Download The Maize MADS Box Gene ZmMADS3 Affects

The Maize MADS Box Gene ZmMADS3 Affects Node
Number and Spikelet Development and Is
Co-Expressed with ZmMADS1 during Flower
Development, in Egg Cells, and Early Embryogenesis1
Sigrid Heuer, Susanne Hansen, Jörg Bantin, Reinhold Brettschneider, Erhard Kranz, Horst Lörz, and
Thomas Dresselhaus*
West Africa Rice Development Association, B.P. 96, St. Louis, Senegal (Si.H.); and Center of Applied Plant
Molecular Biology (AMP II), University of Hamburg, Ohnhorststrasse 18, 22609 Hamburg, Germany (Su.H.,
J.B., R.B., E.K., H.L., T.D.)
MADS box genes represent a large gene family of transcription factors with essential functions during flower development
and organ differentiation processes in plants. Addressing the question of whether MADS box genes are involved in the
regulation of the fertilization process and early embryo development, we have isolated two novel MADS box cDNAs,
ZmMADS1 and ZmMADS3, from cDNA libraries of maize (Zea mays) pollen and egg cells, respectively. The latter gene is
allelic to ZAP1. Transcripts of both genes are detectable in egg cells and in in vivo zygotes of maize. ZmMADS1 is
additionally expressed in synergids and in central and antipodal cells. During early somatic embryogenesis, ZmMADS1
expression is restricted to cells with the capacity to form somatic embryos, and to globular embryos at later stages.
ZmMADS3 is detectable only by more sensitive reverse transcriptase-PCR analyses, but is likewise expressed in embryogenic
cultures. Both genes are not expressed in nonembryogenic suspension cultures and in isolated immature and mature zygotic
embryos. During flower development, ZmMADS1 and ZmMADS3 are co-expressed in all ear spikelet organ primordia at
intermediate stages. Among vegetative tissues, ZmMADS3 is expressed in stem nodes and displays a gradient with highest
expression in the uppermost node. Transgenic maize plants ectopically expressing ZmMADS3 are reduced in height due to a
reduced number of nodes. Reduction of seed set and male sterility were observed in the plants. The latter was due to absence
of anthers. Putative functions of the genes during reproductive and vegetative developmental processes are discussed.
The development of highly specialized plant organs
from undifferentiated meristematic cells is a complex
process and requires a cascade of regulatory genes
controlling e.g. the differentiation of distinct flower
organs from the apical meristem (for review, see Levy
and Dean, 1998). With the recent discovery of individual genes that, when deregulated, cause homeotic
transformation of flower organs, underlying regulatory mechanisms have started to be illuminated. Many
of these genes code for MADS box transcription factors, acting at early stages in the organ developmental
program (Riechmann and Meyerowitz, 1997; Theißen
et al., 2000). Since the isolation of the first plant MADS
box transcription factor genes, AGAMOUS and DEFICIENS, about 10 years ago (Sommer et al., 1990;
Yanofsky et al., 1990), numerous MADS box genes
have been isolated from various mono- and dicotyledonous flowering plants, but also from ferns and
fungi (Krüger et al., 1997; Münster et al., 1997). MADS
box proteins bind to DNA at specific binding sites
1
This work was supported in part by the Körber foundation
(Hamburg, Germany), by the Deutsche Forschungsgemeinschaft
(grant nos. Kr1256/1– 4 and Dr334/2–1), and by the European
Commission (grant nos. BI04 –CT960390 and BI04 –CT960210).
* Corresponding
author;
e-mail
[email protected]; fax 49 – 40 – 42816 –229.
(CarG boxes) as homo- and/or heterodimers regulating their own transcription and that of target genes
(see West et al., 1998, and references therein).
Intensive studies on mutant plants clearly demonstrated the essential, homeotic role of MADS box proteins in the development of the four distinct flower
organs (sepals, petals, stamen, and carpels) and led to
the formulation of the ABC model (Weigel and Meyerowitz, 1994). Because it was demonstrated that the
petunia (Petunia hybrida) MADS box gene FBP11 is
exclusively expressed in whorl 4 and induces ovule
development on sepals when ectopically expressed,
this model has been extended to the ABCD model
(Colombo et al., 1995).
Detailed analyses of AGL2, 4, and 9 (renamed SEPALLATA 1, 2, and 3) recently showed that these
genes represent a novel class of organ identity genes
(class E). It was demonstrated that SEP3 interacts with
ABC function proteins and that ternary and quartary
complexes are probably the molecular basis for regulation of flower development (Pelaz et al., 2000;
Honma and Goto, 2001; Theißen and Saedler, 2001).
Before ABCDE genes determine organ identity of the
distinct whorls, meristem identity genes regulate the
transition of vegetative meristems into inflorescence
and flower meristems. A third group of genes is expressed after the onset of meristem identity genes but
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Heuer et al.
before organ identity genes are detectable and have
been referred to as intermediate or identity-mediating
genes (for review, see Gutierrez-Cortines and Davis,
2000).
Functional analyses of MADS box genes have been
performed mainly with plants possessing bisexual
flowers, e.g. Arabidopsis and tobacco (Nicotiana sp.),
for which efficient transformation systems and numerous mutants are available. Comparably few studies have been performed with plants developing unisexual flowers, e.g. maize (Zea mays). During maize
ear and tassel development, male and female organs
are initiated, but stamen in ear spikelets and the
gynoeceum in tassel spikelets do not reach maturity
(for review, see Cheng et al., 1983). Some maize MADS
box genes have been isolated and exclusive expression
in developing ears has been shown for ZAG2, where
expression is largely restricted to developing carpels
(Schmidt et al., 1993). Other maize MADS box genes
are expressed in developing male and female inflorescences (Schmidt et al., 1993; Fischer et al., 1995; Mena
et al., 1995, 1996; Cacharrón et al., 1999).
Plant MADS box genes are also expressed in mature flowers where they have been detected for example in the stigma, style, and ovules (Flanagan et
al., 1996; e.g. Colombo et al., 1997). In addition, expression in female and male gametophytes, i.e. embryo sac and pollen, have been reported (e.g. Perry et
al., 1996; Heuer et al., 2000, and references therein).
MADS box gene expression in all organs and cell
types participating in the fertilization process indicate that they might regulate expression of genes
involved in pollen-stigma interaction, pollen tube
growth/guidance, embryo sac maturation, and the
onset of gene expression after fertilization. In addition, expression in zygotic and somatic embryos as
well as in endosperm have been described, so that
participation of MADS box proteins in regulatory
processes concerning embryo and endosperm development can also be assumed (Montag et al., 1995;
Filipecki et al., 1997; Perry et al., 1999; Alvarez-Buylla
et al., 2000).
We are interested in the double fertilization process
of higher plants and addressed the question of
whether MADS box genes are expressed in the cells
of the female gametophyte and at earliest stages of
zygote and embryo development. Here, we present
two novel MADS box genes of maize of which the
expression has been studied in detail in reproductive
as well as in vegetative tissues. To elucidate the function of these genes, we have ectopically expressed one
gene in maize and discuss the obtained phenotype.
RESULTS
ZmMADS1 and ZmMADS3 Represent Putative MADS
Box Transcription Factors
Two novel maize MADS box cDNAs, ZmMADS1
and ZmMADS3, were isolated after screening cDNA
34
libraries of maize egg cells (ECs) and mature pollen
under medium stringent conditions with the conserved MADS box region of various maize MADS
box genes as a probe. Predicted amino acid (AA)
sequences are illustrated in Figure 1 and are accessible at the EMBL and GenBank databases (accession
no. AF112148, ZmMADS1; and accession no.
AF112150, ZmMADS3). Both cDNAs encode proteins
possessing the motifs typical for MIKC-type MADS
box proteins (MADS box, I region, K box, and less
conserved C-terminal end). A putative bipartite nuclear localization signal (KR-[X]12KRR) can be outlined in the MADS box of both proteins (Fig. 1, A and
B; for review, see Dingwall and Laskey, 1991). According to a SWISS-MODEL protein structure prediction (Guex and Peitsch, 1997), ZmMADS1 and ZmMADS3 proteins form an N-terminal ␣-helical
structure (N13-C39) and two, C-terminal adjacent
␤-sheets (␤1, E42-F48; loop, S49-K53; ␤2, L54-A58; data
not shown).
ZmMADS1 and ZmMADS3 Belong to Distinct MADS
Box Subfamilies
Comparison of ZmMADS1 and ZmMADS3 protein
sequences with other MADS box proteins revealed
that ZmMADS1 can be classified as a member of the
TM3 subfamily of MADS box proteins, whereas ZmMADS3 belongs to the SQUAMOSA subfamily (Fig.
2). Alignments with the most homologous proteins
(for accession nos., see “Materials and Methods”) are
illustrated in Figure 1. For ZmMADS1, AA identity is
highest to the rice (Oryza sativa) clone S11905 (75%).
Within the C-terminal end, a highly conserved region
can be outlined in all aligned proteins (Fig. 1A).
ZmMADS3 exhibits 95% overall AA identity to the
maize MADS box protein ZAP1 (⫽MADSD; Mena et
al., 1995). Substitutions are mainly conservative and
both proteins additionally share Glu (Q)-rich clusters. At the very C-terminal end a cluster of nine AAs
is highly conserved among aligned proteins (Fig. 1B).
Using two recombinant inbred (RI) families of maize
(TxCM and COxTx), ZAP1 was mapped to the long
arm of chromosome 2 (2L193). We have used the
same RI families and have mapped ZmMADS3 to the
short arm of chromosome 7 (7S000).
ZmMADS1 and ZmMADS3 Are Expressed in ECs,
Zygotes, and Somatic Embryo-Forming Cells As Well
As in Stem Nodes during Vegetative Development
Single-cell reverse transcriptase (RT)-PCR analyses
showed that ZmMADS1 and ZmMADS3 are both expressed in maize ECs as well as in in vivo and in vitro
zygotes (Fig. 3). In contrast to ZmMADS3, ZmMADS1
transcripts are additionally detectable in synergids,
central cells, and antipodals. Zmcdc2, amplified as a
positive control, was detectable in all cells analyzed
(Fig. 3). Northern-blot analyses (Fig. 4) revealed ex-
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Plant Physiol. Vol. 127, 2001
Maize MADS Box Genes
Figure 1. Predicted AA sequence of ZmMADS1 and ZmMADS3, and alignment to MADS box proteins with high AA identity.
The MADS domain of ZmMADS1 (A) and ZmMADS3 (B) is illustrated in light-gray and the K domain in dark-gray boxes.
Conserved C-terminal regions are boxed. Gaps (⫺) were introduced to improve alignment and identical AAs are indicated
by asterisks. A putative nuclear localization signal is indicated by bold, italic letters. AAs highly conserved among MADS
box proteins are indicated by plus signs. Positions where conservative AA substitutions occur are indicated by dots. A Q-rich
region within the C-terminal end of ZmMADS3 is indicated by bold letters.
pression of ZmMADS1 and ZmMADS3 also in immature pistils as well as in non-pollinated and pollinated mature pistils (2 and 5 d after pollination
[DAP]). However, expression of both genes is undetectable in isolated immature (stage 2) and mature
embryos (Fig. 4). Analyses of distinct maize in vitro
culture systems indicated ZmMADS1 expression in
embryogenic suspension cultures and embryogenic
type II callus (Fig. 4). More sensitive RT-PCR analyses showed that ZmMADS1 is also expressed in embryogenic type I callus and confirmed lack of expression in nonembryogenic suspension cultures.
Expression of ZmMADS3 was not detectable by
northern-blot analysis (Fig. 4), but RT-PCR studies
showed a similar although weaker expression pattern
than that of ZmMADS1 in all embryogenic cultures
analyzed and expression was undetectable in nonemPlant Physiol. Vol. 127, 2001
bryogenic suspension cells (data not shown). Type II
callus and suspension cultures were analyzed in more
detail by RNA in situ hybridization (Fig. 5). Experiments were performed with competent type II callus,
which consists of a central area with large, highly
vacuolated cells and a peripheral part consisting of
smaller, less vacuolated cells (Fig. 5A). In this type of
callus, ZmMADS1 transcripts are mainly detectable in
the peripheral zone (Fig. 5B). At 7 d after the induction
of somatic embryogenesis on hormone-free medium,
ZmMADS1 transcripts accumulate in developing globular structures (Fig. 5, D and E). When somatic embryo and scutellar-like structures were further differentiated, ZmMADS1 transcripts centralized to the
embryo axis and outer cell layers (Fig. 5F). RNA in situ
analyses of embryogenic suspension cultures showed
that ZmMADS1 transcripts accumulate in sub-
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35
Heuer et al.
Figure 2. ZmMADS1 and ZmMADS3 belong to different MADS box
subfamilies. A homology search was performed with ZmMADS1 and
ZmMADS3 full-length cDNA sequences to identify MADS box genes
with sequence homology. Subsequent multiple alignments were performed with protein sequences of ZmMADS1 and ZmMADS3 (gray
boxes), most homologous proteins, and representatives of the MADS
box subfamilies. Note that ZmMADS1 is the only maize protein
within the TM3 subfamily. ZmMADS3 is a member of the SQUAMOSA subfamily and most similar to ZAP1. Names of subfamilies are
given at the junctions. Bar represents 10% AA substitution per site.
The tree is unrooted, bootstrap is 1,000.
peripheral cell layers, most likely constituted from
cells with embryogenic potential (Fig. 5H). No expression was detectable in the central part and the outermost cell layers of the cell aggregates as well as in
nonembryogenic callus (Fig. 5I). Hybridization of the
samples with a ZmMADS1 sense probe never gave
any signal (Fig. 5C). ZmMADS3 transcripts were not
detectable by in situ hybridization due to the low
expression level already pointed out above.
Expression of ZmMADS1 and ZmMADS3 during
Flower Development
The northern-blot analyses performed further revealed that ZmMADS1 and ZmMADS3 are coexpressed during ear and tassel development (Fig. 4).
36
Maize plants develop ear primordia at several stem
nodes, although depending on the variety, only one
or a limited number of ears reach maturity. Analyses
of immature ears isolated from nodes 5 through 7
showed that ZmMADS1 and ZmMADS3 expression is
highest in the ear isolated from node 7 (Fig. 4). This
corresponds to the most advanced stage of development among the ears analyzed and to the node where
the fully developed ear generally appears in inbred
line A188. More detailed in situ hybridization analyses of female flower development showed that transcripts of both genes are first detectable after two
spikelet primordia are differentiated from the female
inflorescence meristem (stage D; Fig. 6A) but not at
earlier stages (stage A/C, data not shown; for comparison of flower developmental stages, see Cheng et
al., 1983). Within single spikelet primordia, transcripts were detectable in the upper and the lower
floret as well as in glumes (Fig. 6, B, C, and F). This
pattern persisted throughout further development
and transcripts were detectable in all flower organs,
including the stamen primordia, which later abort in
the developing ear (Fig. 6, D and G). At more advanced developmental stages, when the silk can be
clearly distinguished (stage I/J), ZmMADS1 and ZmMADS3 transcripts were no longer detectable (Fig.
6E). No signals were obtained after hybridization
with ZmMADS1 and ZmMADS3 corresponding sense
probes (Fig. 6, H and K). Analyses of gene expression
during tassel development indicated that ZmMADS1
and ZmMADS3 are not expressed in tassel primordia
at very early stages of development (stage A/C; data
not shown). In tassels more advanced in development (after stage G/H), ZmMADS1 and ZmMADS3
Figure 3. Expression of ZmMADS1 and ZmMADS3 in female gametophytic cells and zygotes. Single-cell RT-PCR analysis was performed with individual maize egg cells (ECs), synergids (SYs), central
cells (CCs), and antipodal cells (APs), with primers specific for
ZmMADS1 (A) and ZmMADS3 (B), respectively. Zygotes (Z) were
analyzed at 24 h after pollination (hap; in vivo zygotes) and 14 to
29 h after in vitro fertilization (haf; in vitro zygotes), respectively.
Maize suspension cells (BMS) served as a control for vegetative gene
expression. Cells were washed four times after isolation and washing
buffer (WB) of the last wash step served as control for contamination
with cytoplasm of burst cells of the embryo sac, nucellus, or integument cells. Multiplex RT-PCR was performed with Zmcdc2-specific
primers as a control for successful RT-PCR. DNA fragments were
blotted after gel electrophoresis and hybridized to ZmMADS1-,
ZmMADS3-, and Zmcdc2-specific probes. Size of DNA fragments
and gene names are indicated.
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Plant Physiol. Vol. 127, 2001
Maize MADS Box Genes
Figure 4. Temporal and spatial ZmMADS1 and ZmMADS3 expression. RNA gel-blot analyses were performed with 10 ␮g
of total RNA of the tissues indicated and hybridized to ZmMADS1- and ZmMADS3-specific probes. As a loading control,
filters were hybridized to an 18S rRNA probe. Relative RNA amounts were determined with a phosphor imager. Size of RNA
fragments is indicated.
transcripts are most abundant in developing stamen
(Fig. 6, I and J). As reported earlier, ZmMADS1 is
expressed throughout pollen development with the
highest transcript abundance in microspores (Heuer
et al., 2000). Expression of ZmMADS3 was undetectable in the pistil primordia, which is still present in
developing maize tassels at the stage analyzed.
Within vegetative organs, ZmMADS1 is most abundant in leaves (Fig. 4). Low level of expression additionally was found in root tips and internodes (data
not shown). Whereas ZmMADS1 is expressed at a
low level only in nodes 5 and 6 (counted from the
first node above ground), ZmMADS3 is detectable in
all nodes analyzed, displaying a gradient with the
highest expression found in the last stem node immediately adjacent to the tassel (node 12; Fig. 4).
Preliminary results from in situ hybridization experiments performed with transverse and longitudinal
node sections indicate that ZmMADS3 is not expressed in vascular and parenchymatic cells, but in
cell layers consisting of small, non-vacuolated cells
probably representing meristematic cells (data not
shown).
Plant Physiol. Vol. 127, 2001
Ectopic ZmMADS3 Expression Affects Plant Height and
Male Spikelet Development
To gain insight into putative functions of ZmMADS3, immature maize embryos were transformed
with a full-length ZmMADS3 sense construct and a
ZmMADS3 antisense construct under the control of
the constitutive rice actin promoter. Taking the high
sequence identity of ZmMADS3 and ZAP1 into account, the antisense construct used for these experiments encompassed only the 3⬘-untranslated region
of the ZmMADS3 cDNA. Plants regenerated from
these experiments were transferred into the greenhouse for further cultivation and were monitored by
Southern- and northern-blot analyses until the F3
generation. Transgenic plants that integrated the antisense construct did not display a phenotype over
the generations analyzed, which might be due to the
short length of the antisense construct, and were not
analyzed further. The plant T0#12 (Fig. 7A) contained
five copies of the sense construct and full-length
transgene expression at a low level was determined
by northern-blot analyses of leaves, where ZmMADS3 is not detectable in WT plants (data not
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37
Heuer et al.
Figure 5. Expression of ZmMADS1 during somatic embryogenesis. RNA in situ hybridization
experiments were performed with type II callus
before (A–C) and after (D–F) induction of embryogenesis, with a maize embryogenic (G and
H) and nonembryogenic suspension culture (I).
Samples were hydridized to ZmMADS1 specific
probes in antisense (B, E, F, H, and I) and sense
orientation (representative experiment shown in
C). Arrows point at hybridization signals in developing globular structures (E), and at later
stages to a cluster of cells at the embryo axis and
outer cell layer (F). In embryogenic suspension
cultures, signals are restricted to subperipheral
cell layers (arrows in H). A signal was never
obtained in nonembryogenic suspension cultures (I). Bars represent 100 ␮m.
shown). The transgenic plant was strongly reduced
in height and developed no ear, whereas the basal
region of the apical tassel developed into ear-like
structures (Fig. 7A). The apical region of the tassel
showed no differentiation into male spikelets. Seeds
could not be obtained after pollination of the female
spikelets located at the tassel with WT pollen, which
prevented analyses of the progeny of this plant. The
plant T0#6 (Fig. 7B) contained two integrated copies
of the transgene and expression in leaves was higher
than in T0#12 (data not shown). The plant was male
sterile and strongly reduced in height compared with
control plants transformed with the selection marker
only (Fig. 7B, left). Leaf development was not affected (Fig. 7B). After pollination with WT pollen,
only 11 kernels developed that germinated normally.
The phenotype observed in T0 was confirmed in the
progeny: Tassels of representative plants of the T2
and T3 generation are presented in Figure 7C. Progeny plants that lost the transgene due to segregation
were always cultivated as control plants and developed normally (Fig. 7C, left). Plants ectopically expressing ZmMADS3 showed different levels of female and male sterility and were reduced in height
(Fig. 7, B and C). Seed set was reduced, but the grains
obtained after self-pollination and pollination with
A188 pollen germinated normally. The reduction in
height reflected a reduced number of nodes because
transgenic plants developed only eight to nine nodes,
in contrast to 12 nodes generally developed by WT
plants in the greenhouse. Tassels of transgenic plants
were smaller with a reduced number of branches in
comparison with control plants (Fig. 7, C–E). More
detailed analyses of the tassel of transgenic plants
showed that the outer glume appeared normal (Fig.
38
7, E, F, and H), whereas the inner glume was reduced
to a small, leaf-like structure (Fig. 7, F and H). No
differentiation of lemma, stamen, lodicules, and palea was apparent in the lower and the upper male
floret of transgenic plants (Fig. 7H).
DISCUSSION
ZmMADS3 Is Allelic to ZAP1 and Represents the
ZAP1b Gene
We have characterized two novel maize MADS box
cDNAs, ZmMADS1 and ZmMADS3, members of the
TM3 and SQUAMOSA subfamiliy of MIKC-type
MADS box proteins (Theißen et al., 2000), respectively. The high conservation of functional/structural units within the MADS and K box of ZmMADS1 and ZmMADS3 suggests that both proteins
are located within the nucleus, that they can bind to
DNA, and that they are capable of dimer formation.
As was determined for the human SRF core homodimer, the primary DNA-binding element is an
antiparallel coiled coil of two amphipathic ␣-helices,
one from each monomer (Pellegrini et al., 1995).
Dimerization of the monomers is permitted by interaction of the ␤-sheets forming a four-stranded antiparallel ␤-sheet. These structural domains are conserved in the ZmMADS1 and ZmMADS3 proteins.
ZmMADS3 exhibits 95% AA identity to the maize
MADS box protein ZAP1, which has been mapped at
2L193. A duplicated gene of ZAP1 (ZAP1b) has been
predicted based on RFLP mapping analyses (Mena et
al., 1995). We have mapped ZmMADS3 on 7S000, the
same position determined for ZAP1b. Therefore, we
propose that ZmMADS3 represents the ZAP1b gene.
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Plant Physiol. Vol. 127, 2001
Maize MADS Box Genes
Figure 6. ZmMADS1 and ZmMADS3 expression during spikelet development. RNA in situ
hybridization experiments were performed with
ZmMADS1- (A–E and J) and ZmMADS3- (F, G,
and I) specific RNA probes in antisense orientation. Representative sense control experiments
are shown in H and K. ZmMADS1 is expressed
in meristems of upper (uf) and lower (lf) ear
florets and in glume (gl) primordia at developmental stage D (A and B). At stage G and H (C
and D), ZmMADS1 expression is additionally
detectable in developing lemmas (l), stamen (st),
and the gynoeceum (gyn; gr, gynoecial ridge). At
stage I/J (E), ZmMADS1 is no longer detectable.
ZmMADS3 is expressed in an identical temporal
and spatial pattern but signals were always less
intense (F and G). During tassel development,
ZmMADS1 is expressed in lodicules (lo),
glumes, lemmas, and stamen (J). The ZmMADS3
expression pattern is identical but signals were
not obtained in gynoeceum primordia (I).
It was shown by Mena et al. (1995) that ZAP1 expression is excluded from mature stamen and carpels that
clearly distinguishes ZAP1 from ZmMADS3, which is
detectable in mature pistils. ZAP1 is not represented
in the cDNA library of ECs as was determined by
PCR with ZAP1-specific primers (data not shown).
As a consequence of its ancestral allotetraploid origin
(Leitch and Bennett, 1997), other maize MADS box
genes are reported to represent duplicated genes,
namely ZAG1/ZMM2, ZAG2/ZMM1, ZAG3/ZAG5,
and ZMM8/ZMM14, and likewise have developed
distinct expression patterns (Mena et al., 1995;
Theißen et al., 1995; Cacharrón et al., 1999).
ZmMADS1 and ZmMADS3 Expression Pattern Implies a
Function during Fertilization and Early Embryogenesis
Many of the MADS box genes described so far have
important functions during inflorescence development and flower organ differentiation, and only relatively few data are available for MADS box gene
expression in mature flowers. Transcripts of some
MADS box genes have been detected in mature
ovules (for review, see Riechmann and Meyerowitz,
1997), but so far AGL15 and AGL18 are the only
MADS box genes shown to be expressed in the cells
Plant Physiol. Vol. 127, 2001
of the embryo sac, without further specification of the
cell type (Perry et al., 1996, 1999; Alvarez-Buylla et
al., 2000). Therefore, ZmMADS1 and ZmMADS3 represent the first MADS box genes for which an expression in plant ECs and zygotes has been shown. Tight
temporal regulation of cell cycle regulatory genes
(cyclins) in maize zygotes demonstrated de novo
gene transcription before the first cell division of the
zygote takes place (Sauter et al., 1998). Changes of
transcript abundance in cDNA populations derived
from maize in vitro zygotes additionally has been
shown for distinct genes expressed in maize ECs
(Dresselhaus et al., 1999). These analyses showed that
zygotic gene activation in plants occurs already at the
one-cell stage and therefore earlier than in animals.
Transcription factors accordingly must be present
regulating this transcription activity. Co-expression
of ZmMADS1 and ZmMADS3 in ECs and zygotes
theoretically facilitates heterodimerization/interaction of the proteins (provided that RNA and proteins
are co-expressed). However, exclusive expression of
ZmMADS1 in the CC, SYs, and APs suggests ZmMADS1 interaction with yet unidentified partners.
Both genes are, although at highly different levels
of transcript abundance, expressed also during somatic embryogenesis of distinct in vitro culture sys-
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39
Heuer et al.
Figure 7. Ectopic expression of ZmMADS3 in transgenic maize plants. Immature maize embryos were transformed with a
pAct1::ZmMADS3::nosT full-length sense construct. Transgenic plants of the T0 generation with five (plant T0#12 shown in
A) and two integrated copies of the transgene (plant T0#6 shown in B, left) were reduced in height in comparison with
wild-type (WT) plants (B, right) and were male sterile. Progenies of plant T0#6 were reduced in height and developed small,
completely (C, left: plant T2#6.6.8), or partially sterile tassels (C, middle: plant T2#6.7.2). A progeny plant without the
transgene is shown for comparison (C, right). The phenotype was confirmed in the T3 generation: No anthers dehisced from
sterile spikelets (E) when control plants were at anthesis (D). Sterile transgenic spikelets developed an outer glume (og),
whereas the inner glume (ig) appeared as a small leaf like structure (arrows in F). Longitudinal 2-␮m sections of the regions
indicated in D and F were stained with Toluidine blue (G and H). In spikelets of WT plants, lemmas (le), lodicules (lo), palea
(p), and part of the filaments (f) and anthers (a) are visible (G). In transgenic spikelets, only the outer glume was differentiated,
whereas the other organs were missing or not fully differentiated and leaf-like structures (arrows) developed instead (H). Bars
represent 2 mm (D–F) and 300 ␮m (G and H), respectively.
tems analyzed. Before somatic embryos develop from
callus, ZmMADS1 is expressed in cells in the periphery of the callus and is subsequently detectable in
developing embryos, where transcripts are finally
restricted to specific cells at the periphery and the
embryo axis. This expression pattern is distinct from
that observed for other MADS box genes, which are
expressed in external cell layers of the radicular part
in heart stage somatic embryos (CUS1) or are not
restricted to specific regions (AGL15), respectively
(Filipecki et al., 1997; Perry et al., 1999). Neither
ZmMADS1 nor ZmMADS3 transcripts are detectable
in mature zygotic embryos indicating a specific function during early stages of embryo development. Because transgenic seeds germinated normally, ZmMADS3 overexpression has no obvious effect on
zygotic embryo and early seedling development.
ZmMADS1 and ZmMADS3 Are Expressed at
Intermediate Stages of Flower Development
ZmMADS1 and ZmMADS3 are also co-expressed
during flower development, where expression was
40
detectable in the upper and the lower floret only at
intermediate stages of development. This expression
pattern is distinct from that of other maize MADS
box genes. ZMM8 and ZMM14 are exclusively expressed in the upper floret of maize ear spikelets,
whereas ZAG1 and ZAG2 expression is restricted to
reproductive organ primordia (Schmidt et al., 1993;
Cacharrón et al., 1999).
At later stages of flower development, ZmMADS1
and ZmMADS3 become undetectable, but are again
expressed in mature pistils. Based on the signal intensity observed in northern-blot analyses, we assume that ZmMADS1 and ZmMADS3 are not exclusively expressed in the cells of the embryo sac, but
also in surrounding nucellus and/or integument tissues. This expression pattern is similar to that of
SEP1 (AGL2, Flanagan and Ma, 1994) and largely
identical to that of DEFH200 and DEFH72 (Davies et
al., 1996). These genes are expressed in all four
whorls of floral meristems at intermediate stages,
and later in development in ovules (DEFH200 and
DEFH72), developing embryos, and the seed coat
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Copyright © 2001 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 127, 2001
Maize MADS Box Genes
(SEP1), respectively. In analogy to ZmMADS1 and
ZmMADS3, transcription of DEFH200 and DEFH72 is
overlapping (Davies et al., 1996).
In transgenic maize plants ectopically expressing
ZmMADS3, organ differentiation processes in male
spikelets are prevented (except glumes), but the individual whorls are distinguishable. This phenotype
suggests normal function of meristem identity genes,
but absence of organ identity gene function. In cosuppression and antisense mutants of the intermediate genes FBP2 from petunia and TM5 from tomato
(Lycopersicon esculentum), respectively, organ development was not prevented, but organs were phenotypically abnormal and floral meristems undetermined (Angenent et al., 1994; Pnueli et al., 1994).
Functional analyses of intermediate Arabidopsis
MADS box genes recently showed that SEP1/2/3 triple mutant flowers develop sepals in all whorls of
indeterminate flowers (Pelaz et al., 2000), and that
overexpression of SEP3 in combination with ABC
function genes leads to the transformation of vegetative organs into petaloid and staminoid organs, respectively (Honma and Goto, 2001). These analyses
showed that class E SEP genes interact with ABC
organ identity genes. Lack of organ differentiation in
plants ectopically expressing ZmMADS3 therefore
might suggest that proper ternary and quartary complex formation is prevented. In an alternate manner,
absence of ZmMADS3 expression at a certain developmental stage might be necessary for the function of
organ identity genes. This hypothesis is supported by
the finding that ZmMADS3 expression is absent during intermediate stages of flower development in WT
plants.
ZmMADS1 and ZmMADS3 Are Specifically
Expressed in Stem Nodes
A remarkable characteristic of ZmMADS1 and ZmMADS3 is their expression in nodes. MADS box gene
expression in the stem has been reported frequently
(e.g. Ma et al., 1991; Mandel and Yanofsky, 1995), and
recently the expression of the barley MADS box gene
BM1 in the meristematic cell layer of stem nodes and
the vascular system was reported (Schmitz et al.,
2000). More detailed analysis has so far been performed only with STMADS16, a MADS box gene
from potato (Solanum tuberosum) that is exclusively
expressed in vegetative tissue (Garcı́a-Maroto et al.,
2000; see below).
ZmMADS1 and ZmMADS3 expression overlap in
stem node 5 and 6, but not in the more apical nodes
(7–12). Furthermore, ZmMADS3 displays a gradient
between the nodes and reaches an expression maximum in the uppermost node. Because expression is
highest in nodes where no ear primordia is present
(nodes 8–12), a node-specific function of ZmMADS3
can be assumed. The reduced number of stem nodes
observed in transgenic plants indicates that ZmPlant Physiol. Vol. 127, 2001
MADS3 overexpression influences node development. A similar phenotype was observed in 35S:
STMADS16 transgenic tobacco plants, which also
developed a reduced number of nodes, although
plants were not reduced in height due to an increased
number of internode cells (Garcı́a-Maroto et al.,
2000). However, the number of inflorescence
branches was increased in 35S:STMADS16 plants
(under long-day conditions), whereas number and
size of tassel branches were reduced in most of the
ZmMADS3 transgenic plants analyzed.
The precise function of ZmMADS3 cannot be determined by ectopically expressing the gene in maize
and our future experiments therefore will concentrate on the study of loss of gene function after
screening for ZmMADS1 and ZmMADS3 insertion
mutants. Again, a transgenic antisense approach will
not be a valuable tool due to the high sequence
identity of the ZAP1 and ZmMADS3 genes, and an
even higher gene redundancy within the ZmMADS1
gene group (data not shown). Further experiments
will focus on the determination of dimerization properties of ZmMADS1 and ZmMADS3 proteins and the
identification of target genes. It will be of particular
interest to further characterize the role of ZmMADS1
and ZmMADS3 during the earliest events of fertilization and embryo development.
MATERIAL AND METHODS
Screening of cDNA Libraries, Sequence Analyses, and
Gene Mapping
cDNA libraries of maize (Zea mays) ECs (Dresselhaus et
al., 1994) and mature pollen (Heuer et al., 2000) were
screened with the MADS box region of maize MADS box
genes as described by Heuer et al. (2000). cDNA isolation
and FASTA homology search with ZmMADS1 and ZmMADS3 full-length cDNA sequences were performed as
described therein. Alignment of ZmMADS1 and ZmMADS3 homologous MADS box genes, MIKC-type maize
MADS box genes, and representatives of MADS box gene
subfamilies subsequently were performed at the protein
level with ClustalX version 1.8 (Thompson et al., 1997) and
graphically illustrated with TREEVIEW (Page, 1996). GenBank and EMBL accession nos. of proteins aligned with
ZmMADS1 (accession no. AF112148) and ZmMADS3 (accession no. AF112150) are as follows: AG, X53579; AGL17,
U20186; AGL20 (SOC1), T00879; ANR1, Z97057; AP1,
Z16421; BpMADS3, X99653; DEF, X52023; DEFH125,
Y10750; FDRMADS8, AF141965; GLO, X68831; HvM5,
AJ249144; HvM8, AJ249146; LtMADS1, AF035378; LtMADS2, AF035379; OsFDRMads6, AF139664; OsMADS14,
AF058697; OsMADS15, AF058698; OsRAP1B, AB041020;
OsS11905, AB003328; PrMADS5, U90346: SaMADSa,
U25696; SbMADS2, U32110; SEP1 (AGL2), M55551; SEP2
(AGL4), M55552; SEP3 (AGL9), AF015552; SILKY1,
AF181479; SQUA, X63071; TaMADS11, AB007504; TM3,
Pnueli et al., (1991); TobMADS1, X76188; ZAG1, L18924;
ZAG2, L18925; ZAG3, L46397; ZAG5, L46398; ZAP1,
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2001 American Society of Plant Biologists. All rights reserved.
41
Heuer et al.
L46400; ZMM1, X81199; ZMM2, X81200; and ZmMADS2,
AF112149. The 3⬘-untranslated region of ZmMADS3 was
amplified by PCR using the primers below and used as a
probe in DNA gel blots to identify RFLPs between the
parents of the inbred mapping populations CO159 ⫻
TX303 and CM37 ⫻ T232A (Burr and Burr, 1991). The
resulting polymorhisms were scored within the corresponding loci placed on the Brookhaven National Laboratory map using the Map-Maker program.
Northern-Blot and Single-Cell RT-PCR Analyses
Plant material for northern-blot analyses was collected in
the greenhouse from maize inbred line A188. Node samples include the complete node section plus approximately
0.5-cm apical and basal adjacent internode regions. Immature tassels were approximately 1 to 2 cm in size. Root tips
were isolated from seedlings cultivated under sterile conditions in a growth chamber. RNA was isolated with
TRIzol (Gibco-BRL, Karlsruhe, Germany) according to the
manufacturer’s specification. Northern-blot analyses were
performed according to Heuer et al. (2000) with probes
specific for the 3⬘-end of ZmMADS1 and ZmMADS3, respectively. Filters subsequently were stripped before hybridization with an 18S-rRNA probe. Relative RNA
amounts were quantified with a bio-imager system (BAS1000, Fuji, Tokyo).
ECs, SYs, CCs, APs, and in vitro zygotes were isolated
from maize inbred line A188 (Green and Phillips, 1975)
according to the protocols of Kranz et al. (1991, 1995). In
vivo zygotes were isolated as described by Cordts et al.
(2000). Multiplex RT-PCR analyses of individual cells were
performed with specific primers for the 3⬘ end of ZmMADS1 (5⬘-GAAGGACGACGGGATGGA-3⬘; 5⬘-CACACAACGCGATATCACAT-3⬘) and intron-spanning primers
specific for the 3⬘ end of ZmMADS3 (5⬘-CTGAAGCACA
TCAGATCAAGA-3⬘ and 5⬘-AGAGGTTTTATTCATG-CA
TCC-3⬘) as described by Cordts et al. (2000). Specific amplification of Zmcdc2 served as control for successful
RT-PCR (Cordts et al., 2000).
In Vitro Culture Systems
For the induction of type I callus (low embryogenic
potential), zygotic maize embryos derived from crosses of
maize inbred lines H99 (D’Halluin et al., 1992) and A188
were isolated 11 to 13 DAP and cultivated on modified
N6⫹ medium according to Brettschneider et al. (1997). To
obtain competent type II callus, immature embryos (11–13
DAP) were isolated from inbred line B73 (Iowa State University, Ames), pollinated with A188 pollen, and cultivated
on N6.1.100.25 medium (Songstad et al., 1992). Calli were
sub-cultivated every 2 weeks as described by Brettschneider et al. (1997) for 6 months. Somatic embryo development
from type II callus was initiated by transferring calli to
Murashige and Skoog medium without hormones. Embryogenic and nonembryogenic suspension cultures were
started from competent type II callus and cultivated in
callus maintenance medium (Emons and Kieft, 1991).
42
RNA in Situ Hybridization Experiments
Male and female flowers at various developmental
stages were collected from maize inbred line A188 and B73.
The in situ hybridization procedure basically followed the
protocol provided by Dr. L. Colombo (personal communication). Samples were fixed in ethanol-acetic acidformaldehyde medium (50% [v/v] ethanol, 5% [v/v] acetic
acid, and 4% [w/v] paraformaldehyde) and embedded in
paraffin (Paraplast Plus, Sigma, Taufkirchen, Germany).
Eight- to 10-␮m sections were digested with 1 ␮g mL⫺1
Proteinase K (Roche, Mannheim, Germany) for 30 min at
37°C. Further treatment and hybridization to gene-specific
probes was performed as described by Cañas et al. (1994).
In vitro culture tissues were embedded in butyl-methyl
methacrylat (BMM) according to the protocol of Gubler et
al. (1989). Material was fixed for 2 h in 4% (w/v) paraformaldehyde in PBS buffer (Sambrook et al., 1989) with 3- ⫻
20-min vacuum infiltration. After washing in PBS buffer
(4 ⫻ 30 min), material was dehydrated in an ethanol series
(10%, 30%, and 50% [v/v] ethanol, 30 min each) at room
temperature and incubated in 70% (v/v) ethanol overnight
at 4°C. The material was further dehydrated in 90%, 96%,
and 3⫻ 100% (v/v) ethanol (1 h each at room temperature).
BMM (40 mL of butyl-methacrylate, 10 mL of methylmethacrylate, 250 mg of ethylbenzoine, and 10 mm dithiothreitol) infiltration was performed at room temperature
with 5:1, 3:1, 1:1, 1:3 ethanol:BMM (v/v) for 2 h each step
and in 100% BMM overnight before probes were transferred to Beem capsules with fresh BMM solution. BMM
polymerization was performed at ⫺20°C under long-wave
UV light (8 W, TW6; N.V. Philips, Eindhoven, The Netherlands; at ⫾15-cm distance) for 48 h. Sections (7–9 ␮m) of
BMM-embedded material were transferred to Super-FrostPlus slides and BMM was removed with acetone (10 min
100% [acetone] and 5 min 50% [acetone] in water [v/v]).
After washing in water and 0.05 m Tris-HCl (pH 7.6),
probes were digested with 1 ␮g mL⫺1 Proteinase K (Roche)
in 0.05 m Tris-HCl (pH 7.6) for 20 min at 37°C. Reactions
were stopped with cold water and probes were washed
three times with water and dehydrated in 70% and 100%
(v/v) ethanol before hydridization to gene-specific probes
as described above. Digoxigenin-labeled RNA probes were
synthesized from ZmMADS1 and ZmMADS3 gene-specific
3⬘ ends cloned into pGEM-T-vector (Promega, Mannheim,
Germany). Probes were synthesized from 1 ␮g of plasmid
at 37°C for 3 to 4 h in 40-␮L assays (40 units of T7 or Sp6
RNA polymerase, Roche), 4 ␮L of NTP labeling mix
(Roche), and 20 units of RNasin (Promega) according to the
manufacturer’s protocol (Roche).
Biolistic Transformation and Analyses of Transgenic
Maize Plants
Full-length ZmMADS3 cDNA was cloned in sense orientation into the SmaI and KpnI restriction sites in the
polylinker of the pAct1.cas vector (McElroy et al., 1995).
Immature embryos from maize inbred line A188 were isolated 12 d after hand pollination and cobombarded with
pAct1::ZmMADS3::nosT and p35S::pat::35ST (P. Eckes, un-
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Copyright © 2001 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 127, 2001
Maize MADS Box Genes
published data; Aventis, Frankfurt) containing phosphinotrycin-acetyl-transferase as the selection marker. Experimental procedures followed the protocol of Brettschneider
et al. (1997), except that embryos were bombarded twice
with 28 Hg inch vacuum and 36.46 ng of each plasmid.
Cultivation and plant regeneration was carried out as described by Brettschneider et al. (1997). Sections of male
spikelets for microscopic analyses were prepared as follows: after prefixation in 0.5% (v/v) glutaraldehyde in 0.1
m cacodylate buffer at pH 7.1 overnight at 4°C, spikelets
were fixed in 2.5% (v/v) glutaraldehyde in 0.1 m cacodylate buffer at pH 7.1 for 2 h followed by six buffer rinses.
The spikelets were then postfixed overnight at 4°C with 1%
(w/v) OsO4 in 0.1 m cacodylate buffer followed by four
buffer rinses, dehydrated in an acetone series, and embedded in Spurr resin. Semithin sections of 2 ␮m were stained
with 0.1% (w/v) Toluidine blue in 2% (w/v) sodium tetraborate buffer.
ACKNOWLEDGMENTS
We wish to thank Lucia Colombo and Peter Wittich and
coworkers for their help with the in situ hybridization
experiments as well as Gislind Bräcker for technical assistance. We acknowledge Irmhild Wachholz for her help
with tissue preparation for microscopic analyses, Benjamin
Burr for providing his RI lines and the analysis of the RFLP
data, and two unknown reviewers for many helpful suggestions to improve the manuscript.
Received January 12, 2001; returned for revision March 21,
2001; accepted May 22, 2001.
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