Genetic control over silica deposition in wheat awns.

Abstract

Awns are long, stiff filamentous extensions of glumes in many grasses. In wheat, awns contribute up to 40% of the grain's photosynthetic assimilates, and assist in seed dispersal. Awns accumulate silica in epidermal hairs and papillae, and silica has been positively associated with yield and environmental stress tolerance. Here, the awns of a set of domesticated wheat genotypes and their direct progenitor, Triticum turgidum ssp. dicoccoides were characterized. In addition, the silica concentration in awns was genetically dissected in a tetraploid wheat population of recombinant inbred lines (RILs) derived from a cross between durum wheat (cv. Langdon) and wild emmer (accession G18-16). Scanning electron micrographs revealed a continuous silica layer under the cuticle. Extended silicification was identified in the epidermis cell wall and in sclerenchyma cells near the vascular bundles, but not in the stomata, suggesting that an active process directs the soluble silica away from the water evaporation stream. The number of silicified cells was linearly correlated to silica concentration in dry weight (DW), suggesting cellular control over silicification. Domesticated wheat awns contained up to 19% silica per DW, as compared with 7% in the wild accessions, suggesting selection pressure associated with the domestication process. Six quantitative trait loci (QTLs) for silica were identified in the awns, with a LOD score of 3.7-6.3, three of which overlapped genomic regions that contribute to high grain protein. Localization of silica in the awns and identification of QTLs help illuminate mechanisms associated with silica metabolism in wheat.

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Physiologia Plantarum 140: 10–20. 2010
Copyright © Physiologia Plantarum 2010, ISSN 0031-9317
Genetic control over silica deposition in wheat awns
Zvi Pelega,†, Yehoshua Sarangaa, Tzion Fahimab, Asaph Aharonicand Rivka Elbauma,∗
aThe Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel
bDepartment of Evolutionary and Environmental Biology and the Institute of Evolution, University of Haifa, Mount Carmel, Haifa 31905, Israel
cPlant Sciences Department, The Weizmann Institute of Science, Rehovot 76100, Israel
Correspondence
*Corresponding author,
e-mail: elbaum@agri.huji.ac.il
Received 10 February 2010;
revised 19 April 2010
doi:10.1111/j.1399-3054.2010.01376.x
Awns are long, stiff filamentous extensions of glumes in many grasses. In
wheat, awns contribute up to 40% of the grain’s photosynthetic assimilates,
and assist in seed dispersal. Awns accumulate silica in epidermal hairs
and papillae, and silica has been positively associated with yield and
environmental stress tolerance. Here, the awns of a set of domesticated wheat
genotypes and their direct progenitor, Triticum turgidum ssp. dicoccoides
were characterized. In addition, the silica concentration in awns was
genetically dissected in a tetraploid wheat population of recombinant inbred
lines (RILs) derived from a cross between durum wheat (cv. Langdon) and
wild emmer (accession G18-16). Scanning electron micrographs revealed a
continuous silica layer under the cuticle. Extended silicification was identified
intheepidermiscellwallandinsclerenchymacellsnearthevascularbundles,
but not in the stomata, suggesting that an active process directs the soluble
silica away from the water evaporation stream. The number of silicified cells
was linearly correlated to silica concentration in dry weight (DW), suggesting
cellular control over silicification. Domesticated wheat awns contained up to
19% silica per DW, as compared with 7% in the wild accessions, suggesting
selection pressure associated with the domestication process. Six quantitative
trait loci (QTLs) for silica were identified in the awns, with a LOD score of
3.7–6.3, three of which overlapped genomic regions that contribute to high
grain protein. Localization of silica in the awns and identification of QTLs
help illuminate mechanisms associated with silica metabolism in wheat.
Introduction
Silicon dioxide [SiO2] and other silicate minerals are the
major components of most soils, and therefore, silicon
is ubiquitous in the plant environment. While silicon
is not considered as an essential element for higher
plants, many studies have demonstrated the beneficial
roll of silica in increasing plant resistance to biotic
and abiotic stresses, including infections, pests, lodging,
Abbreviations – BSE, back-scattered electron; DArT, diversity array technology; DW, dry weight; EDS, energy-dispersive
spectroscopy; LOD, logarithmic of the odds; MIM, multiple interval mapping; PCA, principle component analysis; PEV,
proportion of explained variability; QTL, quantitative trait loci; RIL, recombinant inbred line; SEM, scanning electron
microscope; SiO2/DW, silica per awn dry weight; SiO2/SUR, silica per awn surface; SUR, surface.
†Present address: Department of Plant Sciences, University of California, Davis, Davis, CA 95616, USA
drought, nutrient imbalance, and in improving yields
(Epstein 1999, Liang et al. 2007, Ma et al. 2006, Savant
et al. 1999, Yoshida 1981). Thus, better understanding
of the biochemical and biophysical mechanisms associ-
ated with SiO2accumulation in plants could shed light
on its physiological roles.
Plants are exposed to silicon in the soil solution, in the
form of monosilicic acid [Si(OH)4] (Jones and Handreck
1967, Knight and Kinrade 2001). Grasses, in contrast to
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Physiol. Plant. 140, 2010

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most dicotyledonous plants, tend to actively concentrate
silicic acid in their sap (Mitani and Ma 2005). The acid
is transported with the water stream to the epidermis
where it polymerizes to hydrated amorphous silicon
oxide (silica, SiO2·nH2O, also known as the mineral
opal). Three silicon transporters (LSi1, LSi2 and LSi6),
associated with silica uptake and distribution, have been
identified in rice (Oryza sativa L.), a typical silicon
accumulator(Ma et al. 2006,2007a,Yamaji et al. 2008).
Homologous silicon transporters have been indentified
in maize and barley (Chiba et al. 2009, Mitani et al.
2008, 2009).
Awns (Fig. 1A), which are long, stiff filamentous
extensions of glumes in many grasses, accumulate high
concentrations of silica (Grundbacher 1963). Awns play
a major role in plant propagation by providing means
for ripe seeds to reach an appropriate germination
site (Elbaum et al. 2007, Zohary 1959). About one-
third of the awn’s volume consists of chlorenchymatous
tissue, which is encapsulated by sclerenchyma (Fig. 1B).
The chlorenchyma contributes 10–40% of the CO2
assimilates in the grain (Tambussi et al. 2007). Silica
is deposited in awns under the cuticle and in silica cells,
papillae and hooked hairs embedded in the epidermis
(Grundbacher 1963).
Wild emmer wheat, Triticum turgidum ssp. dicoc-
coides (K¨ orn.) Thell., is the allo-tetraploid (2n = 4x =
28; genome BBAA) progenitor of both domesticated
tetraploid durum wheat [T. turgidum ssp. durum (Desf.)
MacKey]andhexaploid(2n = 6x = 42;BBAADD)bread
wheat (Triticum aestivum L.) (Feldman 2001). Wild
emmer is distributed throughout the Near Eastern Fer-
tile Crescent, across a variety of ecological conditions,
from hot and dry (with as little as 230 mm annual rain-
fall) to cool and humid (with over 1300 mm annual
rainfall) habitats (Harlan and Zohary 1966). Hence,
the wild emmer gene pool offers a rich allelic reper-
toire for the improvement of numerous economically
important traits, such as protein and mineral nutri-
ent concentrations in the grain, disease resistance and
drought tolerance (Feldman and Sears 1981, Nevo et al.
2002, Peleg et al. 2009a, 2009b). The genetic bottle-
necks associated with plant domestication (i.e. founder
effect; Ladizinsky 1998) and subsequent selection in
man-madeagro-ecosystems(i.e.evolutionunderdomes-
tication)haveresultedinmanygeneticandphysiological
differences between the domesticated wheat and its wild
progenitors. Here, the variation of silica in the awns of
domesticated wheat vs its wild progenitor is examined.
Such a comparison offers an indispensable opportunity
for understanding the physiological and genetic basis
of crop adaptations. The aim of the current study was
to (1) test the hypothesis that the amount of silica in
awns differs between wild and domesticated wheat,
(2) identify the major location of silicification in wheat
awns and (3) genetically dissect the silica concentration
in the awns using a mapping population.
Materials and methods
Plant material
A collection of 48 wheat genotypes, including 16
domesticated breadand durum wheat (8 T. aestivum and
8 T. turgidum ssp. durum) cultivars and 32 wild emmer
wheat (T. turgidum ssp. dicoccoides) accessions, were
characterized (Table S1). The wild emmer accessions
A BC

Fig. 1. (A) A mature spike of wild emmer wheat with one dispersal unit disconnecting from the rachis. The long awns attached to the grain glumes
are marked by an arrow. (B) A SEM image of a cross-section of an awn from emmer wheat. The thick sclerenchyma is indicated by an arrow, on top
of which silicified papillae and hairs grow, and the chlorenchyma is marked by triangles. One stoma can be identified at the epidermis adjacent to this
tissue (encircled). (C) The surface of a wild awn. The density and dimensions of the silicified papillae and hairs are similar in the wild and domesticated
genotypes.
Physiol. Plant. 140, 2010
11

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used in the current study represent a wide range of
ecogeographical conditions across the Near Eastern
Fertile Crescent (Israel, Jordan, Syria, Lebanon, Turkey,
Iran and Iraq). A population of 152 F7 recombinant
inbred lines (RILs) that was developed by the single-seed
descent approach from a cross between durum wheat
(cv. Langdon, hereafter referred to as LDN) and wild
emmer wheat (accession G18-16) (Peleg et al. 2008)
was used for the quantitative trait loci (QTL) mapping of
silica concentration in wheat awns.
A field trial was carried out in the experimentalfarm of
The Hebrew University of Jerusalem in Rehovot, Israel
(34◦47?N, 31◦54?E; 54 m above sea level) during the
winter of 2006–2007. The soil at this location is brown-
red degrading sandy loam (Rhodoxeralf) composed of
76% sand, 8% silt and 16% clay. Seeds were disinfected
(3.6% sodium hypochloric acid for 10 min) and placed
for vernalization on a moist germination paper for
3 weeks in a cold room (4◦C) in the dark. Seedlings
were then transplanted into an insect-proof screen-
house protected by a polyethylene top. A randomized
block design, with five or six replicates (for RILs and
wheat accessions, respectively), was employed. Each
plot consisted of five plants planted 8 cm apart in a
40-cm-long single row. The two plants at the edges
of each plot served as borders, and the other three
were harvested. Water was applied during the winter
months (December–April) to mimic the natural pattern
of rainfall in the eastern Mediterranean region. The field
was treated with fungicides and pesticides to avoid the
development of fungal pathogens or insect pests, and
weeded manually once a week.
Phenotypic characterization
Silica measurement
Awns from mature, dry spikes were cut at the base. The
length and the base diameter of five awns from each line
were measured, and their collective dry weight (DW)
was taken. These data were used to estimate the surface
area (SUR) per DW, assuming a cone shape. To evaluate
the silica concentration of the awns, about 1 g of dry
awns was oven dried (80◦C for 48 h), weighed, and then
ashed at 600◦C for 24 h. The ash was washed twice
with 1 M HCl and twice with double-distilled water to
remove soluble minerals. The remaining silica deposit
was dried, and weighed, and the deposit per unit of DW
(SiO2/DW) was calculated.
Scanning electron microscopy
Samples of mature and dry awns were prepared via three
methods. To examine the surface of the awns, pieces of
about 2 mm in length were cut from the base, and the
samples were mounted as is on an aluminum stub using
double-sided carbon tape. To study the awns in cross-
section, thin 50-μm sections were obtained as described
by Elbaum et al. (2007). Lastly, smooth cross-sections
of 11 genotypes (dic119, dic140, 1082, G18-16, J29,
LDN, Kofa, 580D, Svevo, Beit Lechem and Sariganak98;
Table S1) were imaged using back-scattered electrons
(BSEs) to assess the silica distribution. For this, pieces of
about 1 cm cut from the base of awns were placed in a
70% ethanol solution overnight. The samples were dried
by washing them twice for 10 min each in 90% then
96% ethanol, twice for 20 min each in 100% ethanol
andtwicefor10 mineachin100%propyleneoxide.The
samples were immersed overnight in 50% (v/v) propy-
lene oxide in Epon (SPI-Pon™ 812 Epoxy Embedding
Kit, SPI-CHEM, hard mixture), after which the propy-
lene oxide was evaporated for 2 h at room temperature,
and the samples were moved to pure Epon solution for
overnight incubation. The Epon solution was replaced
twice, after 1 h incubation each time, and then the sam-
ples were placed in fresh Epon for final curing at 60◦C
for 3 days. The blocks were sectioned transversely and
the cross-sections of the awns were cut by a microtome
(Reichert-Leica Ultracut E) equipped with a glass knife.
ImageswerescannedinaPhilipsXL-30environmental
scanning electron microscope (SEM) in low-vacuum
mode, using the BSE detector, and the energy-dispersive
spectrometer (EDS). Image processing was performed
with the IMAGEJ program (National Institutes of Health;
http://rsb.info.nih.gov/ij).
Statistical analysis
The JMP®ver.7.0 statistical package (SAS Institute, Cary,
NC) was used for all statistical analyses. One-way ANOVA
was used to test the differences between wild and
domesticated wheat. All phenotypic variables recorded
in the RIL population were tested for normal distribution.
A factorial model was employed for the analysis of
variance, with RILs and blocks as random effects
and trial as fixed effect. The associations among the
six traits were studied using correlation analyses and
principal component analysis (PCA). PCA was based
on a correlation matrix and was presented as biplot
ordinations of RILs (PC scores). Two components were
extracted using Eigenvalues >1 to ensure meaningful
implementation of the data by each factor.
QTL analysis
A genetic linkage map of 2317 cM was previously
developed from the mapping population of 152 RILs
12
Physiol. Plant. 140, 2010

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based on 197 microsatellites and 493 DArT (Diversity
Array Technology) markers (Peleg et al. 2008). DArT
markers presented in the above map by clone ID
numbers were renamed with the prefix ‘wPt’, ‘rPt’ or ‘tPt’
(corresponding to wheat, rye or Triticale, respectively)
followed by number. A skeleton map comprised
307 markers scattered along the 14 chromosomes
of tetraploid wheat (one marker per 7.5 cM) was
used for QTL mapping. QTL analysis was performed
with the MultiQTL package (http://www.multiqtl.com)
using the general interval mapping for an RIL-selfing
population as described by Peleg et al. (2009a). QTL
detection was carried out with a structured multistep
scheme embedded within the software (Korol et al.
2001). First, the entire genome was screened for
geneticlinkage,usingsingle-traitanalysis.Next,multiple
intervalmapping(MIM) was applied,whichincorporates
interfering effects ofother
chromosome(s) into the model to reduce the residual
variation (Kao et al. 1999). MIM was applied when more
than one QTL was detected per trait. The hypotheses
that a single locus or two linked loci on the considered
chromosome have an effect on one or two quantitative
traits were first tested by running 5000 permutation tests
(Churchill and Doerge 1994). The hypothesis that one
locus on the chromosome has an effect on a given
trait (H1) was compared with the null hypothesis (H0)
of no effect of the chromosome on that trait. Once
the genetic model was chosen, 5000 bootstrap samples
were run to estimate the standard deviation of the main
parameters: locus effect, its chromosomal position, its
LOD score, and the proportion of explained variability
(PEV). Finally, to evaluate the genome-wide significance
of estimates obtained on a chromosome x trait basis, an
approach based on controlling the false discovery rate
was used to correct for multiple comparisons (Benjamini
and Hochberg 1995). The effect of epistatic interaction
wasexaminedforeachtraitbycomparingH0(ε = 0),i.e.
additive effects of the QTL, and H1(ε ?= 0), i.e. assuming
epistasis (Ronin et al. 1999).
Correspondence between QTLs of different traits
was determined using the hypergeometric probability
QTLsona separate
function (Larsen and Marx 1985) according to Paterson
et al. (1995):
?l
P =
m
??n−l
s
s−m
?n
?
?
, (1)
where n is the comparable number of intervals; m is
the number of ‘matches’ (QTLs of two traits with >50%
overlap of their confidence intervals) declared between
QTLs; l is the total number of QTLs found in the larger
sample and s is the number of QTLs found in the smaller
sample.
Results
Comparison between awns of wild and
domesticated wheat
Awnswerecollectedfrommaturespikesofdomesticated
and wild wheat genotypes that were grown together
under similar environmental conditions.On average, the
domesticated genotypes had significantly lighter weight,
shorter and thinner awns (Table 1, Table S1). The wild
genotypes had, on average, 2.5 times less silica per awn
dry weight (SiO2/DW) than the domesticated cultivars.
This result was expected because silica is known to be
deposited on the surface of the awns, and the wild awns
are larger than domesticated awns (having smaller sur-
faceperDW).Tocorrectforthesmallersurface,thesilica
per awn surface (SiO2/SUR) was calculated (assuming
a cone shape), and even then, the wild genotypes had
about half the silica found in domesticated varieties. Fur-
ther analysis revealed that the silica per awn (SiO2/awn)
was higher in the wild lines than in the domesticated
ones, but the difference was not significant (P = 0.057;
Table 1). An interesting finding was that the variation in
awn length, as expressed by the standard deviation, was
much smaller in the wild awns than in the domesticated
ones.
The PCA of 48 wild and domesticated wheat
genotypes extracted two major principal components
(Eigenvalues >1) that collectively accounted for 91.0%
of the variation. Principal component 1 (PC1, x-axis;
Table 1. Comparison of awn length, thickness and dry weight, and concentration of SiO2/DW,SiO2/awnand SiO2/SUR inwild emmer vs domesticated
wheat genotypes. Averages and standard deviations of 32 wild and 16 domesticated lines were calculated. The probability of the groups being
identical was tested by Student’s t-test.aCalculated based on SiO2/DW and DW/awn.bCalculated from the geometrical dimensions of five awns,
assuming a cone shape.
nAwn length (cm)Awn thickness (mm) DW/awn (mg)SiO2/DW (mg g−1)SiO2/awna(mg) SiO2/SURb(μg mm−2)
Wild emmer
Domesticated
t ratio
Prob >|t|
32
16
17.8 ± 1.8
10.4 ± 3.9
9.73
<0.001
0.80 ± 0.15
0.58 ± 0.09
4.91
<0.001
25.5 ± 10.37
7.9 ± 4.7
7.13
<0.001
49 ± 10
119 ± 39
9.93
<0.001
1.2 ± 0.4
0.9 ± 0.4
1.96
0.057
5 ± 1
9 ± 3
6.91
<0.001
Physiol. Plant. 140, 2010
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-5
-4
-3
-2
-1
0
1
2
3
4
5
-5-4-3-2 -1012345
SiO2/SUR
Awn thickness
Awn DW
SiO2/awn
Awn length
SiO2/DW
PC1 (65.5%)
PC2 (25.5%)
Fig. 2. Principal component analysis (based on correlation matrix) of
continuous plant traits. SiO2/DW, SiO2/SUR, silica per awn (SiO2/awn),
awn DW, awn thickness and awn length recorded from 32 wild emmer
(circle) and 16 domesticated wheat (triangle) genotypes. Biplot vectors
are trait factor loadings for PC1 and PC2.
Fig. 2) explained 65.5% of the variation among
genotypes and was loaded positively with DW per awn,
awn thickness and awn length, and negatively with
SiO2/DW and SiO2/SUR. PC2 (y-axis; Fig. 2) explained
25.5% of the genotype variation, and was positively
loaded with SiO2/DW, SiO2/SUR and SiO2/awn. The
PCA showed a strong association between DW per
awn and awn thickness, as well as between SiO2/DW
and SiO2/SUR. This association was supported by
the high and positive correlations between these
variables (r = 0.81, P < 0.0001 and r = 0.92, P <
0.0001, respectively).
The location of silica in awn cross-sections
To determine the location of the silica deposition,
selected awns were examined by SEM. We selected
11 lines that represent a large variation in the amount
of silica expressed as SiO2/DW (Table S1). The same
lines also represent large variation in the SiO2/SUR.
The silicified features exposed on the surface of the
awns were found to be similar among genotypes with
high SiO2/DW as compared to low SiO2/DW. A typical
awn of wild emmer and awn surface is shown in
Fig. 1. Nevertheless, large variations were detected in
the cross-sections: silica was identified in the cuticle of
all of the samples measured by EDS (Fig. 3). However,
additional silica deposition was detected in awns with
high SiO2/DW at the epidermal cell wall, and further
inside, at the sclerenchymatous tissues near the vascular
bundles. SiO2/DW was found to correlate linearly
with the maximal number of silicified cell layers in
the sclerenchyma next to the main vascular bundle
(r = 0.93, P = 0.001; Fig. 4). Correlations between the
metric thickness of the silicified layer to SiO2/DW and
SiO2/SUR, and between the number of silicified cell
layerstoSiO2/SURwerealsotested,withlowerstatistical
significance. We therefore concluded that SiO2/DW
represents a good measure for silicification in awns.
The high correlation to the number of cells in the
silicified layer indicates that silicification is controlledby
cellular processes. If silicification was a result of passive
diffusionandpolymerizationthen,wewouldexpecthigh
correlationto the metric thickness of the silicified region.
QTL mapping of SiO2concentration in wheat awns
Genetic dissection of silica concentration in the awns
was conducted in an RIL population from a cross
between a domesticated durum wheat cultivar (LDN)
and a wild emmer wheat accession (G18-16). The
amount of silica in the awns and its distribution in
the cross-section were rather similar between the two
parents (Fig. 5, Table S1). The RIL mapping popula-
tion exhibited transgressive segregation for SiO2/DW
(Fig. 5B). A total of six significant QTLs were associated
with SiO2/DW, with LOD scores ranging between 3.7
and 6.3, explaining 5.9–14.5% of the variance (Table 2,
Fig. 6).HigherSiO2/DWwasconferredbythewildallele
(G18-16) at five loci (2B, 3B, 5B, 6B and 7B) and by the
domesticated allele (LDN) at one locus (3A). Two QTLs
were mapped to seemingly homoeologous positions on
chromosome group 3, raising the possibility that this
genomic region controlled silica concentration also in
the wheat ancestor. No significant two-locus epistasis
was found between any of the QTLs.
Discussion
The beneficial role of silica in plants, especially under
stress, has been known for many years (reviewed by
Jones and Handreck 1967). However, biochemical
mechanisms of its absorption, transportation and
deposition in higher plants have only recently been
revealed (Currie and Perry 2007, Epstein 2009, Mitani
et al. 2009). A better understanding of the functional
roles of SiO2in plant growth, development and defense
mechanisms against biotic and abiotic stresses may help
improve yields. In the current study, the phenotypic
variation between awns of domesticated wheat and
its direct wild progenitor was characterized, the main
silicification locations in the awns were identified, and
14
Physiol. Plant. 140, 2010

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Wild emmerDomesticated wheat

A
B
S
VB
BSE
mode
SH
SH

D
C
EDS
mode
Fig. 3. Silica distribution measured under SEM. Images taken in BSE mode (A and B), and by EDS detector tuned to measure silicon X-ray emissions
(C and D). Insert shows the complete cross-section of the wild awn, with the square marking the region of the main vascular bundle, which is shown
in the close-up. The wild plant (A and C) collected 3% SiO2/DW, which is deposited at the cuticle. The domesticated genotype (B and D) collected
18% SiO2/awn. Silicification occurs at the sclerenchyma around the vasculature (VB), up to five cell layers below the cuticle. The stoma (S) shows low
silicification, SH –silica hair. The width of the close-up images is 310 μm.
5
4
3
2
1
00
50100150200
Silicified cell layers
Fig. 4. Correlation between SiO2/DW and the number of silicified
sclerenchyma cell layers.
their silica concentration was associated to other traits
via genetic mapping.
Awn morphology and patterns of silica deposition
The awns of wild and domesticated wheat differed
in morphological appearance. Generally, awns of the
wild emmer were larger than domesticated wheat awns
and showed less variability in length (Table 1). This
may be because of their role in dispersing the seeds,
assuming that large awn dimensions are beneficial for
seed dispersal as has been suggested based on lab
experiments (Kuli´ c et al. 2009). It is known that silica
is deposited at the awn surface– in epidermal silica
cells and in surface features such as hairs and papillae
(Grundbacher 1963). Nevertheless, no differences were
detected in the silica-deposition pattern on the external
awn surface of lines containing low and high silica. We
explain this by our observation that silicification occurs
throughoutthe cuticle in the wild and domesticatedlines
(Fig. 5A), and not only in surface features. Examination
of awns with high SiO2/DW in cross-section revealed
additional deposition of silica at the epidermis cell wall,
and further inside the awn, in the sclerenchymatous
tissues near the vascular bundles (Fig. 3). The fact that
SiO2/DW was linearly correlated to the number of
silicified cell layers (Fig. 4) shows that the deposition is
a phenomenon occurring in the bulk tissue (as opposed
to a surface trait) and supports our choice to create a
genetic map of silica based on SiO2/DW. In the wild
plant, the silica has a role in seed dispersal, by creating a
ratchet that allows the dispersal unit to slide only in the
direction of the seed. Plants grown without silica carried
much smoother awns, reducing the friction force that
the awns can apply on a surface (Rafi et al. 1997). For
this purpose, a thin silica layer is enough.
In contrast to these results, a comparison between
another cereal crop, maize (Zea mays L. ssp. mays) and
Physiol. Plant. 140, 2010
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5
10
15
20
25
40 5060708090100
EDS mode
BSE mode
Wild parent
(G18-16)
Domesticated parent
(Langdon)
A
B
G18-16 LDN
Number of RILs
SiO2/DW (mg g-1)
Fig. 5. (A) Silica distribution in a cross-section of the awn of the wild
(G18-16) and domesticated (Langdon) parents of the RIL population.
The silica is found mainly at the cuticle in both varieties. (B) Phenotypic
distribution of SiO2/DW in 152 RILs. Data are means of three replicated
plots. Arrows indicate the values of the parental lines Langdon (LDN)
and G18-16.
its wild progenitor, teosinte (Z. mays L. ssp. parviglumis),
showed differential surface silicification patterns in
the glumes (Dorweiler and Doebley 1997). The wild
subspecies deposited silica across the entire surface of
the glumes, whereas in the domesticated subspecies, the
silicification occurredmainly in distinct cells (silica short
cells). The silicification pattern was thought to affect the
stiffness of the glumes: the wild glumes are stiffer, and
thus able to protect the seed from predators, whereas
the domestic glumes are softer, allowing easy threshing
(Dorweiler and Doebley 1997). Silicification in wheat
awns may repel herbivores, thus defend the seeds.
TheaverageSiO2perawntendtobehigherinthewild
germplasm than in the domesticated wheat. SiO2/awn
was calculated by estimating the DW per awn according
toasampleoffiveawns,whichmayhaveinsertedalarge
error. Nevertheless, it may suggest that the same silica
amount is allocated to awns during their development.
This contradicts the common understanding that sees
Table 2. Biometrical parameters of QTLs affecting SiO2/DW in a
tetraploid wheat (Langdon × G18-16) RIL population.aLOD scores that
were found significant when comparing hypotheses H1(there is QTL in
the chromosome) and H0(no effect of the chromosome on the trait),
using 1000 permutations test (Churchill and Doerge 1994).bThe PEV
of the trait.cThe adaptive effect of an allele calculated as one-half
the mean difference between homozygotes with and without the allele.
dFavorable parental allelecontributing tohigher silicainawns –Langdon
(L) and G18-16 (G).
respectively.
∗∗and
∗∗∗Significance at P ≤ 0.01 or 0.001,
Chr.
Position
(cM)
Nearest
marker LODaPEVb dc
Favorable
alleled
2B
3A
3B
5B
6B
7B
101 ± 16
3 ± 12
9 ± 17
149 ± 10
91 ± 17
168 ± 20
gwm1249
gwm133
gwm493
wPt-11579
gwm771
cfa2040
4.9∗∗∗
6.3∗∗∗
4.8∗∗∗
5.6∗∗∗
4.3∗∗
3.7∗∗
0.087
0.109 −118 ± 36
0.083 105 ± 22
0.145139 ± 28
0.069
0.059
108 ± 19G
L
G
G
G
G
96 ± 21
81 ± 42
silica as a surface phenomenon, and our results, which
show that silicification is under cellular control. In
addition, our attempt to map SiO2/awns genetically
resulted in no statistically significant QTLs. We thus
conclude that the apparent silica quanta allocated to
each awn does not represent a genetically controlled
measure.
Therecentlydiscoveredsilicontransportersinrice(Ma
et al.2006,2007a,Yamajiet al.2008)haveshedlighton
SiO2absorption. Evidence for a physiological function
of silica is based on relations between silica deposition
at key points and increased tolerance to various
stresses. For example, deposition at the endodermis
has been correlated with increased tolerance to high
concentrations of sodium ions (Gong et al. 2006),
cadmium ions (Nwugo and Huerta 2008) and drought
(Luxet al. 2002).Cuticleand epidermisdepositionshave
beencorrelatedtoreducedtranspirationandinterference
withfungalattack(Dallagnolet al.2009,Kimet al.2002,
Rodrigues et al. 2003, Yoshida 1981). As a solute, silica
has been associated with enhancing the plant defense
response (Cai et al. 2008, Fauteux et al. 2006, Fawe
et al. 1998).
Examination of domesticated wheat genotypes (highly
silicified) in cross-section revealed a silicification
gradient from the xylem to the cuticle (Fig. 3). This
gradient did not parallel the main water evaporation
stream, from the xylem to the stomata. It is therefore
suggested that active processes are associated with the
distribution of silica inside the awn, its transport toward
the cuticle and its localization in the sclerenchyma cells.
Silica may play a mechanical role in strengthening the
cell wall of the sclerenchyma cells, or these cells may be
aconvenientsilicasink,whichdoesnotinterferewiththe
16
Physiol. Plant. 140, 2010

Page 8

Xgwm32
Xgwm133
Xgwm720
Xgwm1159
Xgwm1121
XwPt-8104
0.0
2.6
5.2
13.9
18.2
21.6
XwPt-2756
47.7
XwPt-1092
Xgwm155
Xgwm1217
XwPt-5173
71.0
78.9
80.0
86.4
XwPt-11559
XwPt-1339
XwPt-1888
Xgwm494
Xgwm1229
XwPt-9160
99.8
104.8
107.8
112.0
117.0
118.8
SiO2/DW
Xgwm389
0.0
Xgwm493
XwPt-0267
XwPt-9410
XwPt-8828
XwPt-0250
XwPt-0013
XwPt-2766
XwPt-1612
XwPt-8592
XwPt-6973
XwPt-6945
Xgwm685
Xgwm77
Xgwm376
Xgwm1015
Xgwm1029
Xgwm1005
17.1
28.7
31.1
35.2
38.5
40.4
41.6
46.9
48.5
54.7
56.8
66.8
69.2
71.9
73.1
85.6
87.6
Xgwm853
113.0
XwPt-0384
XwPt-9577
124.0
129.8
XwPt-9049
Xgwm705
139.7
146.2
XwPt-2491
XwPt-8363
XwPt-8752
XwPt-4194
XwPt-5943
163.7
169.2
170.4
175.5
183.7
Xgwm1266
XwPt-8959
XwPt-0177
XwPt-11583
XwPt-1151
XwPt-0280
Xgwm181
193.9
203.1
207.1
209.6
211.6
213.9
217.3
SiO2/DW
2B 3A 3B
Xgwm614
XwPt-8788
0.0
2.0
XwPt-11514
XwPt-8404
XwPt-4664
XwPt-11518
XwPt-7932
XwPt-8097
XwPt-7757
XwPt-6199
Xgwm410
16.7
24.1
29.6
32.1
35.9
38.4
48.9
50.9
57.1
Xgwm374
Xgwm1177
Xgwm55
XwPt-6576
72.5
76.9
79.6
81.9
Xgwm1249
102.7
XwPt-1294
XwPt-3651
116.5
120.5
XwPt-0694
128.0
XwPt-6894
150.4
XwPt-2929
XwPt-6643
XwPt-2724
XwPt-2135
XwPt-6522
Xgwm4828
159.8
166.2
168.9
170.8
176.0
182.9
SiO2/DW
Xgwm234
0.0
Xgwm443
9.5
XrPt-6127
19.8
Xgwm1180
XwPt-1951
Xbarc128a
Xgwm1108
Xgwm371
Xgwm831
Xgwm1191
Xgwm499
34.2
38.7
43.0
49.6
54.8
59.9
62.0
66.5
Xwmc415b
77.0
XtPt-3719
XwPt-6022
XwPt-3661
Xgwm1043
Xgwm777
XwPt-0498
87.7
89.8
95.6
98.4
104.0
109.8
XwPt-1733
Xgwm408
XwPt-5896
121.3
128.2
133.2
XwPt11579
156.7
XwPt-6880
163.9
XwPt-3213
173.2
Xgwm1016b
181.5
Xgwm814
191.2
XwPt-6910
XwPt-0054
XwPt-0837
202.4
208.6
209.8
SiO2/DW
XwPt-6293
XwPt-8641
XwPt-7582
XwPt-0351
XwPt-1437
XwPt-11506
XwPt-3376
XwPt-5256
XwPt-8153
XwPt-7748
XwPt-11542
XwPt-0554
Xgwm390
Xgwm768
XwPt-5333
XwPt-7426
XwPt-11560
XwPt-7846
Xgwm518
Xbarc136
Xgwm193
Xgwm361
Xgwm771
XwPt-11556
0.0
8.1
12.2
15.9
20.3
22.5
24.8
34.1
39.3
41.2
43.2
44.7
52.8
60.6
65.0
67.5
69.5
71.5
76.9
83.4
88.2
89.0
91.1
101.0
Xgwm907
Xgwm1016a
XwPt-8554
113.6
118.4
124.0
Xgwm1076
Xgwm219
133.9
137.7
XwPt-5270
XwPt-2162
XwPt-0696
XtPt-9048
XwPt-4560
159.0
165.3
167.8
169.4
175.3
SiO2/DW
XwPt-8920
0.0
Xgwm263
9.9
Xgwm537
Xgwm400
40.7
47.0
Xgwm951
59.9
Xgwm46
XwPt-2737
XwPt-11565
74.9
80.6
83.6
Xgwm983
95.8
XwPt-11511
XwPt-2305
XwPt-3730
110.4
112.0
114.1
XwPt-8417
XwPt-8233
XwPt-7295
141.2
146.7
153.3
Xcfa2040
163.4
XwPt-5228
XwPt-11540
XwPt-0465
172.1
173.7
182.3
SiO2/DW
5B 6B 7B
Fig. 6. Likelihood intervals for QTLs conferring silica per wheat awn dry weight (SiO2/DW) in RILs of the cross between durum wheat (cv. Langdon)
and wild emmer wheat (accession G18-16).
evaporation stream. In any case, the fact that the solid
silica is deviated from the stomata,the site of highest
evapotranspiration, indicates an active process of silica
concentration. This process may involve a transporter
similartoLSi2,orachelatorlocatedinthesclerenchyma,
which binds silica to the cell wall, as suggested by Iler
(1979).
QTL analysis
There is a great deal of evidence for genetic control
over silica deposition. In barley, 20 varieties were grown
in similar conditions in two successive years. For each
variety, the grain’s silica concentration from the first
year was strongly correlated to the silica concentration
in the second year. This indicates genetic control over
the silicaconcentrationin grains(Ma et al. 2003).Inrice,
silica absorption has been correlated to variation in the
expression levels of the silicon transportersLSi1 and LSi2
(Ma et al. 2007b). Silicon deficiency has been found to
affect yieldinrice plantsthataredefectivein oneofthese
silicontransporters(TamaiandMa2008).Thismayresult
from a direct effect of silica or indirect effects reducing
rice tolerance to biotic or abiotic stresses. However, in
wheat, silica concentration in the flag leaf was not found
to be correlated with the grain yield (Merah et al. 1999).
It was also shown that in wheat, the total plant silica was
not correlated to husk silica, indicating that the silica
Physiol. Plant. 140, 2010
17

Page 9

absorption processes are under different control from
translocation processes (Hutton and Norrish 1974).
Little information is available on the genetic control
and molecular and physiological mechanisms contribut-
ing to the accumulation of SiO2 in wheat awns. QTL
analysis has proven to be a powerful tool, elucidating
the chromosomal location of genes suitable for breed-
ing programs. However, to the best of our knowledge,
there is only one report on QTL mapping of SiO2in rice
panicle, flag leaf and stem (Dai et al. 2005), and none in
awns.
In the present study, QTL analysis was employed to
dissect the genetic basis of SiO2in wheat awns, using a
tetraploid wheat (LDN × G18-16) RILs population. The
concentration of silica relative to awn DW was selected
for mapping, rather than its ratio to awn surface area,
because SiO2/DW was measured directly, while the
surface was estimated from the geometry of the awns
and was prone to higher experimental error. SiO2/DW
was associated to six QTLs (2B, 3B, 3A, 5B, 6B and
7B), with the wild allele conferring higher values in five
cases. This may seem unexpected, as the awns of the
wild emmer usually contain less silica than those of the
domesticwheat.However,comparedwiththeotherwild
emmer accessions, the SiO2/DW of the wild parent was
very high, whereas the SiO2/DW of the domestic parent
was very low among the cultivars. This may indicate a
high potential for silica accumulation in the awns of the
wild parent, which thus contributed most of the QTLs
for this trait.
Relationships between QTLs conferring SiO2 con-
centration in awns and other traits may shed light on
possible mechanisms influencing the accumulation of
SiO2in wheat awns and other cereal species. The rice
silicon transporter LSi1 was mapped to the long arm
of rice chromosome 2 (Ma et al. 2006). Based on the
synteny between rice and wheat genomes, rice chromo-
some 2 is orthologous to wheat chromosome group 6
(Rota and Sorrells 2004). Our results of a QTL for silica
accumulation on wheat chromosome 6B may indicate a
wheat LS1 transporter gene in this locus.
Recently, the same RIL population was used by us to
map QTLs associated with productivity, drought-related
morpho-physiological traits, and protein and mineral
nutrient concentrations in the grain (Peleg et al. 2009a,
2009b). In the current study, SiO2/DW QTLs were found
to co-localize with other desirable traits, including the
drought-related traits: water-use efficiency (δ13C; 5B,
6B), osmotic potential (5B, 6B), flag leaf rolling index
(2B, 6B) and culm length (6B) (Peleg et al. 2009a), grain
concentration of: Fe (3A), Zn (6B), P (5B), Mg (3A, 5B),
K (2B, 6B), S (6B) and Ca (2B) (Peleg et al. 2009b)
and resistance to powdery mildew (3A) (unpublished
data). However, these overlaps failed to show statistical
significance.
Three of the six QTLs affecting SiO2/DW (on
chromosomes2B,5B and6B)werefoundtooverlapwith
three of the ten genomic regions conferring grain protein
concentration (Peleg et al. 2009b). The likelihood of
such an association occurring by chance is P = 0.03
(Larsen and Marx 1985, Paterson et al. 1995). It is
worth noting that in all three cases, the wild allele
conferred higher values for both traits; however, no
phenotypic association was found between the two
traits (r = 0.01, P = 0.13). Peleg et al. (2008b) reported
on genetic (significant overlap between QTLs) and
phenotypic associations between grain protein, zinc
and iron concentrations, suggesting the involvement of
common physiological mechanisms in the translocation
and accumulationof these nutrients.In the currentstudy,
however, a significant genetic association is shown
between the protein concentration in grains and SiO2
concentration in awns. These results suggest a possible
common mechanism controlling the translocation of
protein (nitrogen-containing compounds) and SiO2from
vegetative parts to the spike, where they are transported
to and deposited in different parts, presumably under the
control of separate mechanisms and genes.
Conclusions
The results of the current study show that domesticated
wheat accumulate more silica per DW in their awns
than their wild progenitors. The high roughness and
toughness of the wild awns may result from their
larger dimensions, the lignification pattern and surface
silicification. Crop plants have undergone dramatic
morpho-physiological changes during domestication
and man-made selection (Ladizinsky 1998). Thus, the
higher amount of silica in domesticated wheat awns
could be an indirect result of selection for other traits.
Silica was shown to deposit at the periphery of awns,
as well as inside the sclerenchyma-thickened cell wall.
This deposition may isolate the chlorenchyma, enabling
higher photosynthetic rates with lower transpiration and
thus leading to an increase in grain yield. In order to
examine these hypotheses, silica’s roles in living tissues
need to be explored.
Acknowledgements – The authors would like to thank
E. KleinforusefuladviceandL.Nakar,Z.Hajbi,L.Zaltzman
and M. Chazav for technical assistance. This study was
supported by The Israel Science Foundation grant #1089/04
and the Kimmelman Center for Structural Biology. Z.P. is
indebted to the Israel Council for Higher Education post-
doctoral fellowships award. R.E. thanks the Charles Clore
18
Physiol. Plant. 140, 2010

Page 10

foundation and the Koshland Foundation for a scholarship.
A.A. is the incumbent of the Adolpho and Evelyn Blum
Career Development Chair of Cancer Research. The work
in A.A.’s laboratory was supported by Mrs Louise Gartner
(Dallas, TX) and Mr and Mrs Mordechai Segal (Israel).
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Table S1. Geographical origin and phenotypic charac-
terization of wild emmer and domesticated wheat lines
used in this study.
Please note: Wiley-Blackwell are not responsible for
the content or functionality of any supporting materials
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for the article.
Edited by J. K. Schjørring
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