The Mucilage Proteome of Maize (Zea mays L.) Primary Roots
Wei Ma,†Nils Muthreich,‡Chengsong Liao,†Mirita Franz-Wachtel,§Wolfgang Schu ¨tz,§
Fusuo Zhang,†Frank Hochholdinger,*,‡and Chunjian Li*,†
Department of Plant Nutrition, China Agricultural University, Beijing 100193, PR China, Center for Plant
Molecular Biology, Department of General Genetics, Eberhard Karls University Tuebingen,
72076 Tuebingen, Germany, and Proteome Center Tuebingen, Interfaculty Institute for Cell Biology,
University of Tuebingen, 72076 Tuebingen, Germany
Received December 18, 2009
Maize (Zea mays L.) root cap cells secrete a large variety of compounds including proteins via an
amorphous gel structure called mucilage into the rhizosphere. In the present study, mucilage secreted
by primary roots of 3-4 day old maize seedlings was collected under axenic conditions, and the
constitutively secreted proteome was analyzed. A total of 2848 distinct extracellular proteins were
identified by nanoLC-MS/MS. Among those, metabolic proteins (∼25%) represented the largest class
of annotated proteins. Comprehensive sets of proteins involved in cell wall metabolism, scavenging
of reactive oxygen species, stress response, or nutrient acquisition provided detailed insights in functions
required at the root-soil interface. For 85-94% of the mucilage proteins previously identified in the
relatively small data sets of the dicot species pea, Arabidopsis, and rapeseed, a close homologue was
identified in the mucilage proteome of the monocot model plant maize, suggesting a considerable
degree of conservation between mono and dicot mucilage proteomes. Homologues of a core set of 12
maize proteins including three superoxide dismutases and four chitinases, which provide protection
from fungal infections, were present in all three mucilage proteomes investigated thus far in the dicot
species Arabidopsis, rapeseed, and pea and might therefore be of particular importance.
Keywords: Maize • mucilage • proteome • shotgun • metabolism
Plant roots can sense water and nutrients by continuously
producing and secreting compounds into the rhizosphere.1,2
A wide variety of carbon compounds is released from living
roots to the soil, including higher molecular weight compounds
such as polysaccharide mucilage and enzymes, and low mo-
lecular weight compounds such as sugars, amino acids, organic
acids, fatty acids, and growth factors.3–5The root mucilage
which covers the root cap is an amorphous and uneven gel,
which ranges in thickness from 50 µm to 1 mm.6As the root
extends through the soil, mucilage and associated root cap cells
are left behind along the root-soil interface.7The gel-like
mucilage contributes to many interactions between the plants
and soil. Mucilage facilitates soil aggregation,8,9provides carbon
for microbes,10–12and facilitates root movement in the soil by
decreasing the frictional resistance.13,14Moreover, mucilage
contributes to water holding15and reduces the surface tension
of water, thus, changing the water relations of the rhizo-
sphere.16Furthermore, it ameliorates toxic effects of elements
like Al3+,17Cd2+,9,18and Cu2+.18,19Finally, mucilage contributes
to a coherent sheath (rhizosheath) formation in many grasses
and some dicots, which is ecologically significant and plays an
important role in water and nutrient uptake.8,9Because of the
large amount of secretion, the maize mucilage has been used
as a model system for decades to investigate the synthesis and
secretion of the cell wall matrix.15
Chemical analyses of mucilage collected from roots of
axenically grown maize revealed a protein content of 1-6% in
addition to the presence of several polysaccharides and
monosaccharides.20–23These results indicate that the secretion
of such carbon compounds is a natural process and not
induced by environmental stress. Compounds found in the
mucilage surrounding the root tip are not only secreted by the
root cap, but also by border cells, which detach and differenti-
ate from the root cap.24A large number of border cells are
present in the majority of higher plant species including
In recent years, considerable progress has been made in
understanding the mechanisms and ecological significance of
the secretion of small molecular weight compounds, such as
organic acids induced by either phosphorus deficiency or
aluminum toxicity and phytosiderophores induced by iron
deficiency in grass species.26–29In contrast, only little is known
about the root mucilage proteome. Plant roots can secrete a
* To whom correspondence should be addressed. Chunjian Li, Depart-
ment of Plant Nutrition, China Agricultural University, Yuanmingyuan West
Road 2, Beijing 100193, PR China. Phone, +86 10 6273 3886, fax, +86 10
6273 1016; e-mail, email@example.com. Frank Hochholdinger, Center for Plant
Molecular Biology, Department of General Genetics, Eberhard Karls Uni-
versity Tuebingen, 72076 Tuebingen, Germany. Phone, +49 7071 29 77024;
fax, +49 761 295042; e-mail, firstname.lastname@example.org.
†China Agricultural University.
‡Eberhard Karls University Tuebingen.
§University of Tuebingen.
2968 Journal of Proteome Research 2010, 9, 2968–2976
Published on Web 04/21/2010
2010 American Chemical Society
battery of defense1and signal proteins,30which play a strategic
role in the plant responses to biotic and abiotic stress.31
Because of the low protein content and the difficulty of protein
purification from root mucilage, the lack of powerful tools for
protein identification allowed studying only a limited number
of mucilage proteins thus far. While in pea root tip exudates
124 proteins were identified by multidimensional protein
identification,32in Arabidopsis and rapeseed, 52 and 16 proteins
have been identified by MudPIT and LC-MS/MS, respectively.31
In the present study, 2848 distinct proteins of the maize primary
root mucilage proteome were identified and functionally
characterized. This data provides a first comprehensive insight
into the physiological and molecular functions involved in root
interaction with the soil environment.
Plant Culture and Mucilage Collection. Seeds of the maize
(Zea mays L.) hybrid Nongda 108 were surface-sterilized with
30% H2O2for 30 min and then rinsed seven times for 1 min in
sterile deionized water according to Berthelin and Leyval.33The
seeds were germinated on moist filter paper in the dark at 25
°C. After 3 days (d), seedlings with 1-2 cm long primary roots
were transferred into a plastic tray with small holes on the
bottom which allowed roots to grow through. This tray was
used as a lid for a plastic container so that the seedling roots
could contact with 1 mM axenic CaCl2solution34containing
Micropur microorganism inhibitor (Katadyn Products, Inc.,
Wallisellen, Switzerland). Micropur inhibitor contains sodium
dichloroisocyanurate (NaDCC) and silver chloride and was
applied in order to prevent microbial degradation of the root
exudation during and after collection.35Twelve hours later,
mucilage was collected from the tip of the primary roots (2-3
cm long at this stage) using a sterilized drawn glass Pasteur
pipet. There was no yellowing of the root tips observed during
and after mucilage collection. Potential bacterial contamination
of the mucilage was tested by streaking extracted root mucilage
on nutrient agar plates according to Chaboud20and incubating
the agar plates overnight. All tests for bacterial contamination
of the mucilage were negative. Proteins were collected in three
biological replicates with about 540 seedlings each. Biological
replicate mucilage collection was repeated three times within
12 h. These mucilage samples were pooled. In this study, the
maize extracellular proteins of the primary root mucilage
proteome were defined as mucilage proteins released by
primary root tips and border cells. The proteins released into
the 1 mM axenic CaCl2solution were not included. Seedling
growth and mucilage collection were carried out in a laminar
flow cabinet (1400 L × 700 W × 370 H), which provided an
Protein Purification and SDS-PAGE. Prior to protein analy-
ses, the mucilage samples were centrifuged at 14 000g three
times for 10 min at 4 °C to remove the border cells from the
mucilage according to Read and Gregory.23The supernatant
which contained the mucilage was then precipitated three
times with 10% (v/v) trichloroacetic acid (TCA) containing 0.1
mM phenylmethanesulfonyl fluoride (PMSF) and subsequently
purified with acetone five times as described in Damerval et
al.36Dried protein pellets were dissolved in SDS extraction
buffer containing 50 mM Tris, pH 8.0, 1 mM EDTA, 2.5% (w/v)
SDS, 5% (w/v) mercaptoethanol, 15% (v/v) glycerol, and 0.05%
(w/v) bromophenol blue. The protein concentration was as-
sessed with the 2-D Quant Kit (GE Healthcare, Amersham
Biosciences, Piscataway, NJ). For SDS-PAGE electrophoresis,
30 µg of protein extract was denatured by incubation at 100
°C for 5 min, then loaded onto a 12% SDS gel and run at 100
V using a 13 cm electrophoresis chamber (GE Healthcare;
Amersham Biosciences, Piscataway, NJ). Gels were stained with
Coomassie blue as described by Neuhoff et al.37
In-Gel Tryptic Digestion. The gel lane was vertically sliced
into 16 pieces. Each fragment was cut into 1 mm3cubes and
washed three times with 50% 10 mM NH4HCO3/50% acetoni-
trile. After dehydration with 100% acetonitrile, gel pieces were
incubated with 10 mM DTT in 20 mM ammonium bicarbonate
for 45 min at 56 °C to reduce disulfide bonds. Alkylation of
cysteines was performed by incubating the samples with 55
mM iodoacetamide in 20 mM ammonium bicarbonate for 30
min at 24 °C in the dark. Gel pieces were washed two times
with 50% 10 mM ammonium bicarbonate/50% acetonitrile,
then dehydrated with 100% acetonitrile, and finally dried in a
vacuum concentrator. Protein digestion was performed over-
night at 37 °C with 12.5 ng/µL trypsin in 20 mM ammonium
bicarbonate. For protein extraction, gel pieces were incubated
first with 30% acetonitrile/3% TFA, a second time with 80%
acetonitrile/0.5% AcOH, and a third time with 100% acetoni-
trile. Supernatants were combined and desalted using RP-C18
StageTip columns.38,39Eluted peptides were analyzed by
NanoLC-MS/MS and Data Analysis. All digested peptide
mixtures were separated and analyzed by online reversed-phase
(RP) nanoscale capillary liquid chromatography (nanoLC) using
an Eksigent nanoLC-2D system (Axel Semrau, Sprockho ¨vel,
Germany) coupled to a LTQ-Orbitrap-XL mass spectrometer
(ThermoFisher, Bremen, Germany) equipped with a nanoelec-
trospray ion source (Proxeon Biosystems, Odense, Denmark).
Binding and chromatographic separation of the peptides
took place in a 15 cm fused silica emitter of 75 µm inner
diameter (Proxeon Biosystems, Odense, Denmark) in-house
packed with reversed-phase ReproSil-Pur C18-AQ 3 µm resin
(Dr. Maisch GmbH, Ammerbuch-Entringen, Germany). The
peptide mixtures were injected onto the column with a flow
of 500 nL/min for 20 min and subsequently eluted with a flow
of 200 nL/min from 1.6% to 64% acetonitrile in 0.5% acetic acid
in a 107 min gradient.
The mass spectrometer was operated in the data-dependent
mode to automatically switch between MS and MS/MS (MS2)
acquisition. Survey full scan MS spectra (from m/z 300 to 2000)
were acquired in the orbitrap with a resolution of 60 000 at
m/z 400 (after accumulation to a target value of 106charges in
the linear ion trap) using the lock mass option for internal
calibration of each spectrum.40The 10 most intense ions were
sequentially isolated for fragmentation in the linear ion trap
using collisionally induced dissociation with normalized col-
lision energy of 35% at a target value of 5000. Target ions
already selected for MS/MS were dynamically excluded for 90 s.
The resulting fragment ions were recorded in the linear ion
trap with unit resolution.
Peak lists for database searching were generated from the
raw data using MaxQuant.41Proteins were identified by
automated database searching (Mascot, Matrix Science)42
against an in-house curated version of the maize ZmProt
database (www.maizegdb.org) which contains 34 958 unique
maize protein sequences as of 02/05/2009. This database was
complemented with frequently observed contaminants like
porcine trypsin and human keratins. Carbamidomethyl-cys-
teine was used as a fixed modification, and variable modifica-
tions were oxidation of methionine and protein N-acetylation.
Mucilage Proteome of Maize Primary Roots
Journal of Proteome Research • Vol. 9, No. 6, 2010
We required full tryptic specificity (cleavage at Arg-Pro and Lys-
Pro as well as Asp-Pro was included), a maximum of two
miscleavages, and mass accuracies of 10 ppm for the parent
ion and 0.5 Da for fragment ions. The minimum peptide size
that was required was six amino acids. The maximum false
discovery rates (FDR) were set to 1% for both, peptide and
Data Search and Functional Analysis. All proteins were
functionally annotated via the MIPS FunCats (http://mips.gsf.
de/proj/thal/db/tables/tables_gen_frame.html) catalogue. Ho-
mology analyses of pea,32rapeseed,31and Arabidopsis31secre-
tory proteins with the maize mucilage proteome were per-
formed by downloading the protein data sets to a local stand
alone blast server and performing blast analyses of all data sets
against the 2848 maize proteins identified in this study (cutoff
value: E < 10-10). The presence of putative signal peptides was
predicted using the SignalP v3.0 (www.cbs.dtu.dk/services/
SignalP-3.0/) algorithm. Transmembrane helices were analyzed
by the TMHMM server v. 2.0 (www.cbs.dtu.dk/services/TM-
HMM).43Glycophosphatidylinositol (GPI) modifications were
predicted with the “big-PI Plant Predictor” algorithm (http://
Collection and Purification of Proteins from Maize
Primary Root Exudates. The goal of this study was to generate
a comprehensive reference map of proteins secreted by maize
primary root in order to better understand the composition
and function of the maize mucilage proteome. The maize
primary root mucilage proteome is defined as the complement
of all proteins that are released by root cap cells and detached
border cells into the mucilage which covers the root cap (Figure
1A). To analyze the composition of the maize mucilage pro-
teome, seedlings were germinated on moist paper for 3 days
and then transferred into a hydroponic system. Maize primary
roots continuously secrete mucilage. Therefore, between 12-24
h after transfer into the hydroponic system, mucilage was
collected from the tips of primary roots which had a length of
2-3 cm with a drawn glass Pasteur pipet three times per root.
Mucilage of ∼540 maize primary roots was combined for
analysis. Prior to protein isolation, mucilage samples were
centrifuged and only the supernatant was subjected to protein
analyses, in order to ensure that only secreted proteins were
analyzed and border cells and cellular debris were excluded
from further analyses. Protein extraction from mucilage yielded,
on average, 0.95 µg per root. To purify and size fraction the
maize primary root mucilage proteome, ∼30 µg of mucilage
proteins was separated on a 12% SDS-PAGE gel (Figure 1B).
Identification and Functional Annotation of the Maize
Primary Root Mucilage Proteome. To comprehensively iden-
tify proteins of the maize mucilage proteome, the SDS-PAGE
gel which was used to separate proteins extracted from maize
mucilage (Figure 1B) was sliced into 16 fragments. After elution
of trypically digested proteins from the SDS-PAGE gel slices,
47 007 tryptic peptides were identified by nanoLC-MS/MS
analyses. The sequences and additional features of these 47 007
peptides are summarized in Table S1. In total, 3462 distinct
maize proteins were derived from these peptide sequences by
automated database searching (Mascot, Matrix Science)42
against an in-house curated version of the maize ZmProt
database (www.maizegdb.org). We chose the identification of
at least two different peptide sequences per protein as mini-
mum requirement for further analyses. This criterion was met
by 2848 of the 3462 proteins which were subsequently studied
in more detail. Table S2 summarizes the features of these 2848
proteins including the most likely protein accessions for each
group of peptides, protein descriptions, and number of distinct
peptides per protein. Moreover, the number of unique peptides
(peptides that were unique to one protein ID), sequence
coverage, molecular weight, and amino acid length of the
leading protein in the protein group are provided. Finally,
the posterior error probability (PEP) rate of all identified
proteins (calculated according to Ka ¨ll et al.),45and the
summed peptide intensity for the identified protein group
are indicated. Protein descriptions for the 2848 proteins were
provided by maizegdb.org. For 1703 of the 2848 proteins that
were identified based on g2 peptide sequences, these peptide
sequences can be attributed to several closely related maize
proteins that share these peptide sequences. Because of the
shotgun approach, it is not possible to determine if only one
or all of these related proteins are present in the sample.46
While Table S2 provides only the accession of the most likely
protein, Table S3 summarizes all possible protein accessions
related to the 1703 groups of peptides. Subsequently, all of the
2848 proteins were assigned to functional categories via the
MIPS FunCats (http://mips.gsf.de/proj/thal/db/tables/tables_
gen_frame.html) catalogue. Approximately 41% of the 2848
proteins represented by at least two peptide fragments are of
unknown function (Figure 2, Table S2). The functionally
annotated proteins were distributed among 11 categories
(Figure 2, Table S2). Most of these proteins were related to
metabolism (24.6%), but also among others to the categories:
proteins with binding function (6.7%), disease/defense (4.8%),
protein synthesis (4.3%), signal transduction (4.2%), and protein
fate (4.1%). The remaining 614 of 3462 proteins that were
represented by only one peptide identification were not further
analyzed. Remarkably, ∼95% (583/614) of these proteins were
of unknown function (Table S4).
Mucilage Proteins Cover Many Aspects of Basal and
Secondary Metabolism. The largest proportion of the function-
ally annotated maize root cap mucilage proteins was related
to metabolism. We therefore performed a MapMan47,48analysis
to visualize the metabolic pathways represented by secreted
maize root proteins (Figure 3). Each green square represents a
distinct protein that was mapped to a defined metabolic
pathway, while gray circles indicate subpathways for which no
protein was indentified in the maize mucilage proteome.
Secretory proteins identified in this study are related to basal
Figure 1. (A) Maize primary root tip (left) covered with mucilage.
Pipette tip (right) illustrates the viscosity of mucilage. (B) Muci-
lage protein extract isolated from 2-3 cm maize primary root
and subsequently separated on a 12% SDS gel. For analysis, the
gel was sliced into 16 fragments.
Ma et al.
2970 Journal of Proteome Research • Vol. 9, No. 6, 2010
and secondary metabolism. Proteins involved in cell wall
metabolism, energy production (electron transport, H+-AT-
Pases, TCA circle, glycolysis), amino acid and lipid metabolism
are abundant in the maize mucilage proteome. In addition,
proteins involved in the secondary metabolism of terpenes,
flavonoids, phenylpropanoids, and phenolics are abundantly
represented in the mucilage proteome. Such secondary me-
tabolites could play important functions in the interaction of
maize roots with the environment.49
Comparison of the Monocot Maize and Dicot Arabidopsis,
Rapeseed, and Pea Mucilage Proteomes. In previous studies of
proteins secreted into the mucilage, only a limited number of
proteins from pea (124 distinct proteins),32Arabidopsis (52
distinct proteins),31and rapeseed (16 distinct proteins)31were
identified. Mucilage proteins were defined in all these studies
as proteins released from root cap and border cells. To
determine homologous proteins between the 2848 maize
mucilage proteins identified in the present study and pea,
Arabidopsis, and rapeseed mucilage proteins, we downloaded
these data sets from GenBank and blasted them against the
2848 proteins identified in this study from maize. Secretory
proteins were defined as homologues between maize and the
three other species if blastp analyses provided an E-value <
10-10in pairwise comparisons. While for pea all 124 secreted
proteins were still available in GenBank, only 13 of 16 rapeseed
proteins, and only 50 of 52 Arabidopsis proteins were still
deposited in GenBank. In a first comparison, we determined
the coverage of the pea, Arabidopsis, and rapeseed mucilage
proteins by homologues of the considerably larger maize data
set that comprised 2848 proteins. In total, between 85% and
Figure 2. Summary of functional classes that were attributed to the 2848 maize mucilage proteins identified in this study in percent.
Figure 3. Visualization of metabolic pathways represented by proteins identified in the maize root secretory proteome via Mapman
software. Each green square represents a protein, while gray circles indicate that no proteins matched a particular subpathway.
Mucilage Proteome of Maize Primary Roots
Journal of Proteome Research • Vol. 9, No. 6, 2010
94% of the mucilage proteins identified in the dicot species
pea (Table S5a), Arabidopsis (Table S5b), and rapeseed (Table
S5c) had at least one close homologue in the protein secretome
of the monocot maize plant (Table 1). Therefore, we compared
the overlap of the maize homologues that were identified for
the three dicot species (Figure 4). For 124 distinct pea mucilage
proteins, 309 maize homologues were identified. Similarly, for
the 50 Arabidopsis and 13 rapeseed secreted proteins, 283 and
117 maize homologues were identified, respectively (Figure 4).
In total, for 594 of the 2848 maize mucilage proteins (21%) a
homologue in at least one of the much smaller dicot data sets
was identified. Hence, this study provides 2254 novel maize
mucilage proteins that have not been previously associated with
the mucilage proteome in any other plant species. The maize
proteins for which a homologue was identified in the three
dicot species display a significant overlap between pea, Ara-
bidopsis, and rapeseed (Figure 4). Remarkably, homologues for
12 maize mucilage proteins were identified in all three dicot
species (Figure 4) suggesting a conserved role of these proteins
in all species studies thus far. For seven of these 12 proteins,
extracellular secretion is predicted by TargetP (Table 2).
Structural Analysis of Maize Secretory Proteins. Extracel-
lular secreted proteins are typically characterized by a cleavable
N-terminal signal peptide which targets them to the ER, the
first organelle involved in the protein translocation from the
cytoplasm to the extracellular environment.50This signal
peptide is characterized by a positively charged region at the
N-terminus, a central hydrophobic region followed by a polar
region containing the cleavage site. Secretory pathway signal
peptides among the 2848 secreted maize proteins were pre-
dicted with TargetP v. 1.1.51In total, 464 of the 2848 maize
mucilage proteins (16%) were predicted to contain a secretory
pathway signal peptide (Table S2). Another feature of extra-
cellular proteins is the absence of transmembrane domains.
Secretory proteins are translocated from the ER to the Golgi
complex where they are packaged into vesicles. These vesicles
then move to the plasma membrane where they are released
to the extracellular matrix. Transmembrane proteins would in
this last step not be released to the extracellular environment
but instead be integrated into the plasma membrane. Trans-
membrane helices for the 2848 proteins were predicted via the
TMHMM program.43Surprisingly, TMHMM predicted for 383
of the 2848 proteins having at least one transmembrane helix,
which would exclude them from being a secretory protein.
However, 247 of the 383 putative transmembrane proteins were
also predicted to contain a secretory N-terminal signal peptide
by TargetP. Hence, in these instances, the predicted N-terminal
transmembrane domain was most likely an N-terminal signal
peptide. In addition, 59 of the remaining 136 putative trans-
membrane proteins contained only one transmembrane helix.
In many instances, this transmembrane helix is located at the
N-terminus of the proteins and might thus also be a signal
peptide. Finally, for 43 proteins which were in part also
predicted to contain transmembrane helices, the “big-PI Plant
Predictor” algorithm44suggested glycosylphosphatidylinositol
(GPI) anchors. GPI anchors are glycolipids that can be post-
translationally attached to the C-terminus of a protein. GPI-
linked proteins are secreted via the endoplasmic reticulum (ER)
and Golgi apparatus to the extracellular space where they
remain attached to the exterior leaflet of the cell membrane.
Secretion of Maize Root Extracellular Proteins. As young
roots penetrate the soil environment, exudates produced by
the root cap and border cells are continuously released into
the rhizosphere.52Plant roots exude a large variety of com-
pounds into the rhizosphere including mono and polysaccha-
rides, amino acids, secondary metabolites, and proteins.1
Despite its biological relevance, the composition and function
of the root mucilage proteome remains largely unknown. In
Table 1. Coverage of the Published Plant Mucilage Proteome
Data Sets by Homologues of the Maize Mucilage Proteome
Identified in This Study
aNumbers in % indicate for how many proteins of the pea,32
Arabidopsis,31and rapeseed31mucilage proteome homologues have been
found in the maize mucilage proteome. Absolute number of proteins is
indicated in parentheses.bSequences of only 50 of 52 proteins initially
identified in the Arabidopsis mucilage proteome31are still available at
rapeseed mucilage proteome31are still available at NCBI.
cSequences of only 13 of 16 proteins initially identified in the
Figure 4. Overlap of unique maize proteins identified in this study
that display homology with secreted proteins identified in rape-
seed, pea, and Arabidopsis. In total, 117, 283, and 309 maize
proteins displayed homology to rapeseed (Basu et al.),31Arabi-
dopsis (Basu et al.),31and pea (Wen et al.)32mucilage proteome,
respectively. Remarkably, for 12 maize proteins, a homologue
was found in all three dicot mucilage proteomes (see Table 2).
Table 2. Maize Proteins for Which a Homologue Was
Identified in All Three Dicot Mucilage Proteome Studied thus
Far (Pea,aArabidopsis,band Rapeseedb)
protein IDprotein descriptions
Superoxide dismutase [Cu-Zn]
Seed Chitinase A
Cysteine proteinase 1
40S ribosomal protein S27a
Superoxide dismutase [Cu-Zn] 4AP
Superoxide dismutase [Cu-Zn] 2
Basic endochitinase A
Putative uncharacterized protein
Ubiquitin fusion protein
aWen et al.32 bBasu et al.31 cPredicted with TargetP v. 1.1 (Emanuelsson
Ma et al.
2972Journal of Proteome Research • Vol. 9, No. 6, 2010
previous studies, protein content in mucilage of axenically
grown maize has been estimated in the range of 1-6%.20–23
Maize root cap cells are hypersecretory cells.53In particular,
the lateral root cap cells of maize are rich in cisternae,54which
can exudate large amounts of mucilage.55Moreover, detached
border cells also remain metabolically active for more than a
week after detachment into the soil52and continue to secrete
mucilage for days after detachment.56In the present study,
proteins were collected under axenic conditions, in order to
avoid the induction of secreted proteins by microorganisms
or environmental stress. Hence, the present data set comprises
proteins constitutively secreted into the mucilage by living root
cap and border cells.57Most border cell specific proteins are
exported into the external environment as soon as they are
synthesized.58In mucilage secreted by pea root tips, the
amount of protein remained stable at approximately 1.3 µg of
soluble protein per pea root tip once a full set of border cells
was present.32This is similar to the amount of 0.95 µg of protein
in mucilage of maize root tips determined in this study.
Identification of the Maize Primary Root Mucilage Pro-
teome. In the present study, a comprehensive analysis of
proteins secreted into the mucilage of maize primary root tips
identified 2848 unique proteins with >2 different peptide
fragments per protein group by using shotgun sequencing via
nanoLC-MS/MS. This data set significantly exceeds previous
mucilage protein data sets from rapeseed (16 proteins) based
on 2-DE separation of proteins31and from Arabidopsis (52
For about 59% of the maize mucilage proteins, a function was
provided by the protein descriptions of the maizegdb.org
database. Among the annotated proteins, the largest proportion
of proteins (24.6%) was related to metabolism. Remarkably, for
85-94% of the mucilage proteins identified from pea,32Ara-
bidopsis,31and rapeseed,31a maize homologue was identified
(Table 1). This suggests a considerable conservation of the
mucilage proteomes between evolutionary distant monocot
and dicot species. On the other hand, due to the larger size of
the data set identified in this study, it is likely that on average
more than one maize homologue can be found for mucilage
proteins of the relatively small data sets of the previously
published mucilage proteomes of dicot model plants. The little
overlap of mucilage proteins that was previously found between
Arabidopsis, rapeseed,31and pea32might, therefore, not reflect
the divergence of the mucilage proteomes between dicot
species32but might rather be attributed to the relatively small
set of proteins identified in these studies.
Carbohydrate Metabolizing and Cell Wall Proteins in the
Maize Mucilage Proteomes. Plant cell walls are mainly com-
posed of polysaccharides such as cellulose, hemicelluloses, and
pectins which contain variable amounts of structural proteins.59
It has been previously demonstrated in pea that the root
mucilage proteome displays certain overlap with the cell wall
proteome of maize60but that it is not strictly synonymous. In
line with this observation, numerous proteins involved in cell-
wall metabolism have been identified (Supporting Information
Table S6). Remarkably, ∼46% of the cell wall proteins identified
in this study have not been previously associated with functions
in the root mucilage proteome.31,32For instance, several
expansins have been identified in the maize root mucilage
proteome for which no homologues were identified in dicot
root mucilage proteome studies.31,32Expansins are known to
have cell-wall loosening activity during cell expansion.61More-
over, RTH3 which encodes a monocot-specific, COBRA-like
cell-wall protein62has also been associated in this study with
the mucilage proteome of maize. Furthermore, several proline-
rich cell wall proteins and ?-glucosidases have been identified
in this study for which no homologues were identified in the
dicot mucilage proteome.31,32Many cell wall proteins are
modified post-translationally. Arabinogalactan proteins (AGPs)
are hydroxyproline-rich glycoproteins that are highly glycosy-
lated and abundant in the plant cell walls and plasma
membranes.63–66AGPs form an abundant class of plant cell
surface proteoglycans with high water-holding capacity, and
inherent stickiness.67AGPs were identified in the mucilage layer
by immunofluorescence localization.68It is hypothesized that
classical AGPs act as pectin plasticizers.69Fasciclin-like ara-
binogalactan proteins (FLAs) are a subclass of AGPs that have,
in addition to predicted AGP-like glycosylated regions, putative
cell adhesion domains known as fasciclin domains.66Five FLAs
were identified in the secreted proteome of maize primary
Reactive Oxygen Species Scavenging Enzymes in the
Maize Mucilage Proteome. Reactive oxygen species (ROS) are
continuously produced under normal conditions as toxic
byproduct of aerobic metabolism, but also act as signaling
molecules in plants.70Major ROS-scavenging mechanisms of
plants include superoxide dismutase (SOD), ascorbate peroxi-
dase (APX), and catalase (CAT). Additional ROS-scavageing
enzymes are glutathione reductase (GR), gluthatione peroxidase
(GPX), dehydroascorbate reductase, monodehydroascorbate
reductase (MDAR), and other peroxidases.70In total, 43 iso-
forms of these enzymes except dehydroascorbate reductase
were found in the maize root mucilage proteome (Table S7).
While SOD, APX, GR, MDAR, and other peroxidases were also
found in dicot mucilage proteome,31,32GPX and CAT were
exclusively identified in the present study of the maize mucilage
proteome. SOD acts as the first line of defense converting
superoxide into hydrogen peroxide.70Not surprisingly, SOD was
therefore identified in all four mucilage proteome data sets
identified so far from pea,32rapeseed,31Arabidopsis,31and
maize (Table 2).
Maize Mucilage Proteins Related to Stress Response. The
constitutively secreted proteome of maize primary roots con-
tains a considerable number of proteins responding to abiotic
(heat, salt, and drought), and biotic stress (Table S2). Proteins
related to abiotic stress include a number of heat shock proteins
and chaperonins, temperature-sensitive H2B, salt-inducible
and water-stress proteins, and early proteins responsive to
dehydration. Pathogen responsive proteins include the previ-
ously mentioned peroxidases, but also several 14-3-3 proteins,
glucanases, and chitinases. Chitinases play an important role
in the biocontrol of plant pathogenic fungi,71,72and belong to
the core enzymes of plant mucilage proteomes and were
identified in all four plant mucilage proteomes dissected thus
far (Table 2).
Plant hormones play important regulatory roles in all aspects
of development. Some plant stress hormones are responsible
for activating cellular and environmental responses mediated
by proteins regulated by these hormones including ethylene-
responsive, jasmonate-induced, brassinosteroid biosynthesis-
like, or abscisic stress ripening proteins identified in this study.
Proteins Related to Nutrient Acquisition. Apart from the
function of plant roots as organs for nutrient uptake, roots are
also able to release a wide range of compounds into the root
environment, including proteins.4,29Several proteins identified
in the maize primary root mucilage proteome make nutrients
Mucilage Proteome of Maize Primary Roots
Journal of Proteome Research • Vol. 9, No. 6, 2010
available for plant uptake such as legumin-like and germin-
like proteins, and acid phosphatase. Phosphorus (P) is one of
the major limiting factors for plant growth in many soils. Beside
exudation of carboxylates to mobilize sparingly soluble P
sources in the soil, secretion of acid phosphatase may con-
tribute to P acquisition by hydrolysis of organic P esters in the
rhizosphere, which can compose up to 30-80% of the total
soil P.29Iron (Fe) is another major limiting factor for plant
growth in terrestrial ecosystems. About one-third of the world’s
soils are Fe-deficient for plant growth because the Fe is poorly
soluble.28A group of proteins contributes to iron binding,
including ferredoxin-dependent glutamate synthase, aconitate
hydratase, frataxin, hemoglobin-like protein, aldehyde oxidase,
cytochrome, ferritin, heme-binding protein, and ZmNAS1
protein. Some proteins, such as zinc ion binding protein, NO3-
high affinity nitrate transporter, potassium ion binding, and
molybdenum cofactor biosynthesis protein, can be found in
maize extracellular proteome to contribute to plant nutrition.
Cytosolic Marker Proteins in the Mucilage Proteome.
Among the mucilage proteins of maize, a large array of cytosolic
markers including, for example, 78 distinct ribosomal proteins,
36 translation initiation and elongation factors, 16 histones, and
35 enzymes of glycolysis covering all 10 enzymatic reactions
of glycolysis (Table S8) have been identified. In the previously
studied dicot mucilage proteome data sets, only four of the 10
enzymatic steps of glycolysis were represented.31,32If these
cytosolic proteins have a function in the maize mucilage or
represent leakage that occurs during cell separation from the
root cap remains to be elucidated in the future.
Root exudation plays an important role in plant growth, by
helping plants to create a favorable rhizospheric environment.
Root exudates, by controlling rhizosphere community structure,
significantly influence plant health, development, and produc-
tivity. In the present study, 2848 distinct mucilage proteins of
primary root tips were identified. This comprehensive data set
provides novel insights into the composition and function of
plant mucilage proteomes and helps to better understand the
biological processes proceeding in the root-soil interface.
Acknowledgment. We thank the National Natural
Science Foundation of China (No: 30671237), the State Key
Basic Research and Development Plan of China (No. 2007CB-
109302) and the Innovative Group Grant of National Natural
Science Foundation of China (No: 30821003) for financial
support to C.L. This project was supported in part by grant
HO2249/8 of the Deutsche Forschungsgemeinschaft (DFG) to
F.H. The Proteome Centrum Tu ¨bingen is supported by the
Ministerium fu ¨r Wissenschaft und Kunst, Landesregierung
Baden-Wu ¨rttemberg. We would like to thank Marc Lohse (Max
Planck Institutefor Molecular
Germany) for providing maize mapping files for the Mapman
Supporting Information Available: Table S1, se-
quences and description of all peptides identified in this study.
Table S2, protein groups represented by at least two distinct
peptides. Table S3, multiple protein IDs identified by a unique
set of peptide fragments. Table S4, protein groups that were
represented by only one peptide identification and were not
further analyzed. Table S5a, pea mucilage proteins blasted
against the 2848 maize secretory proteins. Table S5b, Arabi-
dopsis mucilage proteins blasted against the 2848 maize
secretory proteins. Table S5c, unique rapeseed mucilage pro-
teins blasted against the 2848 maize secretory proteins. Table
S6, proteins involved in cell wall metabolism identified in the
maize root mucilage proteome. Table S7, enzymes involved in
the scavenging of reactive oxygen species identified in the
maize root mucilage proteome. Table S8, enzymes of all 10
steps of glycolysis were identified in the maize root mucilage
proteome. This material is available free of charge via the
Internet at http://pubs.acs.org.
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