The Plant Cell, Vol. 7, 407-416, April 1995 O 1995 American Society of Plant Physiologists
The brown midrib3 (bm3) Mutation in Maize Occurs in the
Gene Encoding Caffeic Acid O-Methyltransferase
Florence Vignols, Joan Rlgau, Miguel Angel Torres, Montserrat Capellades, and Pere Puigdomènech '
Departament de Genètica Molecular, Centro de Investigación y Desarrollo, Consejo Superior de lnvestigaciones Científicas,
Jordi Girona 18, 08034 Barcelona, Spain
The brown midrib mutations are among the earliest described in maize. Plants containing a brown midrib mutation ex-
hibit a reddish brown pigmentation of the leaf midrib starting when there are four to six leaves. These mutations are
known to alter lignin composition and digestibility of plants and therefore constitute prime candidates in the breeding
of silage maize. Here, we show that two independent brown midrib3 (bm3) mutations have resulted from structural changes
in the COMT gene, which encodes the enzyme O-methyltransferase (COMT; EC 18.104.22.168), involved in lignin biosynthesis.
Our results indicate that the bm3-7 allele (the reference mutant allele) has arisen from an insertional event producing
a COMT mRNA altered in both size and amount. By sequencing a COMTcDNA clone obtained from bm3-7 maize, a retro-
transposon with homology to the B5 element has been found to be inserted near the junction of the 3 coding region
of the COMT gene intron. The second bm3 allele, bm3-2, has resulted from a deletion of part of the COMT gene. These
alterations of the COMT gene were confirmed by DNA gel blot and polymerase chain reaction amplification analyses.
These results clearly demonstrate that mutations at the COMT gene give a brown midrib3 phenotype. Thus, the gene
genetically recognized as bm3 is the same as the one coding for COMT.
Lignins are universal components in plants that form cross-
links with carbohydrates, such as hemicellulose in the cell wall
(for review, see Lewis and Yamamoto, 1990). The resulting
amorphous matrix provides the plant with multiple advantages,
such as mechanical strength, resistance to pathogens, and
waterproof qualities. However, due to their chemical proper-
ties, lignins are also responsible for the low agronomic and
industrial value of a number of species. Because lignins are
strongly linked to other cell wall constituents, they are difficult
to extract or to degrade (Sarkanen and Ludwig, 1971). Thus,
lignins appear to be an undesirable constituent that increases
manufacturers' costs. Once extracted, lignin byproducts pro-
duce an adverse impact on the environment. Agronomically,
lignin polymers have been shown to lower fiber digestion in
ruminants, and it has been proposed that the degree of lignifi-
cation is inversely proportional to forage crop digestibility
(Grand et al., 1985; Cherney et al., 1991). Lignin biosynthesis
has been studied extensively both because it is intrinsically
interesting scientifically and because it can be used to decrease
lignin content in plants of economic interest.
Lignins are biosynthesized through a complex pathway that
involves various enzymatic activities and polymerization steps.
The first enzymes required for lignin biosynthesis are those
from the phenylpropanoid pathway, such as phenylalanine
ammonia-lyase and cinnamate 4-hydroxylase (Lewis and
1 To whom correspondence should be addressed.
Yamamoto, 1990). Later, a series of hydroxylations and methyla-
tions involving various hydroxylases and an Omethyltransferase
(OMT) provides cinnamic acid derivatives. These compounds
are finally converted to respective alcohols by cinnamoyl-
coenzymeA reductase and cinnamyl alcohol dehydrogenase
(CAD) (Lewis and Yamamoto, 1990). Different approaches de-
signed to produce plants containing low lignin content include
backcross selection, chemical mutagenesis (Cherney et al.,
1991), and production of antisense plants for various enzymes
involved in the lignin pathway (Dwivedi et al., 1993; Halpin et
al., 1994; Ni et al., 1994). More logically, special attention has
been devoted to plants carrying naturally occurring mutations
and exhibiting this property.
The first brown midrib mutant was described in maize as
early as 1924, in Minnesota (Jorgenson, 1931; Cherney et al.,
1991). Plants possessing abrown midfib mutation exhibit a red-
dish brown pigmentation in the leaf midrib, which is initially
visible when there are four to six leaves. As the plant grows
and the stem lignifies, the reddish brown pigmentation is ob-
served to be associated with vascular bundles and lignified
rings in stem cross-sections. Later, the brown midrib pheno-
type gradually disappears from externa1 exposed surfaces of
the mature plant, but it is still detectable in interna1 and ligni-
fied tissues. Based on morphologic and genetic descriptions
in maize, four mutations (brown midribl [bml], bm2, bm3, and
bm4) located on chromosomes 5, 1, 4, and 9, respectively
(Neuffer et al., 1968), were shown to produce this phenotype.
The Plant Cell
Additional analyses of these mutants indicated that the brawn
midrib mutations could be associated with lower quantity and
different quality of lignins (Kuc et al., 1964, 1968; Gentinetta
et al., 1990). Moreover, the mutants appeared to be more eas-
ily digested by ruminants (Barnes et al., 1971; Lechtenberg et
al., 1972). These studies essentially dealt with bm3 maize plants
(and bm1 to a lesser extent) because the properties of this mu-
tant (cell wall content and composition, agronomic values, and
mechanical properties) were shown to be the most appropri-
ate for breeding and feeding purposes (Barnes et al., 1971;
Lechtenberg etal., 1972; Barriere and Argillier, 1993; Barriere
et al., 1994). For these reasons, the brown midrib mutants, and
especially bm3, have been selected as a forage crop. How-
ever, the types of mutations that have occurred in brown midrib
mutants and their relationship with the lignification process
are still not known.
Our group recently characterized the first gene encoding
a lignin-related OMT in maize (Collazo et al., 1992). We showed
that this gene encodes a bifunctional enzyme able to methyl-
ate both caffeic and 5-hydroxyferulic acids to produce ferulic
and sinapic acids, respectively. This caffeic acid OMT (COMT;
EC 22.214.171.124) protein is encoded by a single gene, although an-
other O/WT-related gene (44% homologous to the previous one)
has been described in maize and is probably involved in the
suberin biosynthesis pathway (Held et al., 1993). The lignin-
related COMT gene is expressed in tissues undergoing lig-
nification; these include elongating roots and vascular bundles
in leaves (Collazo et al., 1992). A similar expression has been
described for a number of other lignin-related OMTcDNAs
(Bugosetal., 1991; Gowri etal., 1991; Dumas etal., 1992;Jaeck
et al., 1992; Pellegrini et al., 1993). The obvious importance
of maize brown midrib mutants and the availability of a COMT
probe from maize allowed us to analyze further the effect of
the brown midrib mutation on the expression of the COMTgene.
The experiments described here report the molecular char-
acterization of the lignification-related bm3 mutation in maize.
COMT Gene Expression Is Modified by the bm3
The maize COMT gene is known to be strongly expressed in
roots and to a lesser extent in the stem and in leaves (Collazo
et al., 1992). To determine whether any of the brown midrib
mutations modified COMTgene expression, different tissues
of various brown midrib mutants were subjected to RNA gel
blot analysis. RNA was first extracted from roots harvested from
bml, bm2, and brr>3 homozygote mutants maintained in W23 x
L317 hybrids and hybridized with the 5' coding region (XS1,
Figure 1A) of the wild-type maize COMTgene as a probe. Fig-
ure 1B shows that bm1 and bm2 mutants exhibited the same
expression pattern, as did the wild type. Thus, neither bm1 nor
bm2 mutations appeared to alter COMTmRNA accumulation,
WT bml bm2 bm3
W401 WxL W64A
S e e
f- ja £>
r^ f'l f'J f'l
Figure 1. Expression of the Maize COMT Gene in Different brown
(A) Schematic representation of the MG18 COMTgene (Collazo et al.,
1992). Exons are shown as solid boxes and the single intron as an
open box. The positions of the different probes used in hybridization
procedures are indicated below the scheme. Oligonucleotides used
for PCR experiments are numbered and marked with arrows above
and below the gene. The insertion site of the B5 element in the COMT
is indicated with an open triangle.
(B) COMT mRNA accumulation in roots from brown-midrib mutants
maintained in W23 x L317 hybrids. RNA gel blot analysis was per-
formed from wild-type (WT) or brown-midrib homozygote plants for three
different loci: brown-midribl (bm1), brown-midrib2 (bm2), and brown-
midribS (bm3). Probes XS1 and SS1 shown in (A) or LTR were radiola-
beled and used to hybridize to the blot. The length of the hybridizing
bands is indicated at right in base pairs.
(C) CO/WTmRNA accumulation in roots from brown-midribs mutants
maintained in pure lines. RNA gel blot analysis was performed from
bm3 homozygote plants maintained in W64A and W401 pure lines
(bm3-2). WT refers to the W64A and W401 wild-type plants, and bm3-1
refers to the brown-midribs homozygote from (B) maintained in W23 x
L317 hybrid (WxL). The length of the hybridizing bands is indicated
at right in base pairs.
The bm3 Gene in Maize
m cDNA - ........ KCOCCOC(ICCCOTCT~CCCC~COA~C~O~TC~~CC~CCT~~~~~C~~AC~~TCCTCA 480
b m 3 p r o t . ........ 3
P V C K W L
P N E D
V 9 M A
Q D K V L
M E 9 W Y Y L
- ........ B
P V C K
T P N E D O
A L M N
Q D K V
W Y Y L
CUT Cm - A~-~IcC~~~~OTAC~A~C~~~C
......................................................... end 1360
W p r O t
A V L D O
F N K A Y Q M T
bnu cDNA . AW~CTATOWCCQTCO~~CQ~CCO~~~~T~~~AQTATOWT~~AOC~
3 5 2
LTi( - A~CTAT
-K E L
E W P c N Q W 3
bm3 cDNA . CITA~QbATATln3cCT~
Figure 2. Comparison of COMT cDNA Sequences Obtained from WildType W64A and the bm3-7 Mutant of Maize.
The sequence alignment involves COMT sequences of the wild-type MC1 (OMT cDNA), bm3-7 mutant (bm3 cDNA), and the LTR sequence of
the B5 element (wxB5 LTR). Nucleotide 520 from which bm3 cDNA strongly diverges from MCl COMTcDNA is marked with a thin vertical arrow.
The position of the COMTgene intron in the corresponding genomic clone MG18 is indicated with a solid arrowhead. The “end at position 1360
indicates the size of the wild-type COMTcDNA. A 6-bp sequence that could represent a putative polyadenylation signal in bm3 cDNA is under-
lined. The stop codon in the bm3 COMT sequence is marked with an asterisk. Protein sequence alignment between the wild-type COMT (OMT
prot) and bm3-derived chimeric protein (bm3 prot) is shown below the nucleotide sequences. The two double-underlined sequences in wxB5
LTR represent the two 6-bp inverted repeat sequences of the LTR of the 85 element. Vertical double dots indicate a sequence identity. The Gen-
Bank, EMBL, and DDBJ accession number of the bm3-7 COMT cDNA is X80669.
indicating that the COMTgene was not affected by these mu-
tations. In the bm3 mutant, however, the length of the band
hybridizing to the XS1 probe was clearly shorter and the in-
tensity of the signal greatly reduced (Figure 1B). This analysis
indicated that ~ 6 0 0 nucleotides of the COMTmRNA were lack-
ing in this mutant. An identical result was obtained when the
same experiment was carried out with RNA extracted from bm3
mesocotyls or leaves (data not shown). These results sug-
gested that transcription of the COMT gene terminated early
or that a post-transcriptional regulation event removed a por-
tion of the COMT mRNA.
To determine which part of the COMTgene was lacking in
the COMTmRNA, the same blot was hybridized with a probe
corresponding to the 3’coding region of the COMTgene (SS1,
Figure 1A). With such a probe, we observed a similar hybrid-
ization pattern for the wild type and bm7 and bm2 mutants
(Figure 16). For the bm3 mutant, a different result was obtained.
In this plant, the smaller band previously revealed with the
XS1 probe could not be detected, suggesting that at least the
SS1-corresponding region in the COMTgene was lacking in
the COMT mRNA (Figure 1B).
All of these analyses were also performed with bm3
homozygote mutants maintained in pure W64A and W401
maize lines (bm3-2, Figure 1C). In bm3 roots of both W64A
and W401 lines, no mRNA corresponding to COMT was de-
tected with either the XS1 (Figure 1C) or SS1 (data not shown)
probe. The same results were observed when RNA analysis
was carried out on other tissues (data not shown). All of these
results suggested that at least two events affecting the COMT
locus may have occurred in bm3 mutants.
A bm3 Mutation Produces a Chimeric COMT mRNA
To characterize further the bm3 mutation, we cloned and se-
quenced the bm3 cDNA corresponding to the smaller COMT
mRNA detected in the bm3 mutant maintained in the W23 x
L317 hybrid (so-called bm3-7 mutant). A part of the bm3-7 COMT
cDNA nucleotide sequence, shown in Figure 2, appears iden-
tical to the wild-type COMTcDNA (MC1 cDNA; Collazo et al.,
1992), up to nucleotide 519 (Figure 2). Beyond this point, the
bm3-7 cDNA nucleotide sequence strongly diverges from the
wild-type COMTcDNA and is identical to the long terminal re-
peat (LTR) of the 65 element, a retrotransposon originally
described as interrupting the waxy gene in maize (Varagona
et al., 1992). The sequence of the 65 LTR in the COMTmRNA
provides a new stop codon and a putative polyadenylation
site producing a shorter mRNA, which is consistent with data
previously obtained by RNA gel blot analysis. As a conse-
quence, the presence of the 65 LTR drastically changes the
open reading frame of the chimeric transcript (Figure 2). The
deduced chimeric protein appears shorter than the COMT
410 The Plant Cell
protein (209 amino acids rather than 364) and lacks some
of the conserved domains described for all of these proteins
(Collazo et al., 1992).
To check for the presence of the LTR of the B5 element in
CO/WTmRNA from the£>m3-7 mutant, the region of bm3-1 cDNA
corresponding to the LTR was used as a probe to hybridize
to the previous RNA gel blot. Figure 1B shows that an mRNA
was detected in bm3-1 roots (Figure 1B) that corresponded to
the one previously detected with the XS1 probe (Figure 1B).
No COM7mRNA was detected with such a probe in either the
wild type or plants carrying other mutations. We also checked
for the presence of this transcript in bm3-1 mesocotyls and
leaves and obtained an identical result (data not shown). These
experiments confirmed the presence of the LTR-corresponding
portion of the B5 element in bm3-1 COMT mRNA.
Two bm3 Mutations Arise from Retrotransposition and
Deletion in the COMT Gene
The data obtained by RNA gel blot analysis and bm3-1 cDNA
sequencing indicated that COMT mRNA was modified in the
bm3-1 mutant maintained in the W23 x L317 hybrid or was
undetectable in the bm3-2 mutant maintained in pure lines.
Thus, it was of interest to correlate these data with changes
occurring in bm3 alleles by DNA gel blot analysis. Probes were
chosen to different regions of the COMT"gene and used to com-
pare the structure of both the W23 x L317 hybrid (wild type
and bm3-1 mutant) and W401 (wild type and bm3-2 mutant)
alleles. The probes from the coding region were the same as
those used for RNA gel blot analysis (XS1 and SS1). An INT
probe corresponding to the intron (Figure 1A) present in the
COMT gene (Collazo et al., 1992) was also used.
Figure 3 demonstrates that bm3 alleles are polymorphic in
the COMT locus because different hybridizing bands were ob-
served, depending on the probe used. In the wild type of the
W23 x L317 hybrid, the same band (15kb) was detected using
the three probes (Figure 3). In the corresponding bm3-1 mu-
tant, both XS1 and INT probes detected a polymorphic 5.5-kb
fragment, whereas SS1 recognized a shorter 3.5-kb fragment.
This result indicated that a change occurred in bm3-1 that pro-
duced a restriction fragment length polymorphism (RFLP). It
also suggested that this modifying event was produced at the
junction of the 3' coding region of the intron, which is consis-
tent with data obtained bybm3-1 cDNA sequencing. The result
obtained for bm3-1 could be related to B5 element insertion.
Using the region of the bm3-1 cDNA corresponding to the LTR
as a control probe, a 5.5-kb hybridizing band was detected.
It corresponded to the 5.5-kb hybridizing band detected with
both XS1 and INT probes (data not shown).
The same experiment was carried out with the bm3-2 mu-
tant and the corresponding W401 alleles from which no COMT
transcript was found (Figure 1C). In the W401 wild-type allele,
the three COA/fT-specific probes detected the same 12-kb frag-
ment (Figure 3). In the bm3-2 mutant allele, the hybridization
pattern obtained with both XS1 and INT probes also indicated
a polymorphism because a 13-kb fragment was observed. In
bm3-2, however, the 3' coding region of the COMfgene ap-
peared to be lacking because the 13-kb band that hybridized
with the XS1 and INT probes could not be detected. This re-
sult suggested that another mutational event, different from
the one detected in bm3-1, had occurred in bm3-2. This sec-
ond change also affects the COM7gene at the junction of the
3' coding region of the intron. The lack of a hybridizing band
when using the SS1 probe indicated that this event might be
a deletion of a part of the COMT locus.
The relationship between COM7and bm3 was strengthened
by data obtained by mapping the COMT gene on the Pioneer
Hi-Bred (J ohnston, IA) RFLP map with the MC1 cDNA probe
(Collazo et al., 1992). Two populations of maize were analyzed
(M.L. Katt, Pioneer Hi-Bred Int., personal communication). In
the first population, COMfwas placed 8.5 map units proximal
to BNL05.46 and 2.1 map units distal to BNL15.45. In the sec-
ond population, it was located 21.4 map units proximal to
BNL05.46 and 8.3 map units distal to umc47. All of these RFLP
markers are localized in the same region of maize chromo-
some 4 in which the bm3 locus has been shown to be mapped
(Neuffer et al., 1968; Cherney et al., 1991).
WxL W401 WxL W401WxL W401
Figure 3. Analysis of Wild-Type and bm3 Maize Genomes by DNA
Gel blot analysis was performed with 10 ng of genomic Hindlll-digested
DNA from wild-type (WT) and the corresponding bm3 mutants (bm3-1,
bm3-2) of the W23 x L317 hybrid (WxL) and pure W401 (W401) maize
lines. The length of the hybridizing bands is indicated at left in kilobases.
The bm3 Gene in Maize 411
g 1 g 1 1
!> .a >
/ ** V <s \ 00
Figure 4. Analysis of Wild-Type and bm3 Mutant Genomes by PCR.
Different regions of the COMT gene were PCR amplified from wild type
(WT) and bm3 mutant (bm3-1, bm3-2) genomic DNA isolated from W23
x L317 hybrid (WxL) and pure W401 (W401) lines. The primers used
are described in Figure 1A. Amplifications using the same primers
were also performed on genomic clone MG18 (Collazo et al., 1992)
as positive controls. PCR products were separated on 1% agarose
gels, blotted onto nylon membranes, and hybridized with the probe
corresponding to the amplified region of the COM! gene (see Figure
1A). Probes and primers are indicated at left, and the length of the
bands is given at right in base pairs.
Analysis of bm3 Alleles by Polymerase Chain Reaction
The data obtained by DNA gel blot analysis reinforced the pre-
sumption that two mutational changes affecting COMToccurred
in the same region of the gene. To confirm and to characterize
further the types of mutations, we used a polymerase chain
reaction (PCR) approach involving Hindlll-digested DNA from
all lines and various COMT gene-specific primers.
Using two primers surrounding the COMT gene 5' coding
region (primers 6 and 9, Figure 1A), PCR amplification occurred
in all alleles (both wild types and mutants), as shown in Figure
4. Two other primers (primers 1 and 2, Figure 1A) surrounding
the COM7gene intron were then assayed in all lines. Primer
1 corresponded to the inverse sequence of primer 6, and primer
2 was chosen from the site of retrotransposon insertion ac-
cording to the sequence of bm3-1 COMT cDNA. With this
primer set, amplification occurred only in the wild-type alleles
(Figure 4). No PCR bands were detected in either bm3-1 or
bm3-2 mutant alleles, indicating that the region corresponding
to primer 2 was modified in both bm3 mutant alleles. When
primer 11 (Figure 1A), chosen 12 bp upstream of primer 2,
was used along with primer 1, an amplification product of the
expected size was derived from all of the alleles. This result
indicated that both bm3-1 and bm3-2 mutations took place
at the same region of the COMT gene, within the sequence
corresponding to primer 2 downstream of the intron, which
is consistent with the DNA gel blot analysis and the sequence
of the bm3-1 cDNA.
To confirm the insertion of the retrotransposon in the bm3-1
mutant, amplification of the 3' coding region was assayed by
using primer 10 (complementary to primer 11) and primer 13,
chosen 16 bp upstream of the stop codon of the COM7gene
(Figure 1A). Using such primers, the expected product was
obtained only from the wild-type alleles (Figure 4). Because
the 3' coding region of the COMT gene could be detected in
the bm3-1 mutant by DNA gel blot analysis (Figure 3), we
predicted that the absence of the PCR product from the bm3-1
allele was the result of the presence of the B5 retrotranspo-
son. This element, which was described as a 6.1-kb DNA
fragment (Varagonaetal., 1992), might not be amplifiable un-
der our PCR conditions. Alternatively, a Hindlll restriction site
introduced due to this insertion may have contributed to the
lack of amplification between primer 10 and primer 13 and
may explain the RFLP detected between bm3-1 and wild-type
To characterize further the bm3-2 mutation, bm3-2 and cor-
responding W401 wild-type alleles were first analyzed using
a set of primers (primers 10 and 13, Figure 1A) surrounding
the COMT 3' coding region. Figure 4 indicates that these
primers failed to amplify the expected region. This result rein-
forced the assumption that the bm3-2 mutation was produced
in the 3'coding region of the COMT gene. However, an identi-
cal result was obtained from the bm3-1 allele when using these
oligonucleotides (Figure 4). Therefore, we tested another set
of primers (primers 12 and 13, Figure 1A) to understand the
difference between the two mutant alleles observed by DNA
gel blot analysis (Figure 3). Primer 12 was designed from the
3' coding region of the COMT gene, downstream of the site
of retrotransposon insertion according to the sequence of the
bm3-1 COMT cDNA. Amplification between primer 12 and
primer 13 normally produced the expected band from both the
wild-type and bm3-1 alleles, but the band did not occur from
the bm3-2 allele (Figure 4). The lack of a PCR product from
the bm3-2 allele indicated that the region containing primers
12 and 13 had been deleted in the corresponding mutant. This
is consistent with the absence of a hybridizing band in the
DNA gel blot analysis from the bm3-2 allele using the SS1
probe (Figure 3).
The Leaf Midrib Is a Location of High COMT Gene
The aforementioned data indicated that COM7"is the gene af-
fected by mutations in bm3 maize lines. On the other hand,
the typical reddish brown pigmentation in bm3 mutants is as-
sociated with the lignified vascular bundles of the leaf midrib
(Cherney et al., 1991). Thus, we investigated the cellular dis-
tribution of COMT mRNA to check whether it is present in the
same cells in which the brown midrib phenotype is visible. For
412 The Plant Cell
Figure 5. Cellular Expression of the COMT Gene in a Wild-Type Maize Leaf.
Bright-field micrographs of 7- to 8-nm-thick tissue sections are shown. In (A) and (C), sections were hybridized with 35S-UTP labeled MC1 an-
tisense probes, and in (B), the MC1 sense probe was used. The presence of hybridizing transcripts is indicated by the silver grains that appear
as black spots.
(A) Transverse section of a young leaf emerging from the coleoptile.
(B) Same view as shown in (A) but obtained after hybridization with the sense probe as negative control.
(C) Detail of the leaf midrib showing the vascular bundles in which COMT" gene expression occurs.
The bm3 Gene in Maize
this purpose, in situ hybridization was carried out with wild-
type maize leaves in which COMTmRNA is normally detected
by RNA gel blot analysis (Collazo et al., 1992). We used the
coding region of MC1 cDNA as a probe (Collazo et al., 1992).
Figure 5A demonstrates that COMTmRNA accumulated to high
levels in the leaf midrib. The cellular distribution of the COMT
mRNA in the leaf midrib corresponded to the cells undergo-
ing lignification located around the differentiated xylem vessels.
The COMT mRNA was specially abundant in the cells of the
sclerenchyma localized under the epidermal layer (Figure 5C).
This result is consistent with the cellular localization of the
brown midrib phenotype observed in maize (Cherney et al.,
1991). In the leaves of bm3 mutants, subepidermal cells ac-
cumulating chimeric COMT mRNA produce a reddish brown
pigmentation visible from the surface. In stem or in root, the
cells expressing COMT mRNA are interna1 (data not shown),
which explains why the typical reddish brown pigmentation
is not visible.
Here, we report the molecular characterization of bm3 muta-
tions in maize and demonstrate that they were produced in
the gene coding for the COMT, a key enzyme in the lignin bio-
synthetic pathway. Two mutant alleles of bm3 were found,
designated bm3-7 (reference allele) and bm3-2. In the case
of the bm3-7 mutation, a retrotransposon homologous to the
B5 family of maize was found to be inserted in the COMTgene.
This 85 element previously had been described as interrupt-
ing one of the waxy gene introns (Varagona et al., 1992). The
insertion of the 85 element in the COMT gene introduced a
new stop codon and a polyadenylation signal that produced
a shorter COMT mRNA. As a consequence, the COMT pro-
tein synthesized in the bm3-7 mutant is considerably shorter.
Specifically, it lacks some of the conserved domains of the
OMTs that have been described up to now and that are thought
essential for their function (Collazo et al., 1992).
Our work also shows that the second bm3 mutation (bm3-2)
resulted from a deletion that removed at least a portion of the
3'region of the COMTgene. The results obtained by DNA gel
blot and PCR analyses indicated that this bm3-2 mutation was
produced in the same region of the COMTgene in which the
bm3-7 mutation occurred. As a consequence of both muta-
tions, COMTgene expression was dramatically altered. Using
severa1 PCR analyses, we did not observe any change in the
COMT gene promoter that might have suggested a modifica-
tion of gene regulation. However, without further sequence
information, it is not possible to rule out this eventuality. Alter-
natively, the lower leve1 of chimeric COMT transcript detected
in the bm3-7 mutant and the absence of COMT mRNA in the
bm3-2 mutant in which the COMTgene appeared to be trun-
cated could be the result of post-transcriptional regulation due
to the instability of the modified mRNAs.
In both cases, molecular and genetic data demonstrated that
bm3 mutations occur in the COMT gene. These data also en-
abled us to observe the effect of disrupting the COMT gene
encoding a key enzyme for lignin biosynthesis and to explain
the lower and chemically modified lignin content usually ob-
served in these mutants (Kuc et al., 1968; Gaudillere and
Monties, 1989; Barrière et al., 1994). Moreover, these data are
consistent with the lower OMT activity previously described
in such maize lines (Grand et al., 1985; Lapierre et al., 1988).
Thus, the availability of the COMTprobe and its identity to bm3
may enable its use in RFLP or PCR-derived methods for breed-
ing programs that include bm3 genes and allow a study of the
relationship between the different bm3 alleles.
Although OMT activity and lignin content are found to be
lower in bm3 mutants, they are still detectable (Grand et al.,
1985; Cherney et al., 1991). Considering that lignin-related
COMT was shown to be encoded by a single gene in maize
(Collazo et al., 1992), we suggest that other methyltransfer-
ases exhibiting a lower specificity and efficiency toward COMT
substrates may be functional in the lignin biosynthesis path-
way. The relative nonspecificity of these enzymes has been
demonstrated in plants (Bugos et al., 1991; Edwards and Dixon,
1991; Collazo et al., 1992), and other OMTs normally involved
in other pathways, such as flavonoid and isoflavonoid path-
ways (Vernon and Bohnert, 1992; Held et al., 1993; Maxwell
et al., 1993), or disease resistance (Schmitt et al., 1991) could
act in an alternative pathway when COMT activity fails. This
hypothesis is supported by recent data concerning a caffeoyl-
coenzymeA 3-O-methyltransferase involved in disease resis-
tance, which has been shown to be effective in the lignification
pathway (Ye et al., 1994). The activity of such enzymes might
explain the low and modified lignin content typically observed
in bm3 mutants.
Antisense technology with genes involved in the lignin path-
way has been used in other species and appears effective in
lowering the lignin content (Dwivedi et al., 1994; Halpin et al.,
1994; Ni et al., 1994). In tobacco, OMT antisense plants were
obtained that exhibited a significant reduction in lignin con-
tent (Ni et al., 1994). In the same species, CAD antisense plants
were produced with a modified lignin structure that probably
would be more accessible to chemical degradation (Halpin et
al., 1994). For such purposes, antisense methodology appears
useful for species in which no mutants related to the lignifi-
cation process have been described. However, our results
indicated that the characterization of lignin-related mutants
might be another useful approach that could be designed for
species, including maize.
Maize mutants, such as bm3, naturally exhibit the proper-
ties of low lignin content and high digestibility (Kuc et al., 1968;
Gentinetta et al., 1990; Barrière and Argillier, 1993; Barrière
et al., 1994) that are usually the objective of antisense meth-
odology. In some cases, CAD antisense tobacco plants were
shown to produce a pigmentation similar to the bm3 mutant
in the leaf midrib (Halpin et al., 1994), indicating that compara-
ble events may occur. Because the COMT mRNA appeared
highly abundant in the leaf midrib in maize, the characteristic
414 The Plant Cell
pigmentation may be explained as the consequence of the
accumulation of an unknown phenolic derivative dueto block-
age of the normal lignin pathway. Studying well-known mutants
related to the lignin biosynthesis pathway to identify mutated
genes thus appears an alternative to other methods. Analyz-
ing the effects of disrupting such genes of interest enables
possible sites and effects of modification of this important path-
way to be evaluated.
All of the brownmidrib mutants analyzed here are homozygous geno-
types. Seed of b w n midribl (bml), bm2, and bm3 homozygote mutants
maintained in W23 x L317 hybrids were provided by E.B. Patterson
(Maize Genetics Stock Center, University of Illinois, Urbana, IL). The
bm3 allele from the corresponding mutant (the reference allele) is re-
ferred to in this study as bm3-7.
Another batch of seed of bm3 homozygote mutants was a gift of
Y. Barrière (Institut National de Ia Recherche Agronomique, INRA, Lu-
signan, France). The original seeds were furnished to the INRA in 1972
by L.F. Bauman (Department of Agronomy, Purdue University, Lafayette,
IN) under code number 95096-102, without further characterization.
Later, the bm3 mutants were maintained in W64A and W401 pure in-
bred maize at the INRA of Lusignan. The bm3 allele from the mutant
maintained in the W401 line, which should be given as bm3-95096-
702, is referred to in this study as bm3-2, for ease of presentation.
All plants were grown on filter paper for 7 days and stored at -8OOC.
Preparation of OMT Probes
Probes XSl and SS1 were obtained by digestion of a caffeic acid
Crmethyltransferase (COMJJ genomic clone MG18 (Collazo et al., 1992)
using Xhol/Sacl and Sal1 restriction enzymes, respectively. Probe INT
corresponding to the COMJgene intron was obtained by polymerase
chain reaction (PCR) amplification of genomic clone MGl8 using
primers 1 and 11 (Figure 1A). LTR is the region of the bm3-1 cDNA
corresponding to the long terminal repeat of the B5 element. This probe
was obtained by PCR amplification of bm3-7 cDNA subcloned in
pBluescript SK+ (Stratagene) using primer 1 (Figure 1A) and
pBluescript T7 primer. MC1 probe is the full-length COMJcDNA en-
coding COMT from W64A (Collazo et al., 1992).
RNA lsolation and RNA Blot Analysis
Total RNA was isolated using lithium chloride precipitation and phenol-
chloroform extractions according to Verwoerd et al. (1989). Total RNA
(10 pg) harvested from 7-day-old roots was separated on a 13% agarose
gel following standard procedures (Sambrook et al., 1989) and blotted
onto a nylon membrane. The hybridization step (16 hr at 65OC) was
carried out in 250 mM Na2HP04, 7% SDS, 1 mM EDTA, 100 pg mL-'
salmon sperm DNA containing radiolabeled probe (Feinberg and
Vogelstein, 1984). Washes were performed three times for 20 min each
in 20 mM NazHP04, lO/o SDS, 1 mM EDTA at 65OC.
cDNA Library Construction, Screening, and DNA Sequencing
Poly(A)+ RNA was isolated from total RNA using the Poly-A-Tract sys-
tem (Promega). The cDNA library was constructed in h ZAPll
(Stratagene) with 2 pg of poly(A)+ RNA from 7-day-old bm3-7 hybrid
roots. The coding XS1 probe was used to screen 300,000 plaques ac-
cording to standard procedures (Sambrook et al., 1989). Positive clones
subcloned in pBluescript SK+ after phagemid in vivo excision were
sequenced using the chain termination method (Sambrooket al., 1989)
and automatic ALF sequencer (Pharmacia, Sweden). Searches in the
data bases (EMBL, GenBank, and DDBJ) and sequence comparisons
were performed with the program package, including FASTA 7.2
(Genetics Computer Inc., Madison, WI).
Genomic DNA lsolation and DNA Blot Analysis
Maize genomic DNA was extracted from 7-day-old leaves, using the
standard protocol of Dellaporta et al. (1983). Approximately 10 pg of
DNA was digested with Hindlll and separated on a 0.8% agarose gel.
DNA gel blot hybridization was performed as described for total RNA.
Washes were performed at 65OC as follows: two washes for 20 min
each in 40 mM NaZHPO4, 1% SDS, 1 mM EDTA, and one wash for
10 min in 20 mM NazHP04, 0.5% SDS, 1 mM EDTA.
Polymerase Chain Reaction Amplifications
The location of the primers used for PCR amplifications is shown in
Figure 1A. PCR analyses were performed with 2 vg of Hindlll-digested
genomic DNA for more efficient amplifications, and with 50 ng of non-
digested DNAfor genomic clone MG18 (Collazo et al., 1992). Primers
were chosen from the sequence of clone MG18 as follows (from 5'to
3'): primer 1, ATGAACCAGGACAAGGTCCTCATG; primer 2, CATCCCG-
TACGCCTTGTTGAACGG; primer 6, inverse complementary to primer
1; primer 9, TAATCGTAATAGCCATGGGCTCCA; primer 10, GACGGC-
GGCATCCCGTTCAAC; primer 11, inverse complementary to primer
10; primer 12, GGCACGGACGCGCGCTTCAAC; primer 13, GCCCAG-
GCGTTGGCGTAGATG. PCR amplification consisted of 30 cycles at
94OC for 2 min, 63OC for 2 min, 72OC for 3 min. PCR products were
subjected to 1% agarose gel electrophoresis, transferred onto a nylon
membrane, and hybridized with the appropriate probe, as described
for total RNA.
In Situ Hybridization
In situ hybridization was performed on 7- to 8-pm sections of 7-day-old
leaves, using procedures described by Stiefel et al. (1990) and Langdale
(1994). Sense and antisense MCl probes were synthesized with 1 pg
of linearized DNA using %-UTP. Labeled transcripts were then hydro-
lyzed by using an alkali treatment. These transcripts were used in a
hybridization step as described by Langdale (1994). Photography was
carried out using bright-field microscopy and Ektachrome 160 ASA
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The brown midrib3 (bm3) Mutation in Maize Occurs in the Gene Encoding Caffeic Acid
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