Three genomes differentially contribute to the
biosynthesis of benzoxazinones in hexaploid wheat
Taiji Nomura*†, Atsushi Ishihara‡, Ryo C. Yanagita‡, Takashi R. Endo*, and Hajime Iwamura§
Divisions of *Applied Biosciences and‡Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan; and§Department
of Bio-Technology, School of Biology-Oriented Science and Technology, Kinki University, Uchita-cho, Naga-gun, Wakayama 649-6493, Japan
Edited by Robert Haselkorn, University of Chicago, Chicago, IL, and approved September 15, 2005 (received for review June 20, 2005)
(Bxs) as defensive compounds. Previously, we found that five Bx
biosynthetic genes, TaBx1–TaBx5, are located on each of the three
genomes (A, B, and D) of hexaploid wheat. In this study, we
the contribution of individual homoeologous TaBx genes to the
biosynthesis of Bxs in hexaploid wheat. We analyzed their tran-
script levels by homoeolog- or genome-specific quantitative RT-
PCR and the catalytic properties of their translation products by
kinetic analyses using recombinant TaBX enzymes. The three
homoeologs were transcribed differentially, and the ratio of the
individual homoeologous transcripts to total homoeologous tran-
scripts also varied with the tissue, i.e., shoots or roots, as well as
with the developmental stage. Moreover, the translation products
of the three homoeologs had different catalytic properties. Some
TaBx homoeologs were efficiently transcribed, but the translation
products showed only weak enzymatic activities, which inferred
their weak contribution to Bx biosynthesis. Considering the tran-
script levels and the catalytic properties collectively, we concluded
that the homoeologs on the B genome generally contributed the
most to the Bx biosynthesis in hexaploid wheat, especially in
shoots. In tetraploid wheat and the three diploid progenitors of
hexaploid wheat, the respective transcript levels of the TaBx
homoeologs were similar in ratio to those observed in hexaploid
wheat. This result indicates that the genomic bias in the transcrip-
tion of the TaBx genes in hexaploid wheat originated in the diploid
progenitors and has been retained through the polyploidization.
biosynthetic genes ? homoeolog ? polyploidization
(Secale cereale), and maize (Zea mays), are involved in the
chemical defense of plants against pathogens and insects (1, 2).
The major Bxs are 2,4-dihydroxy-1,4-benzoxazin-3-one (DI-
BOA) and its 7-methoxy analog (DIMBOA), which are consti-
tutively present in the vacuole as glucosides (DIBOA-Glc and
DIMBOA-Glc). The Bx-glucosides, whose amount reaches the
maximum soon after germination and decreases thereafter to a
constantly low level, are particularly important in defense during
the juvenile stage of plant growth (3, 4).
The biosynthetic pathway of Bxs branches off from that of
tryptophan at indole-3-glycerol phosphate (5–8). Previously, we
isolated five genes responsible for the Bx biosynthesis, TaBx1–
Bx1–Bx5 in maize (5, 11) and the HlBx1–HlBx5 in wild barley,
Hordeum lechleri (12). A TaBX1 enzyme is involved in the
conversion of indole-3-glycerol phosphate to indole, whereas
TaBX2-TaBX5, which are cytochrome P450 monooxygenases
(CYPs) belonging to the CYP71C subfamily, catalyze the suc-
cessive oxidation reactions from indole to DIBOA (Fig. 1).
DIMBOA was suggested to be synthesized by the hydroxylation
and methylation at the C7-position of DIBOA, and a corre-
sponding hydroxylase gene (Bx6) has been cloned in maize (13).
Hexaploid bread wheat (T. aestivum, 2n ? 6x ? 42, genome
constitution AABBDD) is an allopolyploid that was formed
enzoxazinones (Bxs), major secondary metabolites in gra-
mineous plants including wheat (Triticum aestivum), rye
through hybridization and successive chromosome doubling of
three ancestral diploid species (2n ? 14), T. urartu (AA),
Aegilops speltoides (SS?BB), and Ae. squarrosa (DD) (14, 15).
Chromosome mapping using aneuploid lines of hexaploid wheat
revealed that the TaBx1 and TaBx2 genes coexist on homoeolo-
gous group-4 chromosomes, 4A, 4B, and 4D, and that the TaBx3,
TaBx4, and TaBx5 genes are clustered on group-5 chromosomes,
5A, 5B, and 5D (10). The orthologs of TaBx1–TaBx5 also were
found in the three ancestral diploid species (9, 10).
It is of great interest to reveal how the expressions of
all three homoeologs of the majority of genes are assumed to be
expressed in hexaploid wheat (16), it has been known that some
homoeologous genes suffer from epigenetic silencing or that
their gene expressions are altered during the polyploidization of
wheat (16–19). This finding indicates that in hexaploid wheat we
cannot reveal the degrees of contributions of the three genomes
to the expression of a gene simply based on the expression level
of individual homoeologous genes in the three diploid progen-
itors. Several studies have shown the unequal transcriptions of
the three homoeologs of certain genes in hexaploid wheat
(20–22). To our knowledge, however, no information is available
on the differential transcriptions of the respective three homoe-
ologs of multiple genes involved in the same metabolic pathway
in hexaploid wheat. Moreover, there has been no comparative
study both on the transcript levels of the three homoeologs of a
gene and on the properties of their translation products in
hexaploid wheat. The five genes involved in the Bx biosynthesis
of hexaploid wheat are excellent targets for a study to reveal how
the expressions of individual homoeologous genes are regulated
in a complex metabolic pathway in polyploids, because all three
homoeologous loci of those genes have been demonstrated to be
retained in hexaploid wheat (10).
In this study, we investigated the transcript levels of the three
homoeologs of TaBx1–TaBx5, and the enzymatic properties of
their translation products, and estimated the contributions of the
respective three homoeologs (or genomes) to the Bx biosynthesis
in the young seedlings of hexaploid wheat. We also analyzed the
transcript levels of their homoeologs in tetraploid wheat and in
the three diploid progenitors of hexaploid wheat to reveal how
the expression of the TaBx genes changed during the polyploid
evolution of wheat.
Materials and Methods
Plant Materials. A cultivar of hexaploid wheat (T. aestivum, 2n ?
6x ? 42, genomes AABBDD), Chinese Spring (CS), was used for
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: Bx, benzoxazinone; CS, Chinese Spring; DIBOA, 2,4-dihydroxy-1,4-benzo-
RT-PCR; h36, 36-h-old; d5, 5-day-old.
database (accession nos. AB124849–AB124857).
†To whom correspondence should be addressed. E-mail: email@example.com.
© 2005 by The National Academy of Sciences of the USA
November 8, 2005 ?
vol. 102 ?
cDNA cloning, genomic PCR, and quantitative RT-PCR (qRT-
PCR). We conducted the chromosomal assignment of the three
homoeologous cDNAs of the TaBx1–TaBx5 genes by genomic
PCR analysis using six lines of ditelosomic (DT) stocks of CS
(DT4AL, DT4BS, DT4DS, DT5AL, DT5BL, and DT5DL) (23)
and two (5BS-6 and 5BL-11) of the deletion stocks of CS (24).
We also used an extracted tetraploid CS line that had been
CS, 2n ? 4x ? 28, AABB) (25) for qRT-PCR analysis. For
Northern blot analysis we used three diploid progenitors (2n ?
14) of hexaploid wheat, T. urartu (Kyoto University seed stock
accession no. KU199-6, genome AA), Ae. speltoides (accession
no. KU5727, SS) and Ae. squarrosa (accession no. KU20-9, DD).
All seed stocks used in this study were supplied by National
BioResource Project-Wheat (Japan, www.nbrp.jp). Seeds were
germinated and grown as described in ref. 9.
Preparation of Substrates for Enzyme Assay.Indole-3-glycerolphos-
phate was synthesized enzymatically from CdRP [1-(2-
carboxyphenylamino)-1-deoxyribulose 5?-phosphate] by recom-
binant indole-3-glycerol phosphate synthase expressed in
Escherichia coli (for details, see Supporting Text, which is pub-
lished as supporting information on the PNAS web site). 3-Hy-
droxyindolin-2-one, 2-hydroxy-1,4-benzoxazin-3-one, DIBOA,
DIMBOA, DIBOA-Glc, and DIMBOA-Glc were prepared as
described in ref. 9.
Isolation of Three Homoeologous cDNAs. We constructed a cDNA
library of the 30-h-old CS shoots using the Uni-ZAP XR vector
(Stratagene) and screened it with the TaBx1–TaBx5 cDNAs that
had been isolated previously (9, 10). The full-length cDNAs were
labeled with the AlkPhos Direct Labeling Kit (Amersham
Biosciences). The plaque-lifted membrane was hybridized and
washed at 55°C according to the manufacturer’s instructions.
The hybridization signal was generated with ECF (Amersham
Biosciences) and detected with the FLA-2000 (Fujifilm). After
for each TaBx gene and sequenced them.
Chromosomal Assignment of Three Homoeologous cDNAs. To deter-
mine the homoeoalleles from which the three homoeologous
cDNAs were transcribed, we designed primers that would spe-
cifically amplify each of the cDNA homoeologs of TaBx1–TaBx5
primer sequences having interhomoeologous SNP sites at the 3?
ends were unique to the respective homoeologs, and the reverse
ones were common to all homoeologs (see Fig. 5 and Table 3,
which are published as supporting information on the PNAS web
site). To achieve homoeolog- or genome-specific amplification,
we conducted PCR under highly stringent conditions as follows:
1 min at 68°C) in a 20-?l reaction mixture containing 50 ng of
genomic DNA, 0.2 ?M primers, 0.2 mM dNTPs, 1.2 mM MgCl2,
and 0.5 units of Platinum TaqDNA polymerase (Invitrogen).
Aliquots (5 ?l) of the mixture were electrophoresed on 2%
(wt?vol) agarose gels and stained with ethidium bromide. Three
homoeologous cDNAs were assigned to the respective chromo-
some arms (4AS, 4BL, and 4DL for TaBx1 and TaBx2 genes, and
5AS, 5BS, and 5DS for TaBx3–TaBx5 genes) based on the
presence or absence of the PCR products in the CS aneuploid
qRT-PCR Analysis. Total RNA was purified from the shoots and
roots of 36-h-old (h36) and 5-day-old (d5) seedlings of CS and
Tetra CS by using an RNeasy Plant Mini Kit (Qiagen, Valencia,
CA). After DNase treatment, RNA was purified again with an
RNeasy column. Single-stranded cDNA was synthesized from 1
?g of total RNA by using oligo(dT) primer with the Thermo
Script RT-PCR system (Invitrogen) in a 20-?l reaction mixture.
The qPCR was carried out with an Applied Biosystems PRISM
7000 sequence detection system by using 1 ?l (equivalent to 50
ng) of the cDNA mixture as templates under the same conditions
as described above except that the reaction mixture contained
SYBR Green I. Fixed quantities of the cloned cDNAs were used
for generating the standard curves for fluctuations of the
respective cDNA homoeologs.
Expression and Purification of TaBX Enzymes. The TaBx1 cDNA
homoeologs were inserted into pET24b vector (Novagen) to be
expressed in E. coli. The recombinant TaBX1 enzymes were
expressed and purified as described in Supporting Text. The
TaBX2–TaBX5 enzymes encoding membrane-bound cyto-
chrome P450s were expressed in yeast (Saccharomyces cerevi-
siae). The coding sequences of the three homoeologs of TaBx2–
TaBx5 were separately inserted into HindIII site of the vector
pAAH5N (26), and the vector was introduced into S. cerevisiae
AH22 strain. The culture of transformed yeast cells, preparation
of recombinant microsome fractions, and determination of P450
contents were performed as described in ref. 9.
Enzyme Assay. The activity of the recombinant TaBX1 enzymes
was measured in 0.1 M Tris?HCl buffer (pH 7.8) containing 1
mg?ml BSA, 0.04–0.5 ?g of the enzyme, and 10–400 ?M
indole-3-glycerol phosphate in a final volume of 150 ?l. The
enzyme quantity and reaction time were set appropriately so that
the reaction proceeded linearly. After incubation at 37°C, the
reaction was stopped by the addition of 30 ?l of n-propanol, and
the product indole was quantified by HPLC [column, Wakosil II
5C18 HG (4.6 ? 150 mm); elution, 20–80% (vol?vol) MeOH
gradient (0–15 min) in 0.1% (vol?vol) HOAc; flow rate, 0.8
ml?min; temperature, 40°C; detection, 270 nm]. The activity of
TaBX2, TaBX3, TaBX4, and TaBX5 enzymes was measured by
using their respective substrates, indole, indolin-2-one, 3-hy-
droxyindolin-2-one, and 2-hydroxy-1,4-benzoxazin-3-one, as de-
scribed in ref. 9. Apparent Michaelis constants (Km) and max-
imum velocities (Vmax) were determined from [s] vs. [s]?v plot.
Northern Analysis. Total RNA was isolated from the shoots and
roots of h36 and d5 seedlings of diploid species by the AGPC (acid
guanidium–phenol–chloroform) method (27). Total RNA (20 ?g
each) was electrophoresed on a 1% (wt?vol) agarose gel with 1?
Mops buffer (pH 7.0) containing 2.2 M formaldehyde and was
enzymatic reactions catalyzed by the gene products of TaBx1–TaBx5 in
Schematic representation of the Bx biosynthetic pathway showing
Nomura et al.
November 8, 2005 ?
vol. 102 ?
no. 45 ?
blotted onto Hybond N?membrane. The membrane was hybrid-
ized with32P-labeled cDNA probes (TaBx1A, TaBx2A, TaBx3A,
TaBx4A, and TaBx5A) in Church buffer (pH 7.2) (28) at 65°C for
16 h. The membrane was washed at 65°C twice in 2? SSPE buffer
(pH 7.4) containing 0.1% (wt?vol) SDS for 15 min and once in 1?
SSPE buffer (pH 7.4) containing 0.1% (wt?vol) SDS for 30 min.
The washed membrane was autoradiographed with an imaging
plate (Fujifilm) and analyzed with the FLA-2000.
Extraction and HPLC Analysis of Bxs. We extracted Bxs from the
seedlings of CS, Tetra CS, T. urartu, Ae. speltoides, and Ae.
squarrosa every 12 h from 24 to 120 h after seeding. Shoots and
roots excised from the seedlings were frozen in liquid nitrogen
and were separately ground to a fine powder with a mortar and
a pestle, followed by the extraction of Bxs with HOAc-MeOH
(2:98, vol?vol). The extract was passed through a filter (Millex
HG (4.6 ? 150 mm); elution, 22% (vol?vol) MeOH in 0.1%
(vol?vol) HOAc; flow rate, 0.8 ml?min; temperature, 40°C;
detection, 280 nm].
Isolation and Chromosomal Assignment of the Three Homoeologous
cDNAs of the Five TaBx Genes. We screened the CS cDNA library
and isolated three kinds of homoeologous cDNA for each of the
five TaBx genes (Table 1), including the TaBx cDNAs isolated
previously (9, 10). The genomic PCR with the primer combina-
tions TaBx1A5–2?TaBx1Co3–3, TaBx1B5–3?TaBx1Co3–3, and
TaBx1D5–2?TaBx1Co3–3 amplified no PCR products in
DT4AL, DT4BS, and DT4DS, respectively (see Fig. 6A, which
is published as supporting information on the PNAS web site).
This result indicated that the TaBx1A, TaBx1B, and TaBx1D
cDNAs were transcribed from the alleles on 4AS, 4BL, and 4DL,
respectively. The chromosomal origins of the other homoeolo-
gous TaBx cDNAs were revealed in the same manner (see Fig.
6 B–E and Table 1). Two paralogous loci each for TaBx3 (on 5BS
and 5BL) and for TaBx5 (both on 5BS) are located on chromo-
some 5B (10). The fact that the TaBx3B-specific primers ampli-
cDNA was transcribed from the locus on 5BS, not 5BL. The
absence of PCR amplification in 5BS-6 with the TaBx5B-specific
primers (Fig. 6E) indicated that the TaBx5B cDNA was tran-
scribed from the more distal locus of the two TaBx5 loci on 5BS.
The TaBx3 and TaBx5 homoeologs at the other paralogous loci
on 5B are probably not transcribed or at least are transcribed
little because no corresponding cDNAs were isolated in the
cDNA library screening.
The cDNA homoeologs of the TaBx genes showed a striking
similarity in nucleotide sequence, ranging between 94.0% and
on the PNAS web site). They were also very similar in deduced
amino acid sequences, ranging from 95.2% to 98.9%. Like the
TaBx2–TaBx5 cDNAs isolated previously (9), the TaBx2–TaBx5
homoeologs isolated in this study had the heme-binding motif
that is conserved near the C-termini of P450 enzymes (29). We
deposited the amino acid sequences to obtain cytochrome P450
monooxygenase numbers (Table 1) from David Nelson (http:??
Catalytic Activity of the TaBX Enzymes. The three recombinant
TaBX1 enzymes expressed in E. coli were successfully purified
from solubilized inclusion bodies. Microsome fractions of the
recombinant yeast with each of the TaBx2–TaBx5 cDNA ho-
moeologs showed the maximum absorption at 448 nm in reduced
CO-difference spectra (data not shown). This result indicated
that all homoeologs of the TaBx2–TaBx5 cDNAs were expressed
as active P450 enzymes.
The kinetic parameters of the TaBX1–TaBX5 enzymes were
determined (Table 2). The Kmvalues of TaBX1–TaBX4 differed
slightly between the three homoeologs, from 1.4-fold between
TaBX4A and TaBX4D to 1.9 fold between TaBX2A and
TaBX2B. Conversely, the Kmvalue of TaBX5A was ?13 and 16
times larger than that of TaBX5B and TaBX5D, respectively. On
average, the Kmvalues of TaBX2 and TaBX3 were smaller than
those of the other TaBX enzymes. The difference in kcatvalue
between the three homoeologs ranged from 2-fold between
TaBX2A?B and TaBX2D to 13-fold between TaBX5A and
TaBX5D. The average kcatvalue was the highest for TaBX1 and
the lowest for TaBX5 enzymes. The kcat?Kmvalues indicated that
TaBX1B, TaBX2D, TaBX3A, TaBX4B, and TaBX5D had the
highest reaction efficiency among the three homoeologs of the
respective TaBX enzymes.
Transcript Levels of the Three Homoeologous TaBx Genes in Hexaploid
Wheat. We investigated the transcript profiles of the three
homoeologs of the TaBx1–TaBx5 genes in h36 and d5 CS
seedlings by the genome-specific qRT-PCR (Fig. 2A). Both in
shoots and roots, the TaBx5 homoeologous genes had the highest
transcript levels, and the TaBx1 homoeologous genes had the
Table 1. Three homoeologous cDNAs of TaBx1–TaBx5 genes
isolated from CS
arm locationCYP no.
*GenBank accession no.
†TaBx1 genes are not cytochrome P450s.
‡Sequence taken from Nomura et al. (10).
§Sequence taken from Nomura et al. (9).
Table 2. Kinetic constants of the TaBX1–TaBX5 enzymes
www.pnas.org?cgi?doi?10.1073?pnas.0505156102Nomura et al.
lowest. The transcript levels of the TaBx genes in roots were
much lower than those in the shoots. The transcripts of the
TaBx1–TaBx5 decreased in amount from 36 h to 5 days after
seeding both in shoots and in roots. The decrease was more
evident for TaBx1 and TaBx2 than for TaBx3–TaBx5 in roots.
The biased transcription of the three homoeologs was ob-
served for all TaBx genes. In the h36 shoots, the transcript levels
were in descending order of B-genome homoeolog, D-genome
homoeolog, and A-genome homoeolog for all TaBx1–TaBx5
genes. In the d5 shoots, the A- and D-genome homoeologs were
transcribed relatively less, and the B-genome homoeologs were
transcribed relatively more, with the exception of relatively
higher levels of TaBx1D and TaBx3A.
In the h36 plants, the ratio of transcripts to the total homoe-
ologous transcripts (simply called ‘‘the ratio’’ hereafter) of the
transcripts of the A-genome homoeologs was higher, and the
ratio of the D-genome homoeologs was lower in the roots than
in the shoots. The ratios of the A- and D-genome homoeologs
in the d5 roots were similar to those in the h36 roots, in contrast
to their time-dependent decrease in the shoots, with the excep-
tion of TaBx2.
Transcript Levels of the Two Homoeologous TaBx Genes in Tetraploid
Wheat. The transcript level of each homoeolog of the TaBx genes
in the Tetra CS seedlings was analyzed by qRT-PCR (Fig. 2B).
TaBx1 showed the lowest in both shoots and roots, with the
exception of lower transcription of TaBx3 than TaBx1 in the d5
shoots. The total amount of the transcripts was generally smaller
than that of hexaploid CS except for the h36 roots.
The transcript profile of the A- and B-genome TaBx homoe-
ologs in Tetra CS was similar to that in CS. In the h36 shoots,
every homoeolog of the TaBx genes was transcribed more in the
B-genome than in the A-genome, and the ratio of the transcript
levels of A-genome homoeologs was higher than that of the
B-genome homoeologs in the roots than in the shoots. In the d5
shoots, the ratio of the transcripts of the A-genome homoeologs
decreased greatly, and the transcripts of the B-genome homoe-
ologs accounted for almost the entire TaBx transcripts. In
contrast, the ratio of the transcripts of A-genome homoeologs in
TaBx3 and TaBx4 remained unchanged in the h36 and the d5
roots, and the transcripts of the A-genome TaBx2 and TaBx5
homoeologs decreased moderately with age in the roots com-
36 h to 5 days after seeding, the TaBx1 and TaBx2 transcripts
decreased more rapidly than the TaBx3–TaBx5 transcripts.
Transcript Levels of the TaBx Homoeologs in Diploid Wheats. We
investigated the transcript levels of the TaBx genes in the three
diploid progenitors of hexaploid wheat to reveal whether the
differential transcript levels of the three homoeologs in
hexaploid wheat reflect the respective transcription of those in
diploid species (Fig. 3). The transcript profiles in the three
diploids resembled those in the three genomes of hexaploid
wheat. In the h36 shoots, the five TaBx genes were strongly
transcribed in all diploid species. For every TaBx gene the
transcript level was in the order of Ae. speltoides (SS?BB) ? Ae.
squarrosa (DD) ? T. urartu (AA). In the d5 shoots, the transcript
levels of the A- and D-genome species decreased to a trace,
whereas the S-genome species retained the high transcript level.
In the h36 roots, the transcript level of every TaBx gene was
the highest in the S-genome species. This result is slightly
different from the cases of CS and Tetra CS in which the
A-genome homoeologs were predominantly transcribed for the
TaBx1, TaBx3, and TaBx4 genes. In the d5 roots, TaBx3 and
TaBx4 were transcribed slightly higher in the A-genome species
of the amounts of transcripts of the individual homoeologs to the total amounts of homoeologous transcripts are plotted.
Transcript profiles of individual homoeologs of the TaBx1–TaBx5 genes in the h36 (Left) and d5 (Right) seedlings of CS (A) and Tetra CS (B). The ratios
Nomura et al.
November 8, 2005 ?
vol. 102 ?
no. 45 ?
than in the S- and D-genome species, just like the A-genome
TaBx3 and TaBx4 homoeologs were transcribed slightly more
than the B- and D-genome homoeologs in CS and Tetra CS. As
observed in the d5 roots of CS and Tetra CS, the transcripts of
TaBx1 and TaBx2 genes decreased to traces in all diploid species.
Fluctuation in the Amount of Bxs. In all wheat species and strains
investigated, the Bxs increased soon after germination and
gradually decreased to a constantly low level with the decrease
in the transcript levels of the TaBx genes (Fig. 4). Either of the
Bxs, DIBOA or DIMBOA, became predominant, depending on
the species, tissue, and developmental stage.
In both shoots and roots on all stages, Ae. speltoides accumu-
lated more Bxs than T. urartu and Ae. squarrosa (Fig. 4 A–C).
This finding is consistent with the fact that the TaBx genes were
transcribed constantly higher in Ae. speltoides than in T. urartu
and Ae. squarrosa (Fig. 3), except for the d5 roots.
In both shoots and roots, the total amount of Bxs in CS was
slightly less than or similar to that in Tetra CS (Fig. 4 D and E).
This result is inconsistent with the fact that the TaBx genes were
Contribution of the Three Genomes of Hexaploid Wheat to the Bx
Biosynthesis Was Estimated from Difference in the Transcript Level
and Catalytic Property. Polyploidization is accompanied by
changes in genome structure and gene expression, including the
loss of gene sequences, silencing, and down-?up-regulation of
some homoeologs (16–19, 30–39). We found that the loci of the
five Bx biosynthetic genes are retained in all three genomes of
hexaploid wheat (10) and isolated three homoeologous cDNAs
each for TaBx1–TaBx5 genes in this study, showing that neither
the loss nor the epigenetic silencing of the TaBx genes had
occurred during the polyploidization.
Because of the high degree of nucleotide identity among the
three homoeologs of all TaBx genes in hexaploid wheat, we
cannot separately detect the transcripts of individual homoeolog
by hybridization-based methods such as Northern or cDNA
microarray analysis. We developed a genome-specific qRT-PCR
method and analyzed the transcript levels of the three homoe-
ologs of each of the five TaBx genes. To our knowledge, this work
presents a previously unreported analysis of the transcript levels
of three homoeologs that encode the enzymes catalyzing se-
quential reactions involving a metabolic pathway in hexaploid
The transcript levels of the three homoeologs were different
for all TaBx genes. In addition, the catalytic activity of the
TaBX1–TaBX5 enzymes differed, depending on the homoeolog.
This result suggests that the ratio of transcript levels of the three
homoeologs (Fig. 2) does not directly represent their contribu-
tion to the Bx biosynthesis. TaBX1B, TaBX2D, TaBX3A,
TaBX4B, and TaBX5D showed the highest reaction efficiency
(kcat?Km) among the three homoeologs and therefore should
have contributed to the Bx biosynthesis much more than ex-
pected from the amount of their transcripts. For example, of the
total transcripts of TaBx1 genes in the h36 seedlings, TaBx1A,
TaBx1B, and TaBx1D, respectively, accounted for 8.2%, 54.1%,
and 37.6% in shoots and for 64.1%, 34.4%, and 1.5% in roots.
The reaction efficiency (kcat?Km) of TaBX1B is ?17 times and
3 times higher than those of TaBX1A and TaBX1D, respectively.
Thus, we can estimate that TaBX1B contributes much more to
the reaction than the other two homoeologous enzymes in both
shoots and roots. Moreover, the extremely low kcat?Kmvalue of
the TaBX5A enzyme (50 and 190 times lower than TaBX5B and
TaBX5D) made the A-genome contribution to the biosynthetic
reaction negligible, although TaBx5A was substantially tran-
wheats with the TaBx1–TaBx5 probes. AA, SS, and DD represent T. urartu, Ae.
used as a loading control.
Northern blot analysis using the h36 and d5 seedlings of diploid
open circle, DIMBOA-Glc. The average values of three to five replications are plotted together with SD (?SD).
Changes in the amounts of Bxs in the seedlings of T. urartu (A), Ae. speltoides (B), Ae. squarrosa (C), Tetra CS (D), and CS (E). Filled circle, DIBOA-Glc;
www.pnas.org?cgi?doi?10.1073?pnas.0505156102Nomura et al.
scribed. We can safely assume that the B-genome generally
contributes the most to the Bx biosynthesis in hexaploid wheat.
There are some studies demonstrating the difference in the
level of transcripts among homoeologs in polyploid plants (20–
22, 35, 38). However, there have been no studies on the proteins
encoded by individual homoeologs. The slight difference in
amino acid sequence caused a great difference in the enzymatic
properties of the homoeologous TaBX enzymes. Thus, both
efficiency of gene transcription and performance of the gene
products need to be considered in the estimation of the contri-
bution of different genomes to certain biochemical processes in
Variation of Genome-Specific Transcripts in Hexaploid Wheat Origi-
nated in the Diploid Progenitors. In allopolyploids no global
genomic bias in gene expression has been reported, and there-
fore genomes in which preferentially expressed homoeoalleles
exist are implied to be different from gene to gene (35).
However, we found that the ratio of the genome-specific tran-
scripts fluctuated in a similar manner in general for all of the
in the same genome seems to be controlled in a coordinated way.
In the shoots, the same TaBx genes in CS, Tetra CS, and three
diploid species showed similar patterns of time-dependent
changes in the transcript levels according to homoeologs (Figs.
2 and 3). In addition, time-dependent decreases in the transcript
level in roots were more drastic in TaBx1 and TaBx2 than in
TaBx3, TaBx4, and TaBx5 in all wheats. These facts suggest that
the genomic bias in terms of the transcription of the TaBx genes
in hexaploid wheat originated in the diploid progenitors and has
been retained through the polyploidization.
Discrepancy Between the Levels of Bxs and the TaBx Transcripts
Implies an Intergenomic Regulation of Gene Expression. In the three
diploid species, the transcript levels of the TaBx genes directly
correlated with the amount of Bxs, although the predominant Bx
was different from species to species (Fig. 4 A–C). In the h36
transcripts were higher in CS than in Tetra CS, but in the d5
shoots and d5 roots the Bx production in CS was similar to that
in Tetra CS. This discrepancy might be explained in terms of the
reduction of translation efficiency caused by the increase of the
TaBx transcripts in hexaploid wheat. Polyploidization might
increase total transcription of genes, but, conversely, it might
cause reduction in translation by some mechanisms, like small
RNA-mediated or homology-dependent gene silencing as ob-
the endogenous gene (40, 41).
We thank Dr. Chihiro Tanaka for instrumental support in DNA se-
quencing; Dr. Toshiyuki Sakaki and Raku Shinkyo for instrumental
support and helpful suggestions in measuring absorption spectra of P450
proteins; and Dr. Shuhei Nasuda for critical reading of the manuscript.
We also thank Radioisotope Research Center, Kyoto University, for
instrumental support in radioisotope experiments. This work was sup-
ported by Japan Society for the Promotion of Science Grant-in-Aid for
Scientific Research No. 16000377 (to T.N.).
1. Niemeyer, H. M. (1988) Phytochemistry 27, 3349–3358.
2. Sicker, D., Frey, M., Schulz, M. & Gierl, A. (2000) Int. Rev. Cytol. 198, 319–346.
3. Nakagawa, E., Amano, T., Hirai, N. & Iwamura, H. (1995) Phytochemistry 38,
4. Ebisui, K., Ishihara, A., Hirai, N. & Iwamura, H. (1998) Z. Naturforsch. 53c,
5. Frey, M., Chomet, P., Glawischnig, E., Stettner, C., Gru ¨n, S., Winklmair, A.,
6. Frey, M., Stettner, C., Pare ´, P. W., Schmelz, E. A., Tumlinson, J. H. & Gierl,
A. (2000) Proc. Natl. Acad. Sci. USA 97, 14801–14806.
Natl. Acad. Sci. USA 94, 13345–13350.
8. Gierl, A. & Frey, M. (2001) Planta 213, 493–498.
9. Nomura, T., Ishihara, A., Imaishi, H., Endo, T. R., Ohkawa, H. & Iwamura, H.
(2002) Mol. Genet. Genomics 267, 210–217.
10. Nomura, T., Ishihara, A., Imaishi, H., Ohkawa, H., Endo, T. R. & Iwamura, H.
(2003) Planta 217, 776–782.
11. Frey, M., Kliem, R., Saedler, H. & Gierl, A. (1995) Mol. Gen. Genet. 246,
12. Gru ¨n, S., Frey, M. & Gierl, A. (2005) Phytochemistry 66, 1264–1272.
13. Frey, M., Huber, K., Park, W. J., Sicker, D., Lindberg, P., Meeley, R. B.,
Simmons, C. R., Yalpani, N. & Gierl, A. (2003) Phytochemistry 62, 371–376.
14. Huang, S., Sirikhachornkit, A., Faris, J. D., Su, X., Gill, B. S., Haselkorn, R.
& Gornicki, P. (2002) Plant Mol. Biol. 48, 805–820.
15. Huang, S., Sirikhachornkit, A., Su, X., Faris, J., Gill, B., Haselkorn, R. &
Gornicki, P. (2002) Proc. Natl. Acad. Sci. USA 99, 8133–8138.
16. Wendel, J. F. (2000) Plant Mol. Biol. 42, 225–249.
17. Kashkush, K., Feldman, M. & Levy, A. A. (2002) Genetics 160, 1651–1659.
18. He, P., Friebe, B. R., Gill, B. S. & Zhou, J.-M. (2003) Plant Mol. Biol. 52,
19. Islam, N., Tsujimoto, H. & Hirano, H. (2003) Proteomics 3, 549–557.
20. Himi, E. & Noda, K. (2004) J. Exp. Bot. 55, 365–375.
21. Mochida, K., Yamazaki, Y. & Ogihara, Y. (2003) Mol. Genet. Genomics 270,
22. Podkowinski, J., Jelenska, J., Sirikhachornkit, A., Zuther, E., Haselkorn, R. &
Gornicki, P. (2003) Plant Physiol. 131, 763–772.
23. Sears, E. R. & Sears, L. M. S. (1978) in Proceedings of the Fifth International
Wheat Genetics Symposium, ed. Ramanujam, S. (Indian Society of Genetics and
Plant Breeding, New Delhi), pp. 389–407.
24. Endo, T. R. & Gill, B. S. (1996) J. Heredity 87, 295–307.
25. Yang, Y. F., Furuta, Y., Nagata, S. & Watanabe, W. (1999) Genes Genet. Syst.
26. Oeda, K., Sakaki, T. & Ohkawa, H. (1985) DNA 4, 203–210.
27. Chomczynski, P. & Sacchi, N. (1987) Anal. Biochem. 162, 156–159.
28. Church, G. M. & Gilbert, W. (1984) Proc. Natl. Acad. Sci. USA 81, 1991–1995.
29. Schuler, M. A. (1996) Crit. Rev. Plant Sci. 15, 235–284.
30. Comai, L., Tyagi, A. P., Winter, K., Holmes-Davis, R., Reynolds, S. H., Stevens,
Y. & Byers, B. (2000) Plant Cell 12, 1551–1567.
31. Lee, H.-S. & Chen, Z. J. (2001) Proc. Natl. Acad. Sci. USA 98, 6753–6758.
32. Ozkan, H., Levy, A. A. & Feldman, M. (2001) Plant Cell 13, 1735–1747.
33. Shaked, H., Kashkush, K., Ozkan, H., Feldman, M. & Levy, A. A. (2001) Plant
Cell 13, 1749–1759.
34. Madlung, A., Masuelli, R. W., Watson, B., Reynolds, S. H., Davison, J. &
Comai, L. (2002) Plant Physiol. 129, 733–746.
35. Adams, K. L., Cronn, R., Percifield, R. & Wendel, J. F. (2003) Proc. Natl. Acad.
Sci. USA 100, 4649–4654.
36. Kashkush, K., Feldman, M. & Levy, A. A. (2003) Nat. Genet. 33, 102–106.
37. Osborn, T. C., Pires, J. C., Birchler, J. A., Auger, D. L., Chen, Z. J., Lee, H.-S.,
Comai, L., Madlung, A., Doerge, R. W., Colot, V., et al. (2003) Trends Genet.
38. Adams, K. L., Percifield, R. & Wendel, J. F. (2004) Genetics 168, 2217–2226.
39. Wang, J., Tian, L., Madlung, A., Lee, H.-S., Chen, M., Lee, J. J., Watson, B.,
Kagochi, T., Comai, L. & Chen, Z. J. (2004) Genetics 167, 1961–1973.
40. Pickford, A. S. & Cogoni, C. (2003) Cell Mol. Life Sci. 60, 871–882.
41. Adams, K. L. & Wendel, J. F. (2005) Curr. Opin. Plant Biol. 8, 135–141.
Nomura et al.
November 8, 2005 ?
vol. 102 ?
no. 45 ?