the protocol described by Oñate-Sánchez
and Vicente-Carbajosa (2008) was followed. RNA samples were
treated with DNAse I, RNAse-free (Roche Applied Science,
Manheim, Germany) to avoid genomic DNA contamination.
First-strand cDNA was synthesized with random hexamers using
the High-Capacity cDNA Reverse Transcription Kit (Applied
Biosystems, Foster City, CA, USA) according to the
manusfacturer’s recommendations. Samples were stored at −20ºC until used.
PCR-amplification was performed in an Eco Real-Time PCR
System (Illumina, San Diego, CA, USA). For each 10 µl reaction,
2 µl of DNA sample was mixed with 5 µl of FastStart SYBR Green
Master (Roche Applied Science) and 0.25 µl of each primer (final
concentration 500 nM) plus sterile water up to final volume. Samples
were subjected to thermal-cycling conditions of 95ºC for 10 min and
40 cycles of 10 s at 95ºC and 30 s at 60ºC for annealing and
extension, respectively. The melting curve was designed to increase from
55ºC to 95ºC, and the melting temperatures for each amplicon and
primer efficiencies (Supplementary Table S2) were estimated using a
calibration dilution curve and slope calculation (E=10(−1/slope)). The
specific primers used are shown in Supplementary Table S2 and they
were designed on the 3′-non-coding region using the Primer3Plus
cgi). The BdGAPDH gene (encoding glyceraldehyde 3-phospate
dehydrogenase; Hong et al., 2008) was used to normalize the data, since
the expression of this gene was previously demonstrated to be
constant throughout the period studied (Hernando-Amado et al., 2012;
González-Calle et al., 2014; Supplementary Fig. S1). Expression levels
were calculated as the number of cycles needed for the amplification
to reach a cycle threshold fixed in the exponential phase of the PCR
(Ct; Pfaffl, 2001). All analyses used three different biological replicates
for each time-point and each one was made in triplicate. Means ±
standard error (SE) of three independent experiments are indicated in
the corresponding figures.
Preparation of embedded material for microscopy
Samples were treated according to a modified version of the
protocol described in Ferrandiz et al. (2000). After-ripened dry seeds and
germinating seeds of B. distachyon (12 h, 27 h and 36 h) were collected
and, after removal of the lemma and palea, were infiltrated with the
FAE solution (formaldehyde: acetic acid: ethanol: water, 3.5:5:50:41.5
by volume) for 40 min in 25 mm Hg vacuum; the seeds were then
incubated at 4ºC for 3 d with gentle shaking. The samples were dehydrated
through a graded series of aqueous ethanol mixtures and
progressively embedded in paraffin after the replacement of ethanol with
HistoClear (National Diagnostics, Hessle Hull, England). Thin
sections of 8 µm were collected on glass slides and de-waxed.
The protocol used was a modification of those described in Marcus
et al. (2010) and Guillon et al. (2011). In a pre-immunolabelling step,
sections of embedded material as described above, were incubated
in phosphate buffer sodium solution (PBS) and treated with 1 mg/
ml proteinase-K (Roche Applied Science). In order for the specific
antibodies to have access to the heteromannans (mannans,
glucomannans, and galactomannans) of the cell walls, β-1,3-1,4-
glucans were removed by incubating the sections with a solution of 4 µg/
ml lichenase [β-1,3-1,4- glucanase; Megazyme] for 2 h at 37ºC, and
then rinsed with de-ionized water. For heteromannan
immunodetection, sections were first incubated at room temperature for 30min
in a blocking solution (3% BSA, 1× PBS, and 5mM sodium azide;
pH7), and then treated with primary anti-heteromannan antibody
LM21 (PlantProbes, Leeds, UK) at a dilution of 1:5 in the same
blocking solution but only containing 1% BSA for 2 h. Sections were
thoroughly washed in PBS containing 5 mM sodium azide and then
incubated for 2 h in the same buffer containing the secondary
rabbit antibody Anti-Rat IgG-FITC (Sigma-Aldrich) at a dilution of
1:100. The sections were extensively washed in PBS buffer and in
water, mounted and examined in a confocal microscope (absorption
494 nm; emission 521 nm; Leica TCS-SP8, Leica, Wetzlar, Germany).
mRNA in situ hybridization analyses
Pre-hybridization was carried out by incubating the sections in
0.2 M HCl, neutralizing them and then treating them with 1 mg/
ml proteinase-K (Roche Applied Science). Samples were then
dehydrated in an aqueous ethanol dilution series and hybridized with
sense and anti-sense digoxigenin (DIG)-labelled RNA probes,
corresponding to DNA fragments (200–300 bp) derived from the
3′non coding regions of the BdMAN2, BdMAN4 and BdMAN6 genes
(Supplementary Table S3), synthesized with the DIG RNA labelling
mix according to the manufacturer’s specifications (Roche Applied
Science). Probes were hybridized at 52ºC overnight followed by two
washes in 2× SSC (150 mM NaCl, 15 mM Na3-citrate) and 50%
formamide for 90 min at the same temperature. Incubation with the
alkaline phosphatase-conjugated anti-digoxigenin antibody (Roche
Applied Science) and colour detection was carried out according
to the manufacturer’s instructions (Ferrandiz et al., 2000). Sections
were dried and examined on a Zeiss Axiophot Microscope (Carl
Zeiss, Oberkochen, Germany), and images were captured and
processed with the Leica Application Suite 2.8.1 build software (Leica).
Protein and polysaccharide histological determinations
B. distachyon dry and germinating seeds were stained with 5% (w/v)
toluidine blue (Merck, Darmstadt, Germany) for checking tissue
integrity (Fig. 1A). Samples were stained with PAS reagent (0.5%
w/v periodic acid-Schiff reagent) (Merck) to detect polysaccharides
and with 1% (w/v) Naphthol Blue Black (Sigma-Aldrich) for
proteins (Iglesias-Fernández and Matilla, 2010). Visualization was done
on a Zeiss Axiophot Microscope (Carl Zeiss) and the images were
captured and processed with the Leica Application Suite 2.8.1 build
Enzymatic β-mannanase activity during Brachypodium
The time course of sensu stricto germination of B.
distachyon seeds occurs in two different steps: first, the coleorhiza
emerges (CE), and in a second step, the root emergence (RE)
takes place (Fig. 1A). The enzymatic activity of MAN upon
germination has been analysed separately in the embryo and in
the de-embryonated seed (endosperm). As shown in Fig. 1B,
dried seeds have no detectable MAN activity, but this
progressively increases with germination, peaking at 24 h in embryos,
containing the coleorhiza (~0.4 × 10–3 units/mg protein), and
decreasing to half this value at 42 h (~0.2 × 10–3 units/mg
protein). In endosperms, MAN activity is much lower than in
embryos, and reaches its maximum level (~0.15 × 10–3 units/
mg protein) at 36 h of germination. Data from Fig. 1B
indicate that MAN activity is maximum in embryos, just before
t50RE=36h), suggesting that MAN is important for
facilitating both coleorhiza and root emergence.
Heteromannans are preferentially localized to the root
tip and the coleorhiza in germinating seed embryos
Mannan polymers have been detected in longitudinal sections of
B. distachyon germinating seeds (at 12 and 27h of imbibition) by
in situ immunofluorescence labelling, using the LM21 antibody
that specifically recognizes mannan polysaccharides (gluco- and
galacto-mannans). To facilitate accession of the antibody to
mannans in plant CWs, the seed sections have been previously
treated with lichenase (β-1,3-1,4- glucanase; Marcus et al., 2010).
As shown in Fig. 2, at 12 h of seed imbibition, seed
mannan polymers are mainly localized to the periphery cells of
the coleorhiza (C) and to the epidermis of the root tip (R)
(Fig. 2A–C). Interestingly, these mannans are barely detected
at later stages of germination (27 h of imbibition; Fig. 2D–F).
Differential interference contrast (DIC) images are shown in
Fig. 2G–I. This observation together with data of MAN
enzymatic activity (Fig. 1B) with a maximum at 24 h in embryos,
may suggest that the disappearance of the mannan polymers
is due to the hydrolysis catalysed by endo-β-mannanases.
In order to get a deeper insight into the MAN function
upon B. distachyon germination, it was decided to annotate
and characterize further the BdMAN family. The already
described MAN family from O. sativa (Yuan et al., 2007)
has been used to perform a TBLASTN against the whole
Brachypodium genome (http://www.phytozome.net). Six
predicted non-redundant MAN deduced proteins, with MW
43–52 KDa, and Ip 4.4–8.8, three of them with predicted
signal peptides, have been identified and named
according to their orthologues in rice (Supplementary Table S1).
The MAN protein sequences from A. thaliana (AtMAN1-7)
and O. sativa (OsMAN1-8) together with those from B.
distachyon (BdMAN1-6) have been used to construct a
phylogenetic unrooted tree by using the neighbor-joining algorithm.
Four major clusters of orthologous groups (MCOGs) have
been defined (A, B, C, D), supported by bootstrapping values
higher than 62% (Fig. 3A) and by the occurrence of common
motifs (Fig. 3B; MEME).
The search for conserved amino-acid motifs using the
MEME software (http://meme.nbcr.net/meme/cgi-bin/
Fig. 2. Mannan polymer immunolocalization at the root tip and the coleorhiza in longitudinal sections of Brachypodium germinating embryos at (A–C)
12 h and at (D–F) 27 h. (G–I) DIC images of D, E and F. (C, F, I) Close-up of the coleorhizae. C, coleorhiza; R, root. Scale bars: (A, D, G), 50 μm; (B, E, H),
25 μm; (C, F, I), 10 μm.
meme.cgi) reveals that all MAN sequences have in common
motifs described as critical for the enzymatic activity, such
as 1, 3, 5, 6/11 and 7 (Fig. 3B, Table 1). The deduced
signature sequence [AWEL(MI)NEPRC] of Arabidopsis and rice
MANs (Yuan et al., 2007), included in motif 1, is also
present in Brachypodium MAN. Besides, members in MCOG A,
BdMAN2, OsMAN2, BdMAN6 and OsMAN6 share motif
12, and BdMAN6 and OsMAN6 also share motifs14 and 18.
In MCOG C, BdMAN1 shares with OsMAN1 motifs 19 and
22, and in MCOG D BdMAN4 shares motifs 17 and 21 with
OsMAN4, but lacks motif 15 shared by the rice paralogues
OsMAN3 and OsMAN4. Similarly, BdMAN5, OsMAN7
and OsMAN8 (MCOG B) have in common motif 11, but
they do not share with OsMAN5 motifs 20 and 13. The
Table 1. Conserved amino acid motifs obtained by means of
MEME (Bailey et al., 2009) from the analysis of the
endo-βmannanase proteins of Brachypodium distachyon, Oryza sativa,
and Arabidopsis thaliana
The conserved amino acids, critical for enzyme activity, are in bold
and the signature sequence for endo-β-mannanase enzymes is
Fig. 3. (A) Phylogenetic dendrogram with deduced protein sequence
of the mannanase gene families form Brachypodium distachyon, Oryza
sativa and Arabidopsis thaliana; bootstrapping values are indicated in
the branches. (B) Schematic distribution of conserved motifs among the
deduced protein sequences in the phylogenetic tree (A), identified by
means of the MEME analysis. Asterisks indicate those motifs important
for enzymatic activity. Motifs in grey share >85% of similar amino acid
residues. This figure is available in colour at JXB online.
Fig. 4. (A) Different stages of Brachypodium distachyon seed
development (4, 6, 8, 10, 12 d after pollination; dap). (B) Expression of
the BdMAN1, BdMAN2 and BdMAN6 genes by RT-qPCR during seed
development. Data are means ± standard error (SE) of three technical
replicates of three biological samples.
MAN protein motifs from A. thaliana have been included for
comparison (Iglesias-Fernández et al., 2011a).
Expression kinetics of selected BdMAN genes during
seed maturation and germination
The expression pattern of the six BdMAN genes has been
explored by RT-qPCR analysis in different organs: young (6
d) and old (12 d) leaves, roots (6 d) and spikes (mix of different
stages; Supplementary Fig. S2). While BdMAN1, 2, 3, 5 genes
are not detected in leaves, BdMAN4 gene expression in old
leaves is ~10 times lower than in young leaves, and BdMAN6
has the same low expression in young and old leaves. In roots,
BdMAN2, BdMAN4 and BdMAN6 are expressed at low
levels, and BdMAN1, BdMAN2, BdMAN4 and BdMAN6
transcripts are detected in spikes (Supplementary Fig. S2).
Since our preliminary data indicate that BdMAN1,
BdMAN2 and BdMAN6 transcripts are abundant in
developing seeds (see Supplementary Fig. S3A), the expression
kinetics of these three genes has been established throughout
seed maturation, at 4, 6, 8, 10 and 12 d after pollination (dap)
(Fig. 4A). Although the gene BdMAN1 is the most highly
expressed during the late phases of seed development (8,
10, 12 dap), the expression patterns of BdMAN1, BdMAN2
and BdMAN6 show a progressive increase from 4 to 10 dap,
reaching all of them their maximum expression at 10 dap
when maturation is almost completed (Fig. 4B).
Data in Supplementary Fig. S3B indicate that genes
BdMAN2, BdMAN4 and BdMAN6 are the most abundantly
expressed ones during seed germination, and their
expression kinetics has been more thoroughly analysed (Fig. 5).
Germinating seeds taken at 12, 24, 30, 36 and 42 h of
imbibition, have been sectioned into embryos and de-embryonated
seeds (endosperm). In germinating embryos, BdMAN2,
BdMAN4 and BdMAN6 transcripts appear early upon
imbibition (12 h), before CE, and their maximum expression is
attained between 24–30 h (t50CE=30 h) and it decreases as
germination progresses (42 h; Fig. 5A). However, the expression
of BdMAN4 in endosperms is high at early imbibition times
(12 h; ~140% relative to BdGAPDH), decreasing thereafter.
The BdMAN2 and BdMAN6 transcripts have low expression
in the endosperms of germinating seeds with a maximum
at 36 h of imbibition (<10 % for BdMAN2 and ~25 % for
BdMAN6 relative to BdGAPDH, respectively), indicating a
possible role in reserve mobilization for these MAN genes,
and perhaps also for BdMAN4 post-germination (Fig. 5B).
BdMAN2, BdMAN4 and BdMAN6 transcripts are
localized to different seed tissues during B. distachyon
To determine the spatial expression of MAN genes within the
B. distachyon germinating seeds, mRNA in situ hybridization
experiments have been done (Fig. 6). Longitudinal sections
of seeds at 27 h of imbibition have been hybridized to specific
antisense and sense (as negative controls) probes for BdMAN2,
BdMAN4 and BdMAN6. The BdMAN2 transcripts are
mainly expressed in the periphery cells of the coleorhiza and
are not detected in the aleurone layer (Fig. 6A–D). BdMAN4
mRNA is localized preferentially to the tip and the apical
meristem of the root, to the coleorhiza and to the aleurone layer
(Fig. 6E–H), and the BdMAN6 transcripts are detected not
only at the coleorhiza, but also throughout the embryo and
faintly also at the aleurone layer (Fig. 6I–L). BdMAN6
transcripts are detected in the aleurone layer at 27 h of imbibition
by mRNA in situ hybridization, but they are scarcely detected
in 24–30 h germinating endosperms by RTqPCR (Fig. 5B),
indicating that its expression could be diluted by the remaining
endosperm during the RNA isolation process. As expected, no
signal has been detected when sections have been hybridized
with the corresponding sense probes for BdMAN2, BdMAN4
and BdMAN6 (negative controls; Figs 6D, 6H and 6L).
Other histological observations of germinating
B. distachyon seeds
Longitudinal sections of B. distachyon seeds (dry and water
imbibed at 27 and 36 h; including seeds before and after root
emergence) have been stained with PAS reagent to detect
insoluble polysaccharides (mainly cellulose and starch) and
with Naphthol Blue Black for proteins. As shown in Fig. 7A–
C, dry seeds have abundant protein bodies (PBs; blue stained)
and thick cell walls (CWs; pink stained) in the root, coleorhiza
and endosperm cells. When the coleorhiza emerges (27 h of
imbibition) the PBs in the coleorhiza (C) and in the
mesocotyl (M) cells start to hydrolyze, while the endosperm cells
are full of reserves at this stage (Fig. 7D–F). After 36 h, when
the seed coat ruptures but before root emergence (RE), the
coleorhiza cells start to elongate and its protein bodies (PBs),
as well as, those of the mesocotyl (M) are almost completely
consumed, while those of the coleoptile (Co) initiate their
degradation (Fig. 7G–I) and those of the endosperm remain
as in the dry seeds (Fig. 7C, 7I). Finally, when root emergence
(RE) takes place, the lateral part of the coleorhiza breaks
(≥36 h of imbibition) and its PBs are fully degraded, while
those in the endosperm cells are almost intact (Fig. 7J–L).
In this work, mannans and endo-β-mannanases (MAN) in
Brachypodium distachyon have been investigated in order to
establish whether they are important in the germination of
these monocotyledonous seeds. Mannans have been
immunolocalized in the embryo root and the coleorhiza in the early
stages of germination and these polymers decrease upon
imbibition while the enzymatic activity of MAN increases. The
MAN gene family in B. distachyon has been annotated and
the gene expression of the six members of this family has been
explored in different vegetative and reproductive organs, and,
more specifically, in germinating seeds. Three of these genes,
BdMAN2, BdMAN4 and BdMAN6, are highly induced in
germinating embryos and their transcripts are localized to the
coleorhiza and the root, and BdMAN4 and BdMAN6 appear
also in the aleurone layer. These facts indicate that the BdMAN
enzymes should be spatially distributed in the seed in the
vicinity of their putative substrates, thus contributing to the
mannan hydrolysis and to the loosening of the coleorhiza cell walls,
thereby facilitating root protrusion (germination sensu stricto).
During seed development, BdMAN1, BdMAN2 and
BdMAN6 genes are expressed, and their mRNAs are abundant
at the middle and late maturation stages. Upon cereal seed
maturation, several tissues undergo a progressive enlargement of
their cells, a process that involves nutrient remobilization and
CW softening to allow cell expansion; to this aim, the
participation of a complex set of hydrolytic enzymes have been described
(Domínguez and Cejudo, 2014). These data indicate that the
BdMAN1, BdMAN2 and BdMAN6 proteins could contribute
to such a process during Brachypodium seed development.
The enzymatic analysis in the embryos of germinating
seeds shows a maximum of MAN activity at 24 h of
imbibition, just before the coleorhiza emergence (CE50=30 h),
and this enzymatic activity progressively decreases to 50%
at 42 h. However, MAN activity in de-embryonated seeds
(endosperms) is low, with a maximum at 36 h of imbibition,
when germination sensu stricto is almost completed [~100%
coleorhiza emergence (CE); ~80% root emergence (RE) at 42 h].
These data point out to a more important role of the MAN
activity in the embryo than in the endosperm during
germination sensu stricto. In rice, MAN activity and expression
of the OsMAN1, OsMAN2 and OsMAN6 genes have been
detected in the aleurone layer only after 48 h of imbibition
when 100% of germination has been achieved. This MAN
activity is associated with reserve mobilization, a clear
postgerminative event (Ren et al., 2008). In barley, the HvMAN1
enzyme has been purified from 10-day-old seedlings and its
catalytic parameters established, although its physiological
role has not been investigated (Hrmova et al., 2006).
The function of the coleorhiza tissue in the grasses has been
classically associated to a protective function of the
growing root during germination, but other physiological
functions are being uncovered, such as our observations of the
hydrolysis of proteins (disappearance of PBs) or the decrease
in mannan content detected within the coleorhiza cells
during Brachypodium germination sensu stricto. Nowadays, and
similarly to what has been proposed for the endosperm of
eudicot seeds (Piskurewicz et al., 2009), the coleorhiza is
being considered to be a key tissue preventing root emergence
in dormant barley seeds (Millar et al., 2006; Barrero et al.,
2009). These authors have hypothesized that root emergence
may not depend only on the softening of the coleorhiza,
driven by CW remodelling enzymes, but also by the expansive
force of the imbibing root cells. Important transcriptional
changes in the barley coleorhiza associated to the dormancy
degree have been found and these differences affect mainly
the expression of CW modifying genes (mannanases among
them), nitrate and nitrite reductase genes etc. (Barrero et al.,
2009). The cytosolic nitrate reductase is an important source
of the hormone nitric oxide (NO) that is involved in
promoting seed germination (Arc et al., 2013). Therefore, the
coexistence at the coleorhiza of NO and mannanases, and perharps
proteases of the CatepsinB3 type (Iglesias-Fernández et al.,
2014), should have an influence in the seed germination of
The Arabidopsis radicle tip has been described as the
primary location of growth-promoting genes and its
surrounding-cells the centre for CW expansion (Bassel et al.,
2014). Moreover, a dual enzymatic activity for MAN
(hydrolase and transglycosylase activities) has been described; the
transglycosylase activity being more related to cell expansion,
as occurs in the radicle before protrusion, and the hydrolytic
activity could be relevant for weakening of the CWs of the
embryo-surrounding tissues (Schröder et al., 2009;
IglesiasFernández et al., 2011a, b). It is remarkable that the BdMAN4
transcripts are localized to the aleurone layer during
imbibition (27 h), when practically no MAN activity is detected in
the de-embryonated (endosperm) seed, suggesting a possible
accumulation of these transcripts and their corresponding
proteins as inactive forms in the aleurone cells. Interestingly,
the BdMAN4 deduced protein sequence has a predicted
signal peptide for the secretory pathway and it is possible that
the BdMAN4 isozyme could be transported later on
during post-germinative reserve mobilization from the
aleurone to the endosperm cells through the apoplastic space.
In Arabidopsis the AtMAN7 and in poplar the PtrMAN6
proteins also contain signal peptides and have been localized
to the apoplast, indicating that the mature MANs could be
mobilized to the outer space (Iglesias-Fernández et al., 2013;
Zhao et al., 2013).
Several authors have proposed that polysaccharides
(β-1,3-1,4-glucans, mannans and others) present at the
endosperm CWs of the Poaceae grains, not only have a
structural function, but also a storage role (Guillón et al., 2012).
Heteromannans, although globally less abundant in these
seeds than the β-glucans, are concentrated not only in the
coleorhiza and in the root but also these polymers are found
in the aleurone layer and storage endosperm of B. distachyon
(Guillón et al., 2011). In this context, our data demonstrate
that MAN activity is important for the weakening of the
coleorhiza cell walls and for the expansion of the root cells,
thus facilitating germination sensu stricto, but it may be also
important for the mannan hydrolysis in endosperm during
post-germinative reserve mobilization.
Supplementary data are available at JXB online.
Supplementary Fig. S1. Transcription levels of the
housekeeping (BdGAPDH gene), presented as Ct mean values, in
different organs, in developing seeds and during seed
germination of B. distachyon.
Supplementary Fig. S2. Transcripts analysis by RTqPCR
of the BdMAN1-6 genes in different organs.
Supplementary Fig. S3. Expression analysis by RT-qPCR
of BdMAN1-6 genes in developing seeds and germinating
Supplementary Table S1. Major biochemical
characteristics of Brachypodium distachyon and Oryza sativa
Supplementary Table S2. Oligonucleotide sequences,
amplicon length and PCR efficiency of the primers used for
Supplementary Table S3. Primers used for the synthesis of
the in situ mRNA hybridization probes.
Financial support from MINECO, Spain (Project BFU2009-11809;
Principal Investigator PC) is gratefully acknowledged. We thank Prof. AJ
Matilla (Universidad de Santiago de Compostela, USC, Spain) for critical
reading of the manuscript. VG-C is the recipient of a pre-doctoral contract
from Universidad Politécnica de Madrid (Spain).
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