The Inhibitor of wax 1 locus (Iw1) prevents the formation of - and OH-diketones in wheat cuticular waxes and maps to a sub-cM interval on
chromosome arm 2BS
Nikolai M. Adamski1, Maxwell S. Bush1, James Simmonds1, Adrian S. Turner1, Sarah
G. Mugford1, Alan Jones1, Kim Findlay1, Nikolai Pedentchouk2, Penny von WettsteinKnowles3, Cristobal Uauy1,4†
1 John
Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK
2
School of Environmental Sciences, University of East Anglia, Norwich Research
Park, Norwich NR4 7TJ, UK
3 Department
of Biology, University of Copenhagen, DK-2200 Copenhagen, Denmark
4
National Institute of Agricultural Botany, Huntingdon Road, Cambridge CB3 0LE,
UK
†
Corresponding author
NMA nikolai.adamski@jic.ac.uk
MSB max.bush@jic.ac.uk
JS
james.simmonds@jic.ac.uk
AST
adrian.turner-crls@jic.ac.uk
SGM sarahgmugford@hotmail.co.uk
AJ
alan.jones@jic.ac.uk
KF
kim.findlay@jic.ac.uk
NP
N.Pedentchouk@uea.ac.uk
PWK knowles@bio.ku.dk
CU
cristobal.uauy@jic.ac.uk
Cristobal Uauy
John Innes Centre,
Norwich NR4 7UH, UK
Phone: +44-(0)1603-450195
Fax: +44-(0)1603-450023
Short running title: Mapping and characterisation of wheat wax inhibitor
Keywords: Triticum aestivum, Triticum turgidum ssp. dicoccoides, Hordeum vulgare,
cuticular wax, primary alcohols, β-diketones, type III polyketide synthases, glaucous,
carbon isotope, fine mapping
Total word count:
7081
Summary:
215
Introduction:
694
Results:
3084
Discussion:
1513
Experimental Procedures:
904
Acknowledgments:
111
Short legends for Supporting Information:
152
References:
1552
Tables:
88
Figure legends:
472
Summary
Glaucousness is described as the scattering effect of visible light from wax deposited on the
cuticle of plant aerial organs. In wheat, two dominant genes lead to non-glaucous
phenotypes, Inhibitor of wax 1 (Iw1) and Iw2. The molecular mechanisms and the exact
extent (beyond visual assessment) by which these genes affect the composition and quantity
of cuticular wax is unclear. We used a genetic approach with detailed biochemical
characterisation of wax compounds to describe the Iw1 locus. Using synteny and a large
number of F2 gametes, Iw1 was fine mapped to a sub-cM genetic interval on wheat
chromosome arm 2BS which includes a single collinear gene from the corresponding
Brachypodium and rice physical maps. The major components of flag leaf and peduncle
cuticular waxes included primary alcohols, -diketones, and n-alkanes. Small amounts of
C19-C27 alkyl and methylalkylresorcinols (ARs/MARs) previously not described in wheat
waxes were identified. Using six pairs of BC2F3 near isogenic lines, we show that Iw1 inhibits
the formation of - and hydroxy--diketones in the peduncle and flag leaf blade cuticles. This
inhibitory effect is independent of genetic background or tissue and is accompanied by
minor, yet consistent increases in n-alkanes and C24 primary alcohols. No differences were
found in cuticle thickness and carbon isotope discrimination in near isogenic lines differing at
Iw1.
Introduction
Wild emmer (Triticum turgidum ssp. dicoccoides), a tetraploid grass species, is the ancestor
of both modern pasta (T. turgidum ssp. durum) and bread wheat (T. aestivum) (Dubcovsky
and Dvorak 2007). Natural emmer populations occur in the Fertile Crescent across a wide
range of habitats and environments. This adaptability partly results from genetic variability for
morphological traits, including glaucousness, the scattering effect of visible light from wax
deposited on cuticles of aerial organs. In addition to the effect on light reflectance (Feldhake
1990, Reicosky and Hanover 1978), cuticular waxes also determine many physical
properties of the plant, such as water relations (Febrero et al. 1998), canopy temperature,
and interactions with fungal pathogens and insects (Ringelmann et al. 2009). While almost all
modern wheat cultivars are glaucous, wild emmer accessions display a wide range of visual
wax phenotypes across individuals, as well as between different tissues within a single plant.
Wheat genetic studies have revealed two genes for glaucousness (W1 and W2) and two
glaucous suppressors (Iw1 and Iw2) located on chromosome arms 2BS (W1 and Iw1) and
2DS (W2 and Iw2) (Liu et al. 2007, Tsujimoto 2001, Tsunewaki and Ebana 1999). The
presence of either Iw1 and Iw2 is sufficient to inhibit W1 and/or W2 (Tsunewaki and Ebana
1999). Although the original nomenclature Iw refers to ‘Inhibitor of wax’, their true effect is
glaucousness inhibition. Also several quantitative trait loci have been identified acting
specifically on certain plant organs (Bennett et al. 2012, Börner et al. 2002, Dubcovsky et al.
1997, Kulwal et al. 2003, Mason et al. 2010, Peng et al. 2000). Despite these studies, none
of these genes have been characterized at the molecular level, and the exact extent (beyond
visual examination) by which they affect the composition and quantity of cuticular wax is also
unclear. Moreover, the visual phenotype of a plant surface is often not correlated with the
wax load. For example, cer-c36 barley internodes with 15-19 µg amorphous wax/cm2 are
glossy, whereas its leaves with the same amount of dense crystalline wax are glaucous (von
Wettstein-Knowles 1969). In Arabidopsis thaliana the increased wax load on WIN1 overexpression lines results in glossier leaves (Broun et al. 2004), and in wheat inconsistent
results between glaucousness and cuticular wax load have been described (Johnson et al.
1983).
Aliphatics (hydrocarbons, esters, aldehydes and alcohols) and triterpenoids, flavonoids and
phenolic lipids are common components of plant waxes. In addition, some genera such as
the Triticeae contain dominating amounts of aliphatic β-diketones and related compounds.
The 20-34 carbon chains characterizing these wax compounds are synthesized by the
sequential action of a fatty acid synthase complex producing C16 and C18 chains that are
further extended by an elongase complex of which there are two types. (i) Fatty acid
elongases (FAEs) consist of a condensing enzyme, plus three additional tailoring enzymes
that prepare the growing acyl chain for the next round of elongation. (ii) Type III polyketide
synthases (KCSs) lack the tailoring enzymes with the result that oxygens are introduced into
the acyl chain during elongation. While much knowledge has accrued in the past decade
about the wax biosynthetic pathways in Arabidopsis having FAE type elongases (Samuels et
al. 2008), only the results of early genetic-biochemical studies in barley have established that
an additional elongase, pkKCS, synthesizes the β-diketone aliphatics and the roles of the
multifunctional gene cer-cqu therein (von Wettstein-Knowles 2012).
Here a genetic approach with detailed biochemical characterisation of wax compounds
characterizes the Iw1 locus. Using synteny and a large number of F2 gametes, Iw1 was fine
mapped to a sub-cM genetic interval on wheat chromosome arm 2BS which includes a single
collinear gene from the corresponding Brachypodium and rice physical intervals. The major
components of flag leaf and peduncle cuticular waxes were primary alcohols (POHs), diketones, and n-alkanes accompanied by small amounts of aldehydes, free fatty acids plus
the phenolic lipids, alkyl and methylalkylresorcinols (ARs/MARs). Iw1 inhibits formation of and hydroxy--diketones in the specified cuticles, accompanied by minor increases in nalkanes and C24POH. Moreover, the inhibitory effect is independent of genetic background
and across tissues. No differences were found in cuticle thickness and carbon isotope
discrimination in BC2F3 near isogenic lines differing at Iw1.
Results
The non-glaucous phenotype in Shamrock is determined by a dominant allele of Iw1
A doubled-haploid (DH) population developed from two winter wheat varieties, Shango
(glaucous, Figure 1A) and Shamrock (non-glaucous, Figure 1B), was used to map the nonglaucous phenotype to a locus on chromosome arm 2BS (Simmonds et al. 2008). The locus
was originally named Viridescence (Vir) because of the bright green colour of the nonglaucous organs (Figure 1B-C). Interestingly, previous studies (Jensen and Driscoll 1962, Liu
et al. 2007) also mapped a dominant inhibitor of glaucousness (Iw1) with the same
phenotype here. As both Iw1 and Shamrock 2BS are derived from wild emmer, we
hypothesized that Vir and Iw1 are the same gene.
To test this, Shamrock was crossed to six additional glaucous hexaploid UK wheat varieties
(Alchemy, Einstein, Hereward, Malacca, Robigus, and Xi19) with different genetic
backgrounds than that of Shango. The resulting F1 plants were non-glaucous, suggesting a
dominant gene action consistent with Iw1. This was further supported by the 1:3 glaucous to
non-glaucous segregation in the F2 populations (Supporting Data S1). In all six crosses the
non-glaucous phenotype mapped to 2BS as in the Shango cross. These results strongly
support our hypothesis that Iw1 determines the non-glaucous phenotype of Shamrock.
Iw1 maps to a sub-cM interval on wheat chromosome arm 2BS
The non-glaucous phenotype had been previously mapped to a ~3 cM interval between SSR
marker Xgwm614 and DArT marker wPt-4453 (Simmonds et al. 2008). To fine map Iw1,
markers were developed from ten expressed sequence tags (EST)s previously mapped to
the distal deletion bin of 2BS (2BS3-0.84-1.00) (Conley et al. 2004) having a clear syntenic
relationship to the sequenced Brachypodium and rice genomes (Supporting Table S1). Using
the original DH population (87 lines), Iw1 mapped between markers JIC007 and JIC012,
corresponding to Brachypodium genes Bradi5g01220 and Bradi5g01130 (Figure 2,
Supporting Table S2).
An additional four markers were developed for genes between JIC007 and JIC012. Three of
these markers (JIC009, JIC010 and JIC011; corresponding to Bradi5g01180, Os04g05030
and Bradi5g01160, respectively, Figure 2) were completely linked to Iw1, whereas JIC015
(Bradi5g01190) mapped to 2BS, but proximal to Iw1. This genetic position is consistent with
the fact that Bradi5g01190 (and Bradi5g01200) are >95% similar to Bradi5g02390 (and
Bradi5g02380), suggesting a very recent duplication of these genes into the Iw1 syntenic
interval. The duplication seems limited to just these two genes because the flanking genes,
Bradi5g02400 and Bradi5g02370, are single copy. The complete linkage of the JIC009-11
markers was consistent across the six F2 mapping populations generated between
Shamrock and the glaucous UK varieties.
To further delimit Iw1 and increase mapping resolution, 2,111 F2 plants from the Shango x
Shamrock (SxS) cross were screened with EST-derived single nucleotide polymorphism
(SNP) markers JIC004 and JIC015 (Bradi5g01410 and Bradi5g01190). A total of 297
recombination events were identified between these markers (7.03 cM), of which 36 mapped
between markers JIC007 and JIC012 (0.85 cM, Figure 2). Phenotypic evaluation of these
lines and their F3 progeny determined that Iw1 remained completely linked to JIC009, JIC010
and JIC011 within this 0.85 cM interval. Twenty nine recombination events were mapped
between JIC007 and Iw1 (0.69 cM), while seven were proximal, between Iw1 and JIC012
(0.17 cM).
To break the linkage between Iw1 and the three markers (JIC009-11), 850 F2 plants from a
cross between tetraploid durum wheat Langdon (iw1/iw1) and TTD140, a wild emmer
accession previously shown to carry Iw1 (Rong et al. 2000), were screened with JIC007JIC012. This identified recombinants across the Iw1 interval, with the marker order remaining
consistent with the SxS population. The phenotype of these lines and their F3 progeny
remained completely linked to JIC009. However, three independent recombination events,
between Iw1 and JIC010/JIC11 (0.18 cM) (Supporting Data S2), broke the linkage seen in
the SxS population. An additional 25 recombinants between JIC007 and Iw1 (1.47 cM)
positioned Iw1 within a 1.65 cM interval between JIC07 and JIC010/JIC011.The syntenic
intervals in rice and Brachypodium include only one gene with corresponding wheat
sequences having evidence of 2BS localization, Bradi5g01180 (Os04g05010), mapping as
JIC009 within the Iw1 interval.
Identification of additional candidate genes from barley genomic contigs
We screened the recently released barley genome with the Bradi5g01180 sequence, and
identified a contig anchored at 4.85 cM, together with a series of other short contigs at the
same position. This contig cluster includes three annotated genes in addition to the barley
Bradi5g01180 homologue (MLOC_77461). Two of them, MLOC_20994 and MLOC_6767 are
non-syntenic to rice and Brachypodium, whereas the fourth (MLOC_59629) is homologous to
Bradi5g01130 which we mapped to wheat 2BS as JIC012 (proximal to Iw1). The two nonsyntenic barley genes have significant hits to the wheat group 2 chromosome arm
assemblies, suggesting potential localisation within the corresponding wheat Iw1 interval.
Iw1 affects epicuticular waxes, without altering the cuticle membrane
In numerous species mutants affect epicuticular wax deposition and/or alter the cuticle
membrane. Cryo-scanning electron microscopy (SEM) was used to examine different tissue
surfaces of Shango and Shamrock plants to determine if surface structure could be
correlated with their visible phenotypes (Figure 1A-B). The peduncles of Iw1 plants were
completely devoid of any visible wax protruding from the surface, whereas plants lacking Iw1
displayed a dense accumulation of tubular/rod shaped wax structures across the entire
surface (Figure 3A-B). This was also true for the abaxial side of the flag leaves, where plants
differing at the Iw1 locus had the same contrasting phenotypes as seen on peduncles (Figure
3C-D). On the adaxial side of flag leaves, both plants with and without Iw1 had platelet
shaped wax deposited on the surface. Plants lacking Iw1, however, also had the tubular/rod
shaped waxes seen on the peduncle and abaxial surface (Figure 3E-F) suggesting that the
adaxial cuticle surface is more complex than that on the abaxial leaf and peduncle surfaces.
These major variations in epicuticular wax density and structure were consistent across Iw1
BC2F3 near isogenic lines (NILs) derived from glaucous UK varieties (Supporting Figure S1).
These results are in accord with observations on related wheat lines (Netting and von
Wettstein-Knowles 1973), except that the presence of tubes occurred only at the base of the
adaxial flag leaf blades.
In contrast, no differences were found in cuticle thickness of flag leaf blades between Xi19
NIL pairs differing at Iw1 when examined with transmission electron microscopy (TEM,
Figure 3G-H). Xi19 Iw1/Iw1 lines had an average cuticle thickness of 0.105 ± 0.012 (adaxial)
and 0.134 ± 0.016 μm (abaxial), not significantly different (P = 0.52) from the thickness of
corresponding surfaces in Xi19 iw1/iw1 lines (0.098 ± 0.016 and 0.144 ± 0.012 μm,
respectively). The peduncle cuticle thickness and morphology were likewise indistinguishable
in Shamrock (0.239 ± 0.005 μm) and Shango (0.244 ± 0.024 μm) field grown plants (P =
0.84, Figure 3I-J). These data suggest that glaucousness differences between lines with and
without Iw1 result from changes in epicuticular wax deposition.
Primary alcohols, -diketones and n-alkanes are major components of wheat cuticular waxes
Wax composition was analysed to probe the differences giving rise to the glaucous versus
non-glaucous phenotype. Thin layer chromatography (TLC) initially disclosed a major
difference between Shango and Shamrock total flag leaf waxes (Figure 4). Namely, the
dominating β-diketones are absent in Shamrock. This correlates with the presence versus
absence of tubular structures on the examined surfaces (Figure 3C-D). A second difference
between the two varieties is the Shango band labeled unknown in Figure 4. Not only does its
Rf not correspond to that of the hydroxy-β-diketones in the Eucalyptus standard, but
diagnostic ions for these aliphatics (see below) were absent when its eluate was subjected to
mass spectrometry (MS).
To quantify the total and relative amounts of the β-diketones and their hydroxy derivatives if
present, the total mg wax/mg leaf was determined from OD273 measurements plus wax and
leaf fresh weights (Table 1). Shango flag leaves bear more than twice the amount of wax as
Shamrock leaves. Of the former wax, circa 67% were β-diketone aliphatics; in Shamrock
these aliphatics were maximally 8%. In the two varieties, the hydroxy-β-diketones account for
0.5 and 2%, respectively, of the total β-diketones, less than 0.03 and 0.01 µg/ mg leaf. Other
components common to both genotypes are n-alkanes, aldehydes, POHs and free fatty acids
(Figure 4) previously reported in other wheat waxes (Koch et al. 2006, Netting and von
Wettstein-Knowles 1973, Tulloch and Hoffman 1973). Although not resolved in the solvent
system used from free fatty acids, waxes of both varieties contain ARs and MARs (Figure 4).
The standard approach to obtain an overview of the major wax constituents is subjecting
silylated wax samples to gas chromatography-mass spectrometry (GC-MS). In the resulting
total ion chromatogram (TIC) traces, non-silylated aliphatics, for example n-alkanes and
aldehydes, are less prominent than silylated POHs, and silylated β-diketone aliphatics are
underestimated versus silylated POHs (Tulloch and Hogge 1978). Furthermore, in β-diketone
containing waxes not all components are resolved by this technique. Between 18.5 and 20
min, three non-symmetrical peaks occur in the Shango (Figure 5A-C), but not in the
Shamrock (Figure 5B-D) TIC traces. Subjecting the C31 standards hentriacontane-14,16dione and 25-hydroxy-14,16-dione to the same conditions resulted in similarly shaped peaks
eluting at the same retention times. The first eluting peak is the non-derivatized β-diketone,
hentricontane-14,16-dione, the second a mixture of silylated isomers of the same compound
while the third contains a mixture of silylated hydroxy-β-diketones (Figure 5A-C). The
presence of a strong ion at m/z 100 arises from cleavages alpha to the carbonyl groups
when neither has been silylated. When one of the carbonyls of the specified β-diketone is
silylated, cleavage adjacent to a carbonyl gives rise to the prominent diagnostic ions m/z 325
and 353 accompanied by M+ and M-15 ions. When a 25 silylated hydroxy group is also
present, cleavage adjacent thereto gives rise to strong m/z ions at 539 and 187, and if
adjacent to the carbonyl bearing carbons the ions m/z 325 and 441 as well as M+ and M-15
ions (Tulloch and Hogge 1978). Given that (i) none of the just mentioned diagnostic peaks is
seen in the Shamrock TIC traces (Figures 5B,D), (ii) that trace components in cuticular
waxes absorbing at 273 nm will result in an overestimation of the amount of the β-diketone
aliphatics in Table 1 (von Wettstein-Knowles 1976), and (iii) that the latter were undetectable
on TLC plates (Figure 3), we conclude that β-diketone aliphatics are most likely absent in the
investigated Shamrock waxes.
Figure 5A also shows that among the non β-diketone aliphatics, C28POHs are the dominant
constituents of the total flag leaf waxes, amounting to 68% of Shamrock wax. This is in
accord with the Shamrock SEM and TLC observations (Figures 3 and 4). Given that plate
structures were not observed on the abaxial surface of Shango leaves, the observation that
the C28POHs are such prominent constituents in TIC traces was unexpected, but similar
situations have been described (Baker 1982). C28POH are essentially absent in peduncle
waxes of both varieties (Figure 5C-D). Next in frequency are C24POHs plus C29 and C31 nalkanes in both flag leaf and peduncle waxes (see also Table 2). Closer examination of the
traces supplemented with the pertinent ion traces identified n-alkanes from C22-C33 and
POHs from C18-C34 chain lengths. Using the just mentioned technique, less abundant C22-C32
free fatty acids and C24-C34 aldehydes were identified. To further strengthen identification and
reveal peaks co-eluting with other compounds, aliquots of the waxes were subjected to
reduction with NaBH4, oxidation with K2Cr2O7 or passed through NaOH columns before
silylation. This resulted in the expected reduction and shifts of peak retention times (data not
shown). The 53 identified wax components are listed in Supporting Table S3.
Alkyl and methylalkylresorcinols are present in wheat flag leaves
In the course of trying to identify minor components of the waxes, one with a strong m/z 282
ion plus an M+ 562 ion was noted. Given that an m/z 282 ion is characteristic for
methylalkylresorcinols, this component was potentially identified as a MAR with an alkyl
chain of 23 (C23MAR). Since the presence of these phenolic lipids had not been previously
reported in wheat waxes, they were further analysed. TLC plates developed in hexane:
diethyl ether (8:2 v/v) and sprayed with 0.05% fast blue revealed purple spots migrating close
to the origin (Rf = 0.05) in Shango and Shamrock flag leaf and peduncle wax samples. When
the solvent was changed to a combination of chloroform and ethyl acetate (8.5:1.5 v/v) in
which the MARs would be expected to be more soluble, the purple spots migrated with an Rf
= 0.42. Treatment of total waxes with K2Cr2O7 oxidised the hydroxyl groups on the benzol
ring and abolished the fast blue staining of MARs on TLC plates. When silica was removed
from the appropriate region of preparative TLC plates and the compounds recovered for
analysis by GC-MS and ion scanning, we identified in addition to a homologous series of
MARs (C19-C27) with the 21 and 23 homologues being most prominent, an analogous
homologous series of ARs with the 23 and 27 homologues being most prominent. These are
characterized by a strong m/z 286 ion and M+ ions differing by 28. ARs have been frequently
reported in wheat grains (Knödler et al. 2010). Figure 6 shows ion scans at m/z 268 and 282
for the ARs and MARs, respectively, in total waxes from flag leaf blades of Shango and
Shamrock.
The effect of Iw1 on the major aliphatics in the cuticular wax of flag leaves and peduncles of
DH and BC2F3 wheat lines
Flag leaf and peduncle wax samples from four DH lines arising from the SxS cross were
collected nine days after anthesis. The results were compared to those of the parents (Figure
5) in Table 2. The wax load on Shamrock flag leaves was 38% of that on Shango, whereas in
peduncles only 13%. Analogous differences were found among the DH lines. Similar to
Shamrock, doubled haploid lines DH93 and DH81 carrying the dominant Iw1 allele had minor
amounts of β-diketones, whereas lines DH119 and DH74, both homozygous recessive, had
significantly higher (P<0.001), but not identical amounts. The ratios of β-diketones to
hydroxy-β-diketones in the three glaucous lines were also similar, albeit higher on the flag
leaves (3.5-4.7:1) than on the peduncles (2.4-3.4:1). The amounts of C28POHs, by
comparison, were not significantly different among all four DH lines and the parents.
Intriguingly, amounts of n-alkanes and C24POHs were consistently greater (P<0.001) in lines
with the Iw1 dominant allele.
Analogous flag leaf and peduncle waxes from five pairs of BC2F3 NILs were also analysed.
Across genotypes, the effects of Iw1 alleles were consistent and similar to those seen in the
SxS DH lines. The effect was independent of the original varieties’ glaucousness, as
exemplified by the two lines with the highest (Alchemy) and lowest (Malacca) wax load
(Table 2). Alchemy with only 30-40% as much β-diketone aliphatics as Shango on the
peduncle and flag leaf, respectively, had similar ratios of β-diketones to hydroxy-β-diketones
as Shango and the DH lines lacking the dominant Iw1 allele. In Malacca, however, the ratio
was dramatically reduced in both cuticle surfaces. In all five varieties the relationship among
the relative amounts of the C24POHs and n-alkanes was consistent. The lines with the
dominant Iw1 allele had more than those without, although in no case did the increased
amount compensate for the reduction in the β-diketone aliphatics (Table 2). The combined
observations infer that the inhibitory effect of the gene works independently of genetic
background, and functions during formation of β-diketone aliphatics in flag leaf and peduncle
wheat cuticles. Moreover, concordant small increases in the n-alkanes and C24POHs
accompany the inhibition.
The effect of Iw1 throughout plant development
The presence of visible wax on glaucous varieties is manifested clearly at anthesis. To
understand the effect of development on the wax profile and the gene’s effect, we conducted
a time course experiment using flag leaves from the Alchemy, Malacca, Robigus and Xi19
NIL pairs differing at Iw1. At early reproductive stage (Zadoks GS31, first node detectable
within leaf sheaths (Zadoks et al. 1974)), plants lacked visible wax and no obvious
phenotypic differences were detected between NIL pairs (Figure 7).
GC-MS analysis showed that C28POHs were the almost exclusive component of the wax
(circa 6 μg/mg leaf) at this stage in all NILs (Figure 7D). At the GS47-49 (boot stage) the
glaucousness phenotype is visible, the flag leaf sheath is opening, but the ear has not yet
emerged. C28POHs still dominated the wax, but interestingly amounted to only circa 2 μg/mg
flag leaf, significantly lower than at GS31. This is most likely due to the differences in leaf
thickness, and hence weight, between early vegetative leaves and the flag leaf which was
sampled at boot stage (no flag leaf exists at GS31). The wide array of other compounds
characterizing Shango and Shamrock (Figure 5) were also detected by GC-MS. In TIC traces
of NILs carrying the dominant allele of Iw1, -diketone aliphatics were absent (Figure 7E-F),
but n-alkanes (Figure 7A-B) and C24POHs (Figure 7C) were significantly higher than in
glaucous lines. This led to insignificant differences in total wax load (Figure 7G) between any
NIL pair as determined by GC-MS despite the clear differences in visual wax across all
varieties in the field.
Five days later at GS51 (first spikelet of ear just visible above flag leaf ligule), the amounts of
-diketone aliphatics had increased and together with C28POHs were major wax components
in glaucous NILs. The increase of β-diketone aliphatics continued through flag leaf
development, yet these compounds remained absent in NILs with the dominant Iw1 allele
(Figure 7E-F). n-Alkanes and C24POHs also increased during flag leaf development in both
sets of NILs as did the difference between each NIL pair. The total amount of C28POHs
remained similar between NIL pairs across all time points, increasing after 18 DPA (Figure
7D).
Iw1 does not affect 13C discrimination () in flag leaves, spike, and grain
To evaluate the effect of the different wax loads on water-use efficiency (WUE), we
determined the carbon isotope discrimination (Farquhar et al. 1989) for tissues from field
grown plants in 2011 and 2012. Flag leaves and spikes were collected at anthesis and grains
at maturity. No significant effect was detected for Iw1 either in 2011 or 2012: the NIL pairs
were not significantly different in any of the three tissues examined in both the Malacca or
Alchemy genetic backgrounds (Figure 8). The  of flag leaves and grains, however, was
significantly greater in 2012 than in 2011 (by ~ 1.5 to 2.5‰, Figure 8A-B, P<0.001).
Discussion
Fine mapping Iw1
The use of synteny has been extensively exploited in wheat genetics to fine map and clone
genes (Krattinger et al. 2009). This approach is proving increasingly powerful with the
generation of new genome sequences from closely related species such as Brachypodium
(International Brachypodium Initiative 2010) and the genomic contigs of barley (International
Barley Genome Sequencing Consortium 2012). The unrestricted access to the wheat
chromosome arm assemblies is also proving a key resource. For the first time, wheat
researchers have the possibility to access genomic contigs of all three homoeologues, a
critical step for more targeted marker design. Likewise, a simple BLAST search provides
evidence of putative chromosome localization. These tools will allow researchers to focus
marker design on those genes with most probability to lie in candidate gene intervals.
For fine mapping of Iw1, we exploited these new resources focusing marker design to the
Iw1 interval (JIC007 and JIC012), including 7 genes in Brachypodium and 21 genes in rice.
The use of the wheat chromosome arm assemblies allowed us to work only on those genes
with evidence of 2BS localization, including the Bradi5g01180/Os04g05010 gene. This
approach allowed discrimination for genes present in only one of the two sequenced
genomes. For example, Bradi5g01160 and Os04g05030 both had evidence of 2BS genomic
sequence and were genetically mapped to the Iw1 interval, whereas an additional eighteen
genes in rice were excluded from further analysis as they had evidence of localization to
other wheat chromosomes. In an analogous manner, we used the recently released barley
genomic contig to identify two additional barley genes (MLOC_20994 and MLOC_6767)
having evidence of wheat group 2 chromosome localisation.
The synteny based approach greatly facilitated the fine mapping of Iw1 to a sub-cM genetic
interval. The absence of β-diketone aliphatics in Brachypodium and rice, however, makes
highly unlikely the existence of a homolog of the Iw1 dominant inhibitor of the β-diketone
biosynthetic pathway in these species. Furthermore, barley, which synthesizes β-diketone
aliphatics, may likewise lack such a gene given the very rare frequency of chromosome 2H
dominant eceriferum mutants (King and von Wettstein-Knowles 2000) or it is located
elsewhere in the genome. This infers that a gene in the sub-cM interval of 2BS has a
different function than its orthologues in Brachypodium and rice. A pertinent example is the
GPC gene determining orthologous NAC transcription factors with divergent functions in
wheat and rice (Distelfeld et al. 2012). Construction of a physical map and the final
identification of Iw1 will help to answer these questions. At present, the combined genetic
and in silico analysis yield three candidate genes for Iw1; Bradi5g01180/Os04g05010,
MLOC_20994, and MLOC_6767.
Iw1 candidate genes
The complete linkage between JIC009-11 and Iw1 in the SxS population was surprising
based on the large number of F2 gametes screened and the expected high levels of
recombination in the distal ends of wheat chromosomes (Lukaszewski and Curtis 1993).
Moreover, the amplification of gene-based markers JIC009-011 in all the germplasm used in
this study suggests that these genes have not been deleted. The complete linkage between
JIC009 and Iw1 across both populations can therefore be interpreted to mean that (i) these
two genes are in very close proximity, (ii) that a deletion event occurred in glaucous varieties
proximal to JIC009, thereby suppressing recombination between glaucous and non-glaucous
lines, or (iii) that the wheat homologue of Bradi5g01180/Os04g05010 is Iw1.
The three candidate genes share no homology and have different conserved domains and
putative functions. The wheat homologue to Bradi5g01180/Os04g05010 encodes for a
protein including a C-terminal cystathionine β-synthase domain. These domains have no
assigned biological function in plants, but gene expression-based analyses in rice and
Arabidopsis suggest that some may play a role in stress response and development
(Kushwaha et al. 2009). The MLOC_6767 gene encodes for a laccase-like multi-copper
oxidase protein which forms part of a large family of proteins with broad functions. In
Arabidopsis, there is evidence of laccase contribution to lignification of stems (Berthet et al.
2011) and oxidative polymerization of flavonoids in the seed coat (Pourcel et al. 2005). The
MLOC_20994 gene encodes for a protein which includes a C-terminal Mu-homology domain
(MHD) which defines a family of conserved endocytic adaptor proteins called muniscins
(Conibear 2010). These proteins have been characterised in yeast (Reider et al. 2009) and
mammals (Uezu et al. 2007) and been shown to interact with phospholipids and to facilitate
vesicle formation. MHD proteins remain uncharacterised in plants. We are currently
performing allelic diversity studies and complementation experiments to determine if any of
these genes encodes for Iw1.
Complete series of ARs and MARs are present in wheat flag leaves
The identification of complete homologous series of both odd numbered ARs and MARs
(C19-C27) raises the interesting question as to how they are synthesized. Similarly to
stillbene synthase, a type III polyketide synthase, which adds 3 C2 units from malonyl-CoA to
p-coumaric-CoA to form a tetraketide intermediate that cyclizes giving resveratrol (Yu and
Jez 2008), alkylresorcinol synthases also form a tetraketide intermediate at the end of C22
acyl chains that cyclizes giving an AR with loss of a carbon; 1,3-dihydroxy,5-C15-benzene
(Cook et al. 2010). If methyl malonyl replaces malonyl-CoA in one of the last three elongation
steps, the 1,3-hydroxybenzene ring will bear a methyl group on carbon 2, 4 or 6 (MAR). Is
there a specific elongation step in which methylmalonyl-CoA replaces malonyl-CoA as in
Pinus strobus (Schröder et al. 1998), or can this occur in any of the three steps to give a
MAR? And why does this not occur at all when ARs are synthesized or never twice? While
these are questions for the future, clearly the type III polyketide synthase(s) giving rise to the
ARs and MARs of wheat cuticular wax is(are) different from the pkKCS giving the triketide
intermediate with the oxygens close to the middle of the carbon chain that do not cyclize in βdiketone synthesis. Furthermore, Iw1 inhibits only synthesis of the latter. This information will
be useful in trying to predict the function of Iw1.
Effect of lw1 on water-use efficiency
At the plant physiological level, WUE refers to the instantaneous water-use efficiency of gas
exchange, A/T, where A is net photosynthesis and T is transpiration. Because A is
dependent on the ratio between the concentration of CO 2 outside and inside the leaf, relative
changes in the proportion of these two parameters, and related changes in  are used to
estimate shifts in plant A/T. By measuring the 13C values of plant biomass, and thus
determining  (Farquhar et al. 1989), we’ve assessed to what extent lw1 affected WUE
during the growing season in 2011 and 2012.
Our results show that lw1 did not affect in flag leaves, spikes, and grains in Malacca and
Alchemy indicating that WUE remained unaffected in these lines. Our inter-annual data
showed clear differences in WUE between 2011 and 2012 (Figure 8), which differed in the
amount of rainfall and relative humidity, both higher in 2012 (Supporting Figure S2). This
suggests that in the UK, a shift in moisture availability has a greater effect on wheat WUE
than the amount and type of organic compounds that comprise cuticular waxes. Our 
results are not consistent with previous research that found a positive link between
glaucousness and in wheat grain and leaves (Merah et al. 2000, Monneveux et al. 2004).
Variation in the amount of cuticular wax load among varieties could be one of the reasons for
these differences. Furthermore, the generic classification of non-glaucousness irrespective of
the genetic source (Iw1, Iw2, w1, w2) hinders comparisons. Future studies based on
glaucous and non-glaucous germplasm with defined genetic makeup, including quantitative
and qualitative wax compositions will help explore the link between glaucousness and WUE
in more detail.
Concluding remarks
Introgression of Iw1 into seven different genetic backgrounds leads to inhibition of β-diketone
aliphatics synthesis and a small increase in the amount of n-alkanes and C24POHs, while
cuticle thickness and water use efficiency were unaltered. How does Iw1 achieve this? That
10 min were used for wax extraction infers that both epi and intracuticular waxes were
recovered, suggesting that the lack of the β-diketone aliphatics does not result from a defect
in their transport to the cuticle surface, but rather a defect in their biosynthesis or regulation
thereof given the dominant nature of Iw1. If inhibition occurs at the branch point of the FAE
and pkKCS pathways one might expect extensive rechanneling of precursors from one to the
other, but this appears unlikely as the increase exhibited by the n-alkanes and C24POHs is
less than 10% of the decrease in the β-diketone aliphatics. The Cer-cqu gene in barley maps
to a similar region on chromosome arm 2HS as Iw1 on wheat 2BS, however, only one of 522
mutations is dominant (King and von Wettstein-Knowles 2000). All 18 Cer-yy induced
mutations are dominant analogous to Iw1, but Cer-yy maps to chromosome 1H (Lundqvist
and von Wettstein-Knowles 1982, von Wettstein-Knowles 1990). Moreover, given that a
number of barley and wheat varieties carry dominant alleles of these inhibitors, identifying
the molecular natures of Iw1 and Cer-yy is of great interest.
Experimental Procedures
Plant material
The previously published Shango x Shamrock doubled-haploid (DH) population (87 lines)
(Simmonds et al. 2008) was used to map EST-derived markers across chromosome arm
2BS. An F2 population generated from these parents was used to assess segregation ratios
and identify recombinants across the region. Plants with recombination between markers
JIC004 and JIC015 (Supporting Experimental Procedures S1; Supporting Figure S3) were
selfed and homozygous F3 recombinants selected and phenotyped. A total of 2,111 SxS F2
plants were screened (4,222 gametes).
An additional 850 F2 plants were screened from a durum wheat cross between glaucous
variety Langdon (iw1/iw1) and non-glaucous wild emmer accession TTD140 which carries
Iw1 (Rong et al. 2000). A similar strategy was conducted as in the SxS recombinant plants.
Additional F2 populations were developed by crossing six glaucous hexaploid UK varieties
(Alchemy, Einstein, Hereward, Malacca, Robigus, Xi19) to Shamrock. To generate the BC2
NILs, heterozygous plants across the Iw1 interval were selected at each generation using
markers Xgwm614 and Xwmc25 (Alchemy, Einstein, Malacca, Xi19) or Xgwm614 and
Xwmc154 (Hereward, Robigus) and crossed again to Shamrock. After the second backcross
self-pollinated, homozygous BC2F2 NILs were selected.
Isolation of waxes
To study the effect of Iw1 on cuticular waxes, five flag leaves and peduncles from
independent replications were collected for Shango, Shamrock, the four DH lines, and the
five BC2 NIL pairs (Alchemy, Einstein, Hereward, Malacca, Robigus) from the 2009-2010
field plots. For the time course analysis, three flag leaves from independent replications were
collected for the four BC2 NIL pairs (Alchemy, Malacca, Robigus, Xi19) grown in 2010-2011.
Samples were collected in the field, placed in pre-weighed 15 mL polypropylene tubes and
frozen on dry ice. Tubes were reweighed to determine the wet tissue weight before freezing
in liquid nitrogen and storing at -80ºC.
Waxes were extracted using 5 mL chloroform (Merck, analytical grade; as are all other
solvents below) in glass tubes with screw-cap polytetrafluoroethylene lids containing
chloroform and triacontane (C30 alkane, Sigma 263842, Poole, UK) as an internal standard
(35 µg/mL for leaves and 10 µg/mL chloroform for peduncles); samples were immersed for
10 min at room temperature and shaken 3 times for 10 s. The extracts were transferred to
new glass tubes and dried down in a Vortex Evaporator (3-2201, Buchler Instruments Inc.,
NJ, USA). Each wax sample was re-suspended in 1 mL of chloroform and transferred to a
pre-weighed Agilent glass vial, dried under nitrogen and then re-weighed to determine the
total amount of wax extracted.
Gas Chromatography-Mass Spectrometry
Wax samples were derivatised in a 100 µL aliquot of a pyridine and TMS-BSTFA (Sigma
15238) mixture (1:1 v/v) at 75ºC for 1 hr; samples were vortexed every 15 min. Commercial
standards [C30 alkane (Sigma 263842), a mix of C7-C40 n-alkanes (Sigma 49452), a mix of
100 µg each of 1-tetracosanol (Sigma L350), 1-hexacosanol (Sigma H2139), 1-octacosanol
(Sigma O3379) and 1-triacontanol (Sigma T3777)] and samples of β-diketones (96%
hentriacontane-14,16-dione) and hydroxy-β-diketones (97% 25-hydroxyhentriacontane14,16-dione) isolated from barley (Hordeum vulgare L. cv. Bonus) spikes (von WettsteinKnowles 1976) were derivatised similarly.
The derivatised fraction was analysed on an Agilent GC 6890N gas chromatograph (Agilent
Technologies, Wilmington, Delaware, USA) coupled to a 5973 Inert Mass Selective Detector.
Automated splitless 3 μL injections were made using an Agilent 7683 automatic sampler.
Conditions of chromatography were: inlet temperature 250ºC, He carrier gas at a flow rate of
0.8 mL/min, nominal inlet pressure of 9.27 psi, the oven temperature program was: from
140ºC (1 min) to 380ºC (at 10ºC/min), then held for 5 min. The column used was a ZB-5HT
Inferno (Zebron; 7HG-G015-02, Phenomenex, Torrance, CA, USA) 30 m x 0.25 mm x 0.1
μm with a 5 m guard column fitted to the front end. The Retention Time Locking feature was
used and the method locked to the retention time of the triacontane internal standard (16.3
min). The mass spectrometer parameters using electron ionisation in positive mode (70 eV),
with a source temperature of 230ºC and a quad temperature of 150ºC were set to the
manufacturer’s recommended defaults. Total ion scans were made from 50-500 amu; all
data were processed via the Agilent GC Chemstation software (D.03.00) in conjunction with
the NIST Mass Spectral Library, V8.0 (National Institute of Standards and Technology,
Gaithersburg, Maryland, USA).
Quantification of wax compounds
Subtracting the percentages of the β-diketone aliphatics from the total wax gives the
percentage attributable to the other components. Relative abundances for these compounds
were calculated from GC-MS TIC peaks by automatic integration using the Custom Report
function in ChemStation. Where compounds such as the C26 FA/C28 aldehyde and C28 FA/C30
aldehyde eluted closely together, so that individual TIC peaks could not be integrated
separately, the major ion for each compound was searched and integrated separately for the
relevant retention time. The derivatized β-diketone peak was integrated manually and then
the characteristic ion for the obscured C30 FA peak was integrated and subtracted from the βdiketone peak. The same approach was used to estimate the C23MAR peak hidden within
the hydroxy--diketone peak. While the data presented do not take into account that not all
wax aliphatics are silylated nor the differential responses of the chemical groups to flame
ionization (Sternberg et al. 1962), they give a reproducible approximation of the quantities of
the wax aliphatics. All together 53 components were identified, with 26 being studied in more
detail as they account for >95% of the total wax load in both Shango and Shamrock flag
leaves and peduncles.
Acknowledgments
This work was supported by the Biotechnology and Biological Sciences Research Council
(BBSRC) [BB/H018824/1, BB/J004553/1, BB/J004596/1, BB/I002545/1], the John Innes
Foundation, NIAB Trust and Earth & Life Systems Alliance. We thank Dr. Moshe Feldman
(The Weizmann Institute of Science, Israel) for providing seeds of TTD-140; Drs. Hana
Simkova and Jaroslav Dolezel (IEB Olomouc, Czech Republic) for providing DNA of flowsorted arms, the IWGSC for pre-publication access to chromosome arm assemblies, Drs.
Nils Stein and Ruvini Ariyadasa (IPK Gatersleben, Germany) for barley contig information,
Dr. Lionel Hill (JIC), Jack McCurley, and Paul Disdle for technical assistance, and the JIC
glasshouse staff (Peter Sawdon, Lionel Perkins, Damian Alger, Barry Robertson) for plant
husbandry.
Supporting Information
Supporting figures:
Figure S1: Scanning electron micrographs of Iw1 BC2F3 NILs of six UK varieties
Figure S2: Temperature and precipitation in March to August of 2010 and 2011
Figure S3: Overview of Iw1 genetic map in Shango x Shamrock
Figure S4: Additional images of transmission electron microscopy
Supporting tables:
Table S1: Markers used for fine mapping of Iw1 in Shango x Shamrock
Table S2: Brachypodium and rice genes in Iw1 syntenic interval
Table S3: Wax components identified in the present study
Supporting data:
Data S1: Segregation of non-glaucous phenotype in seven F2 populations
Data S2: Graphical genotypes of Langdon x TTD140 F3 homozygous recombinants
Supporting experimental procedures:
Experimental Procedures S1: Molecular markers
Experimental Procedures S2: Field trials and plant growth
Experimental Procedures S3: Thin layer chromatography and spectrophotometry
Experimental Procedures S4: Scanning and Transmission Electron Microscopy
Experimental Procedures S5: Bulk δ13C measurements and calculation of 13C
discrimination
Experimental Procedures S6: Statistical analyses
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Figure Legends
Figure 1: Visual differences arising from the presence of different alleles of the Iw1 gene in
single tillers (A-B) and canopies (C). Shango iw1/iw1 (A), Shamrock Iw1/Iw1 (B) and Xi19
BC2F2 NIL pairs (C) in the field. In A, peduncle (p) refers to the visible, uppermost internode;
flag leaf blade and sheath are also indicated.
Figure 2: Shango x Shamrock genetic map of Iw1 and syntenic relationship with the
Brachypodium and rice (Oryza sativa) physical maps. Syntenic markers are joined with lines.
Red bars indicate the Iw1 interval in wheat and syntenic intervals in rice and Brachypodium.
Figure 3: Cryo-scanning electron micrographs of Shamrock (A-C-E) and Shango (B-D-F)
exposed peduncles (A-B) and abaxial (C-D) and adaxial flag leaf blade surfaces (E-F).
Close-ups of transmission electron micrographs of adaxial flag leaf blades from Iw1/Iw1 (G)
and iw1/iw1 (H) BC2F3 Xi19 NILs, and Shamrock (I) and Shango (J) peduncle cuticles. Full
sized images are available as Supporting Figure S4. Bar represents 10 μm in panels A-F and
500 nm in panels G-J. Cell wall (cw) and cuticle (c) are indicated in panels G-J.
Figure 4: Wax lipids in flag leaf blades of Shango (iw1/iw1), Shamrock (Iw1/Iw1), and
eucalyptus visualized after thin layer chromatography in hexane:ether (9:1 v/v) using
primuline. Numbered arrows correspond to n-alkanes (1); β-diketones (2); aldehydes (3);
primary
alcohols
(4);
hydroxy-β-diketones
(5);
unknown
(6);
alkylresorcinols,
methylalkylresorcinols and free fatty acids (7). All samples were run on the same TLC plate.
Figure 5: Gas chromatography-mass spectrometry total ion chromatograms of Shango (A
and C; iw1/iw1) and Shamrock (B and D; Iw1/Iw1) flag leaf blades (A-B) and peduncles (CD). Vertical axis is relative abundance; IS, internal standard (C30 ALK); * indicates non
silylated. Fatty acids (FA), n-alkanes (ALK), primary alcohols (POH), aldehydes (ALD),
methylalkylresorcinol (MAR), and both β- and hydroxy-β-diketones (β-Dik and OH-β-Dik) are
indicated.
Figure 6: Alkylresorcinol (AR) and methylalkylresorcinol (MAR) components of waxes from
flag leaf blades of Shango (A; iw1/iw1) and Shamrock (B; Iw1/Iw1) as disclosed by MS ion
scans at m/z 268 (blue) and 282 (red), respectively. Total ion chromatogram traces in black
(see Figure 5).
Figure 7: Time course analysis of major cuticular wax components of field-grown BC2F3 NILs
across 5 growth stages in 2011 (Zadoks GS31, GS47, GS51, 18 days post anthesis (DPA)
and 42 DPA). Panels include C29 and C31 n-alkanes (A-B), C24 and C28 primary alcohols (CD), β- and hydroxy-β-diketones (E-F) and total wax load (G). Green lines and triangles
represent Iw1/Iw1 NILs; grey circles represent iw1/iw1 NILs. Asterisks indicate significance at
probability <0.05 (*), <0.01 (**), and <0.001 (***).
Figure 8: Carbon isotope discrimination of Alchemy and Malacca BC2F3 NILs in 2011 (A)
and 2012 (B). Green bars represent non-glaucous Iw1/Iw1 NILs, whereas grey bars
represent glaucous iw1/iw1 NILs. No significant differences were detected between NIL pair
across tissue or years. GS: Zadoks growth stage.
Table 1. Spectrophotometric OD273 measurements comparing the relative abundance of diketones in Shango and Shamrock flag leaf epicuticular waxes from field grown plants 20
DPA in 2011. Average of two biological replicates; Dik = β-diketones plus hydroxy-βdiketones;  = -diketones; OH- = hydroxy--diketones.
Variety
Shango
Shamrock
§
Wax (mg§)
Leaf ( mg¶)
Total Dik (mg)
6.39
2.81
959
919
4.28
0.21
Dried weight of the leaf extract.
Estimations based on OD273 measurements.
¶
Fresh wet weight
tr = < 0.01 µg/mg leaf
†
% of Dik†
β
OH-β
99.5
97.9
0.54
2.12
µg Dik/mg leaf
β
OH-β
4.67
0.23
0.03
tr
Table 2. Total wax load (μg wax/mg leaf) on field grown flag leaf blades and peduncles plus
amounts (µg/mg) of the six most important constituents in the parental Shango and
Shamrock lines, four DH lines and four BC2F3 lines nine days post anthesis in 2010.
Tissue
Wheat line
Iw1
+/-
Wax load
(μg wax/mg leaf)
Flag leaf
Shango
Shamrock
DH119
DH93
DH21
DH81
Alchemy
Alchemy
Malacca
Malacca
+
+
+
+
+
12.68
4.76
10.81
5.32
13.06
4.80
7.67
4.43
5.82
4.80
C29
0.10
0.33
0.11
0.38
0.11
0.34
0.12
0.20
0.10
0.21
C31
0.04
0.13
0.05
0.13
0.05
0.14
0.08
0.26
0.04
0.09
Primary
alcohols
C24 C28
0.12 3.29
0.55 3.23
0.06 3.56
0.78 3.45
0.11 2.85
0.41 3.38
0.13 3.29
0.39 3.09
0.15 3.99
0.36 3.68
+
+
+
+
+
6.03
0.81
3.42
1.24
4.26
0.82
1.82
0.27
0.91
0.44
0.04
0.27
0.07
0.46
0.04
0.35
0.02
0.08
0.06
0.16
0.01
0.05
0.01
0.05
0.01
0.04
0.01
0.06
0.01
0.03
0.07
0.37
0.04
0.61
0.05
0.38
0.03
0.10
0.11
0.18
Peduncle
Shango
Shamrock
DH119
DH93
DH21
DH81
Alchemy
Alchemy
Malacca
Malacca
tr = < 0.01; - = not detected
n-Alkanes
tr
tr
tr
β-diketone
Hydroxy-βdiketone
7.09
0.06
5.10
0.06
7.86
tr
2.68
0.71
-
1.59
1.46
1.67
0.83
0.35
-
4.54
0.04
2.32
0.03
3.13
1.23
0.33
-
1.34
0.03
0.96
1.01
0.53
0.40
-