The Maize MADS Box Gene ZmMADS3 Affects …The Maize MADS Box Gene ZmMADS3 Affects Node Number and...

The Maize MADS Box Gene ZmMADS3 Affects NodeNumber and Spikelet Development and IsCo-Expressed with ZmMADS1 during FlowerDevelopment, in Egg Cells, and Early Embryogenesis1

Sigrid Heuer, Susanne Hansen, Jorg Bantin, Reinhold Brettschneider, Erhard Kranz, Horst Lorz, andThomas Dresselhaus*

West Africa Rice Development Association, B.P. 96, St. Louis, Senegal (Si.H.); and Center of Applied PlantMolecular 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 developmentand organ differentiation processes in plants. Addressing the question of whether MADS box genes are involved in theregulation 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 isallelic to ZAP1. Transcripts of both genes are detectable in egg cells and in in vivo zygotes of maize. ZmMADS1 isadditionally expressed in synergids and in central and antipodal cells. During early somatic embryogenesis, ZmMADS1expression 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 embryogeniccultures. Both genes are not expressed in nonembryogenic suspension cultures and in isolated immature and mature zygoticembryos. During flower development, ZmMADS1 and ZmMADS3 are co-expressed in all ear spikelet organ primordia atintermediate stages. Among vegetative tissues, ZmMADS3 is expressed in stem nodes and displays a gradient with highestexpression in the uppermost node. Transgenic maize plants ectopically expressing ZmMADS3 are reduced in height due to areduced number of nodes. Reduction of seed set and male sterility were observed in the plants. The latter was due to absenceof anthers. Putative functions of the genes during reproductive and vegetative developmental processes are discussed.

The development of highly specialized plant organsfrom undifferentiated meristematic cells is a complexprocess and requires a cascade of regulatory genescontrolling e.g. the differentiation of distinct flowerorgans from the apical meristem (for review, see Levyand Dean, 1998). With the recent discovery of individ-ual genes that, when deregulated, cause homeotictransformation of flower organs, underlying regula-tory mechanisms have started to be illuminated. Manyof these genes code for MADS box transcription fac-tors, acting at early stages in the organ developmentalprogram (Riechmann and Meyerowitz, 1997; Theißenet al., 2000). Since the isolation of the first plant MADSbox transcription factor genes, AGAMOUS and DEFI-CIENS, about 10 years ago (Sommer et al., 1990;Yanofsky et al., 1990), numerous MADS box geneshave been isolated from various mono- and dicotyle-donous flowering plants, but also from ferns andfungi (Kruger et al., 1997; Munster et al., 1997). MADSbox proteins bind to DNA at specific binding sites

(CarG boxes) as homo- and/or heterodimers regulat-ing their own transcription and that of target genes(see West et al., 1998, and references therein).

Intensive studies on mutant plants clearly demon-strated the essential, homeotic role of MADS box pro-teins in the development of the four distinct flowerorgans (sepals, petals, stamen, and carpels) and led tothe formulation of the ABC model (Weigel and Mey-erowitz, 1994). Because it was demonstrated that thepetunia (Petunia hybrida) MADS box gene FBP11 isexclusively expressed in whorl 4 and induces ovuledevelopment 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 SE-PALLATA 1, 2, and 3) recently showed that thesegenes represent a novel class of organ identity genes(class E). It was demonstrated that SEP3 interacts withABC function proteins and that ternary and quartarycomplexes are probably the molecular basis for regu-lation of flower development (Pelaz et al., 2000;Honma and Goto, 2001; Theißen and Saedler, 2001).Before ABCDE genes determine organ identity of thedistinct whorls, meristem identity genes regulate thetransition of vegetative meristems into inflorescenceand flower meristems. A third group of genes is ex-pressed after the onset of meristem identity genes but

1 This work was supported in part by the Korber foundation(Hamburg, Germany), by the Deutsche Forschungsgemeinschaft(grant nos. Kr1256/1– 4 and Dr334/2–1), and by the EuropeanCommission (grant nos. BI04 –CT960390 and BI04 –CT960210).

* Corresponding author; e-mail [email protected]; fax 49 – 40 – 42816 –229.

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before organ identity genes are detectable and havebeen referred to as intermediate or identity-mediatinggenes (for review, see Gutierrez-Cortines and Davis,2000).

Functional analyses of MADS box genes have beenperformed mainly with plants possessing bisexualflowers, e.g. Arabidopsis and tobacco (Nicotiana sp.),for which efficient transformation systems and nu-merous mutants are available. Comparably few stud-ies have been performed with plants developing uni-sexual flowers, e.g. maize (Zea mays). During maizeear and tassel development, male and female organsare initiated, but stamen in ear spikelets and thegynoeceum in tassel spikelets do not reach maturity(for review, see Cheng et al., 1983). Some maize MADSbox genes have been isolated and exclusive expressionin developing ears has been shown for ZAG2, whereexpression is largely restricted to developing carpels(Schmidt et al., 1993). Other maize MADS box genesare expressed in developing male and female inflores-cences (Schmidt et al., 1993; Fischer et al., 1995; Menaet al., 1995, 1996; Cacharron et al., 1999).

Plant MADS box genes are also expressed in ma-ture flowers where they have been detected for ex-ample in the stigma, style, and ovules (Flanagan etal., 1996; e.g. Colombo et al., 1997). In addition, ex-pression in female and male gametophytes, i.e. em-bryo sac and pollen, have been reported (e.g. Perry etal., 1996; Heuer et al., 2000, and references therein).MADS box gene expression in all organs and celltypes participating in the fertilization process indi-cate that they might regulate expression of genesinvolved in pollen-stigma interaction, pollen tubegrowth/guidance, embryo sac maturation, and theonset of gene expression after fertilization. In addi-tion, expression in zygotic and somatic embryos aswell as in endosperm have been described, so thatparticipation of MADS box proteins in regulatoryprocesses concerning embryo and endosperm devel-opment can also be assumed (Montag et al., 1995;Filipecki et al., 1997; Perry et al., 1999; Alvarez-Buyllaet al., 2000).

We are interested in the double fertilization processof higher plants and addressed the question ofwhether MADS box genes are expressed in the cellsof the female gametophyte and at earliest stages ofzygote and embryo development. Here, we presenttwo novel MADS box genes of maize of which theexpression has been studied in detail in reproductiveas well as in vegetative tissues. To elucidate the func-tion of these genes, we have ectopically expressed onegene in maize and discuss the obtained phenotype.

RESULTS

ZmMADS1 and ZmMADS3 Represent Putative MADSBox Transcription Factors

Two novel maize MADS box cDNAs, ZmMADS1and ZmMADS3, were isolated after screening cDNA

libraries of maize egg cells (ECs) and mature pollenunder medium stringent conditions with the con-served MADS box region of various maize MADSbox genes as a probe. Predicted amino acid (AA)sequences are illustrated in Figure 1 and are accessi-ble at the EMBL and GenBank databases (accessionno. AF112148, ZmMADS1; and accession no.AF112150, ZmMADS3). Both cDNAs encode proteinspossessing the motifs typical for MIKC-type MADSbox proteins (MADS box, I region, K box, and lessconserved C-terminal end). A putative bipartite nu-clear localization signal (KR-[X]12KRR) can be out-lined in the MADS box of both proteins (Fig. 1, A andB; for review, see Dingwall and Laskey, 1991). Ac-cording to a SWISS-MODEL protein structure predic-tion (Guex and Peitsch, 1997), ZmMADS1 and Zm-MADS3 proteins form an N-terminal �-helicalstructure (N13-C39) and two, C-terminal adjacent�-sheets (�1, E42-F48; loop, S49-K53; �2, L54-A58; datanot shown).

ZmMADS1 and ZmMADS3 Belong to Distinct MADSBox Subfamilies

Comparison of ZmMADS1 and ZmMADS3 proteinsequences with other MADS box proteins revealedthat ZmMADS1 can be classified as a member of theTM3 subfamily of MADS box proteins, whereas Zm-MADS3 belongs to the SQUAMOSA subfamily (Fig.2). Alignments with the most homologous proteins(for accession nos., see “Materials and Methods”) areillustrated in Figure 1. For ZmMADS1, AA identity ishighest to the rice (Oryza sativa) clone S11905 (75%).Within the C-terminal end, a highly conserved regioncan be outlined in all aligned proteins (Fig. 1A).ZmMADS3 exhibits 95% overall AA identity to themaize MADS box protein ZAP1 (�MADSD; Mena etal., 1995). Substitutions are mainly conservative andboth proteins additionally share Glu (Q)-rich clus-ters. At the very C-terminal end a cluster of nine AAsis highly conserved among aligned proteins (Fig. 1B).Using two recombinant inbred (RI) families of maize(TxCM and COxTx), ZAP1 was mapped to the longarm of chromosome 2 (2L193). We have used thesame RI families and have mapped ZmMADS3 to theshort arm of chromosome 7 (7S000).

ZmMADS1 and ZmMADS3 Are Expressed in ECs,Zygotes, and Somatic Embryo-Forming Cells As WellAs in Stem Nodes during Vegetative Development

Single-cell reverse transcriptase (RT)-PCR analysesshowed that ZmMADS1 and ZmMADS3 are both ex-pressed in maize ECs as well as in in vivo and in vitrozygotes (Fig. 3). In contrast to ZmMADS3, ZmMADS1transcripts are additionally detectable in synergids,central cells, and antipodals. Zmcdc2, amplified as apositive control, was detectable in all cells analyzed(Fig. 3). Northern-blot analyses (Fig. 4) revealed ex-

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pression of ZmMADS1 and ZmMADS3 also in imma-ture pistils as well as in non-pollinated and polli-nated mature pistils (2 and 5 d after pollination[DAP]). However, expression of both genes is unde-tectable in isolated immature (stage 2) and matureembryos (Fig. 4). Analyses of distinct maize in vitroculture systems indicated ZmMADS1 expression inembryogenic suspension cultures and embryogenictype II callus (Fig. 4). More sensitive RT-PCR analy-ses showed that ZmMADS1 is also expressed in em-bryogenic type I callus and confirmed lack of expres-sion in nonembryogenic suspension cultures.Expression of ZmMADS3 was not detectable bynorthern-blot analysis (Fig. 4), but RT-PCR studiesshowed a similar although weaker expression patternthan that of ZmMADS1 in all embryogenic culturesanalyzed and expression was undetectable in nonem-

bryogenic suspension cells (data not shown). Type IIcallus and suspension cultures were analyzed in moredetail by RNA in situ hybridization (Fig. 5). Experi-ments were performed with competent type II callus,which consists of a central area with large, highlyvacuolated cells and a peripheral part consisting ofsmaller, less vacuolated cells (Fig. 5A). In this type ofcallus, ZmMADS1 transcripts are mainly detectable inthe peripheral zone (Fig. 5B). At 7 d after the inductionof somatic embryogenesis on hormone-free medium,ZmMADS1 transcripts accumulate in developing glob-ular structures (Fig. 5, D and E). When somatic em-bryo and scutellar-like structures were further differ-entiated, ZmMADS1 transcripts centralized to theembryo axis and outer cell layers (Fig. 5F). RNA in situanalyses of embryogenic suspension cultures showedthat ZmMADS1 transcripts accumulate in sub-

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 indicatedby asterisks. A putative nuclear localization signal is indicated by bold, italic letters. AAs highly conserved among MADSbox proteins are indicated by plus signs. Positions where conservative AA substitutions occur are indicated by dots. A Q-richregion within the C-terminal end of ZmMADS3 is indicated by bold letters.

Maize MADS Box Genes

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peripheral cell layers, most likely constituted fromcells with embryogenic potential (Fig. 5H). No expres-sion was detectable in the central part and the outer-most cell layers of the cell aggregates as well as innonembryogenic callus (Fig. 5I). Hybridization of thesamples with a ZmMADS1 sense probe never gaveany signal (Fig. 5C). ZmMADS3 transcripts were notdetectable by in situ hybridization due to the lowexpression level already pointed out above.

Expression of ZmMADS1 and ZmMADS3 duringFlower Development

The northern-blot analyses performed further re-vealed that ZmMADS1 and ZmMADS3 are co-expressed during ear and tassel development (Fig. 4).

Maize plants develop ear primordia at several stemnodes, although depending on the variety, only oneor a limited number of ears reach maturity. Analysesof immature ears isolated from nodes 5 through 7showed that ZmMADS1 and ZmMADS3 expression ishighest in the ear isolated from node 7 (Fig. 4). Thiscorresponds to the most advanced stage of develop-ment among the ears analyzed and to the node wherethe fully developed ear generally appears in inbredline A188. More detailed in situ hybridization analy-ses of female flower development showed that tran-scripts of both genes are first detectable after twospikelet primordia are differentiated from the femaleinflorescence meristem (stage D; Fig. 6A) but not atearlier stages (stage A/C, data not shown; for com-parison of flower developmental stages, see Cheng etal., 1983). Within single spikelet primordia, tran-scripts were detectable in the upper and the lowerfloret as well as in glumes (Fig. 6, B, C, and F). Thispattern persisted throughout further developmentand transcripts were detectable in all flower organs,including the stamen primordia, which later abort inthe developing ear (Fig. 6, D and G). At more ad-vanced developmental stages, when the silk can beclearly distinguished (stage I/J), ZmMADS1 and Zm-MADS3 transcripts were no longer detectable (Fig.6E). No signals were obtained after hybridizationwith ZmMADS1 and ZmMADS3 corresponding senseprobes (Fig. 6, H and K). Analyses of gene expressionduring tassel development indicated that ZmMADS1and ZmMADS3 are not expressed in tassel primordiaat very early stages of development (stage A/C; datanot shown). In tassels more advanced in develop-ment (after stage G/H), ZmMADS1 and ZmMADS3

Figure 3. Expression of ZmMADS1 and ZmMADS3 in female ga-metophytic cells and zygotes. Single-cell RT-PCR analysis was per-formed with individual maize egg cells (ECs), synergids (SYs), centralcells (CCs), and antipodal cells (APs), with primers specific forZmMADS1 (A) and ZmMADS3 (B), respectively. Zygotes (Z) wereanalyzed at 24 h after pollination (hap; in vivo zygotes) and 14 to29 h after in vitro fertilization (haf; in vitro zygotes), respectively.Maize suspension cells (BMS) served as a control for vegetative geneexpression. Cells were washed four times after isolation and washingbuffer (WB) of the last wash step served as control for contaminationwith cytoplasm of burst cells of the embryo sac, nucellus, or integ-ument cells. Multiplex RT-PCR was performed with Zmcdc2-specificprimers as a control for successful RT-PCR. DNA fragments wereblotted after gel electrophoresis and hybridized to ZmMADS1-,ZmMADS3-, and Zmcdc2-specific probes. Size of DNA fragmentsand gene names are indicated.

Figure 2. ZmMADS1 and ZmMADS3 belong to different MADS boxsubfamilies. A homology search was performed with ZmMADS1 andZmMADS3 full-length cDNA sequences to identify MADS box geneswith sequence homology. Subsequent multiple alignments were per-formed with protein sequences of ZmMADS1 and ZmMADS3 (grayboxes), most homologous proteins, and representatives of the MADSbox subfamilies. Note that ZmMADS1 is the only maize proteinwithin the TM3 subfamily. ZmMADS3 is a member of the SQUA-MOSA subfamily and most similar to ZAP1. Names of subfamilies aregiven at the junctions. Bar represents 10% AA substitution per site.The tree is unrooted, bootstrap is 1,000.

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transcripts are most abundant in developing stamen(Fig. 6, I and J). As reported earlier, ZmMADS1 isexpressed throughout pollen development with thehighest transcript abundance in microspores (Heueret al., 2000). Expression of ZmMADS3 was undetect-able in the pistil primordia, which is still present indeveloping maize tassels at the stage analyzed.

Within vegetative organs, ZmMADS1 is most abun-dant in leaves (Fig. 4). Low level of expression addi-tionally was found in root tips and internodes (datanot shown). Whereas ZmMADS1 is expressed at alow level only in nodes 5 and 6 (counted from thefirst node above ground), ZmMADS3 is detectable inall nodes analyzed, displaying a gradient with thehighest expression found in the last stem node im-mediately adjacent to the tassel (node 12; Fig. 4).Preliminary results from in situ hybridization exper-iments performed with transverse and longitudinalnode sections indicate that ZmMADS3 is not ex-pressed in vascular and parenchymatic cells, but incell layers consisting of small, non-vacuolated cellsprobably representing meristematic cells (data notshown).

Ectopic ZmMADS3 Expression Affects Plant Height andMale Spikelet Development

To gain insight into putative functions of Zm-MADS3, immature maize embryos were transformedwith a full-length ZmMADS3 sense construct and aZmMADS3 antisense construct under the control ofthe constitutive rice actin promoter. Taking the highsequence identity of ZmMADS3 and ZAP1 into ac-count, the antisense construct used for these experi-ments encompassed only the 3�-untranslated regionof the ZmMADS3 cDNA. Plants regenerated fromthese experiments were transferred into the green-house for further cultivation and were monitored bySouthern- and northern-blot analyses until the F3generation. Transgenic plants that integrated the an-tisense construct did not display a phenotype overthe generations analyzed, which might be due to theshort length of the antisense construct, and were notanalyzed further. The plant T0#12 (Fig. 7A) containedfive copies of the sense construct and full-lengthtransgene expression at a low level was determinedby northern-blot analyses of leaves, where Zm-MADS3 is not detectable in WT plants (data not

Figure 4. Temporal and spatial ZmMADS1 and ZmMADS3 expression. RNA gel-blot analyses were performed with 10 �gof 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 RNAfragments is indicated.




shown). The transgenic plant was strongly reducedin height and developed no ear, whereas the basalregion of the apical tassel developed into ear-likestructures (Fig. 7A). The apical region of the tasselshowed no differentiation into male spikelets. Seedscould not be obtained after pollination of the femalespikelets located at the tassel with WT pollen, whichprevented analyses of the progeny of this plant. Theplant T0#6 (Fig. 7B) contained two integrated copiesof the transgene and expression in leaves was higherthan in T0#12 (data not shown). The plant was malesterile and strongly reduced in height compared withcontrol plants transformed with the selection markeronly (Fig. 7B, left). Leaf development was not af-fected (Fig. 7B). After pollination with WT pollen,only 11 kernels developed that germinated normally.The phenotype observed in T0 was confirmed in theprogeny: Tassels of representative plants of the T2and T3 generation are presented in Figure 7C. Prog-eny plants that lost the transgene due to segregationwere always cultivated as control plants and devel-oped normally (Fig. 7C, left). Plants ectopically ex-pressing ZmMADS3 showed different levels of fe-male and male sterility and were reduced in height(Fig. 7, B and C). Seed set was reduced, but the grainsobtained after self-pollination and pollination withA188 pollen germinated normally. The reduction inheight reflected a reduced number of nodes becausetransgenic plants developed only eight to nine nodes,in contrast to 12 nodes generally developed by WTplants in the greenhouse. Tassels of transgenic plantswere smaller with a reduced number of branches incomparison with control plants (Fig. 7, C–E). Moredetailed analyses of the tassel of transgenic plantsshowed that the outer glume appeared normal (Fig.

7, E, F, and H), whereas the inner glume was reducedto a small, leaf-like structure (Fig. 7, F and H). Nodifferentiation of lemma, stamen, lodicules, and pa-lea was apparent in the lower and the upper malefloret of transgenic plants (Fig. 7H).

DISCUSSION

ZmMADS3 Is Allelic to ZAP1 and Represents theZAP1b Gene

We have characterized two novel maize MADS boxcDNAs, ZmMADS1 and ZmMADS3, members of theTM3 and SQUAMOSA subfamiliy of MIKC-typeMADS box proteins (Theißen et al., 2000), respec-tively. The high conservation of functional/struc-tural units within the MADS and K box of Zm-MADS1 and ZmMADS3 suggests that both proteinsare located within the nucleus, that they can bind toDNA, and that they are capable of dimer formation.As was determined for the human SRF core ho-modimer, the primary DNA-binding element is anantiparallel coiled coil of two amphipathic �-helices,one from each monomer (Pellegrini et al., 1995).Dimerization of the monomers is permitted by inter-action of the �-sheets forming a four-stranded anti-parallel �-sheet. These structural domains are con-served in the ZmMADS1 and ZmMADS3 proteins.

ZmMADS3 exhibits 95% AA identity to the maizeMADS box protein ZAP1, which has been mapped at2L193. A duplicated gene of ZAP1 (ZAP1b) has beenpredicted based on RFLP mapping analyses (Mena etal., 1995). We have mapped ZmMADS3 on 7S000, thesame position determined for ZAP1b. Therefore, wepropose that ZmMADS3 represents the ZAP1b gene.

Figure 5. Expression of ZmMADS1 during so-matic embryogenesis. RNA in situ hybridizationexperiments were performed with type II callusbefore (A–C) and after (D–F) induction of em-bryogenesis, with a maize embryogenic (G andH) and nonembryogenic suspension culture (I).Samples were hydridized to ZmMADS1 specificprobes in antisense (B, E, F, H, and I) and senseorientation (representative experiment shown inC). Arrows point at hybridization signals in de-veloping globular structures (E), and at laterstages to a cluster of cells at the embryo axis andouter cell layer (F). In embryogenic suspensioncultures, signals are restricted to subperipheralcell layers (arrows in H). A signal was neverobtained in nonembryogenic suspension cul-tures (I). Bars represent 100 �m.

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It was shown by Mena et al. (1995) that ZAP1 expres-sion is excluded from mature stamen and carpels thatclearly distinguishes ZAP1 from ZmMADS3, which isdetectable in mature pistils. ZAP1 is not representedin the cDNA library of ECs as was determined byPCR with ZAP1-specific primers (data not shown).As a consequence of its ancestral allotetraploid origin(Leitch and Bennett, 1997), other maize MADS boxgenes are reported to represent duplicated genes,namely ZAG1/ZMM2, ZAG2/ZMM1, ZAG3/ZAG5,and ZMM8/ZMM14, and likewise have developeddistinct expression patterns (Mena et al., 1995;Theißen et al., 1995; Cacharron et al., 1999).

ZmMADS1 and ZmMADS3 Expression Pattern Implies aFunction during Fertilization and Early Embryogenesis

Many of the MADS box genes described so far haveimportant functions during inflorescence develop-ment and flower organ differentiation, and only rel-atively few data are available for MADS box geneexpression in mature flowers. Transcripts of someMADS box genes have been detected in matureovules (for review, see Riechmann and Meyerowitz,1997), but so far AGL15 and AGL18 are the onlyMADS box genes shown to be expressed in the cells

of the embryo sac, without further specification of thecell type (Perry et al., 1996, 1999; Alvarez-Buylla etal., 2000). Therefore, ZmMADS1 and ZmMADS3 rep-resent the first MADS box genes for which an expres-sion in plant ECs and zygotes has been shown. Tighttemporal regulation of cell cycle regulatory genes(cyclins) in maize zygotes demonstrated de novogene transcription before the first cell division of thezygote takes place (Sauter et al., 1998). Changes oftranscript abundance in cDNA populations derivedfrom maize in vitro zygotes additionally has beenshown for distinct genes expressed in maize ECs(Dresselhaus et al., 1999). These analyses showed thatzygotic gene activation in plants occurs already at theone-cell stage and therefore earlier than in animals.Transcription factors accordingly must be presentregulating this transcription activity. Co-expressionof ZmMADS1 and ZmMADS3 in ECs and zygotestheoretically facilitates heterodimerization/interac-tion of the proteins (provided that RNA and proteinsare co-expressed). However, exclusive expression ofZmMADS1 in the CC, SYs, and APs suggests Zm-MADS1 interaction with yet unidentified partners.

Both genes are, although at highly different levelsof transcript abundance, expressed also during so-matic embryogenesis of distinct in vitro culture sys-

Figure 6. ZmMADS1 and ZmMADS3 expres-sion during spikelet development. RNA in situhybridization experiments were performed withZmMADS1- (A–E and J) and ZmMADS3- (F, G,and I) specific RNA probes in antisense orienta-tion. Representative sense control experimentsare shown in H and K. ZmMADS1 is expressedin meristems of upper (uf) and lower (lf) earflorets and in glume (gl) primordia at develop-mental stage D (A and B). At stage G and H (Cand D), ZmMADS1 expression is additionallydetectable in developing lemmas (l), stamen (st),and the gynoeceum (gyn; gr, gynoecial ridge). Atstage I/J (E), ZmMADS1 is no longer detectable.ZmMADS3 is expressed in an identical temporaland spatial pattern but signals were always lessintense (F and G). During tassel development,ZmMADS1 is expressed in lodicules (lo),glumes, lemmas, and stamen (J). The ZmMADS3expression pattern is identical but signals werenot obtained in gynoeceum primordia (I).




tems analyzed. Before somatic embryos develop fromcallus, ZmMADS1 is expressed in cells in the periph-ery of the callus and is subsequently detectable indeveloping embryos, where transcripts are finallyrestricted to specific cells at the periphery and theembryo axis. This expression pattern is distinct fromthat observed for other MADS box genes, which areexpressed in external cell layers of the radicular partin heart stage somatic embryos (CUS1) or are notrestricted to specific regions (AGL15), respectively(Filipecki et al., 1997; Perry et al., 1999). NeitherZmMADS1 nor ZmMADS3 transcripts are detectablein mature zygotic embryos indicating a specific func-tion during early stages of embryo development. Be-cause transgenic seeds germinated normally, Zm-MADS3 overexpression has no obvious effect onzygotic embryo and early seedling development.

ZmMADS1 and ZmMADS3 Are Expressed atIntermediate Stages of Flower Development

ZmMADS1 and ZmMADS3 are also co-expressedduring flower development, where expression was

detectable in the upper and the lower floret only atintermediate stages of development. This expressionpattern is distinct from that of other maize MADSbox genes. ZMM8 and ZMM14 are exclusively ex-pressed in the upper floret of maize ear spikelets,whereas ZAG1 and ZAG2 expression is restricted toreproductive organ primordia (Schmidt et al., 1993;Cacharron et al., 1999).

At later stages of flower development, ZmMADS1and ZmMADS3 become undetectable, but are againexpressed in mature pistils. Based on the signal in-tensity observed in northern-blot analyses, we as-sume that ZmMADS1 and ZmMADS3 are not exclu-sively expressed in the cells of the embryo sac, butalso in surrounding nucellus and/or integument tis-sues. This expression pattern is similar to that ofSEP1 (AGL2, Flanagan and Ma, 1994) and largelyidentical to that of DEFH200 and DEFH72 (Davies etal., 1996). These genes are expressed in all fourwhorls of floral meristems at intermediate stages,and later in development in ovules (DEFH200 andDEFH72), developing embryos, and the seed coat

Figure 7. Ectopic expression of ZmMADS3 in transgenic maize plants. Immature maize embryos were transformed with apAct1::ZmMADS3::nosT full-length sense construct. Transgenic plants of the T0 generation with five (plant T0#12 shown inA) and two integrated copies of the transgene (plant T0#6 shown in B, left) were reduced in height in comparison withwild-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 thetransgene is shown for comparison (C, right). The phenotype was confirmed in the T3 generation: No anthers dehisced fromsterile 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 regionsindicated 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). Barsrepresent 2 mm (D–F) and 300 �m (G and H), respectively.

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(SEP1), respectively. In analogy to ZmMADS1 andZmMADS3, transcription of DEFH200 and DEFH72 isoverlapping (Davies et al., 1996).

In transgenic maize plants ectopically expressingZmMADS3, organ differentiation processes in malespikelets are prevented (except glumes), but the in-dividual whorls are distinguishable. This phenotypesuggests normal function of meristem identity genes,but absence of organ identity gene function. In co-suppression and antisense mutants of the intermedi-ate genes FBP2 from petunia and TM5 from tomato(Lycopersicon esculentum), respectively, organ devel-opment was not prevented, but organs were pheno-typically abnormal and floral meristems undeter-mined (Angenent et al., 1994; Pnueli et al., 1994).Functional analyses of intermediate ArabidopsisMADS box genes recently showed that SEP1/2/3 tri-ple mutant flowers develop sepals in all whorls ofindeterminate flowers (Pelaz et al., 2000), and thatoverexpression of SEP3 in combination with ABCfunction genes leads to the transformation of vegeta-tive organs into petaloid and staminoid organs, re-spectively (Honma and Goto, 2001). These analysesshowed that class E SEP genes interact with ABCorgan identity genes. Lack of organ differentiation inplants ectopically expressing ZmMADS3 thereforemight suggest that proper ternary and quartary com-plex formation is prevented. In an alternate manner,absence of ZmMADS3 expression at a certain devel-opmental stage might be necessary for the function oforgan identity genes. This hypothesis is supported bythe finding that ZmMADS3 expression is absent dur-ing intermediate stages of flower development in WTplants.

ZmMADS1 and ZmMADS3 Are SpecificallyExpressed in Stem Nodes

A remarkable characteristic of ZmMADS1 and Zm-MADS3 is their expression in nodes. MADS box geneexpression in the stem has been reported frequently(e.g. Ma et al., 1991; Mandel and Yanofsky, 1995), andrecently the expression of the barley MADS box geneBM1 in the meristematic cell layer of stem nodes andthe vascular system was reported (Schmitz et al.,2000). More detailed analysis has so far been per-formed only with STMADS16, a MADS box genefrom potato (Solanum tuberosum) that is exclusivelyexpressed in vegetative tissue (Garcıa-Maroto et al.,2000; see below).

ZmMADS1 and ZmMADS3 expression overlap instem node 5 and 6, but not in the more apical nodes(7–12). Furthermore, ZmMADS3 displays a gradientbetween the nodes and reaches an expression maxi-mum in the uppermost node. Because expression ishighest in nodes where no ear primordia is present(nodes 8–12), a node-specific function of ZmMADS3can be assumed. The reduced number of stem nodesobserved in transgenic plants indicates that Zm-

MADS3 overexpression influences node develop-ment. A similar phenotype was observed in 35S:STMADS16 transgenic tobacco plants, which alsodeveloped a reduced number of nodes, althoughplants were not reduced in height due to an increasednumber of internode cells (Garcıa-Maroto et al.,2000). However, the number of inflorescencebranches was increased in 35S:STMADS16 plants(under long-day conditions), whereas number andsize of tassel branches were reduced in most of theZmMADS3 transgenic plants analyzed.

The precise function of ZmMADS3 cannot be de-termined by ectopically expressing the gene in maizeand our future experiments therefore will concen-trate on the study of loss of gene function afterscreening for ZmMADS1 and ZmMADS3 insertionmutants. Again, a transgenic antisense approach willnot be a valuable tool due to the high sequenceidentity of the ZAP1 and ZmMADS3 genes, and aneven higher gene redundancy within the ZmMADS1gene group (data not shown). Further experimentswill focus on the determination of dimerization prop-erties of ZmMADS1 and ZmMADS3 proteins and theidentification of target genes. It will be of particularinterest to further characterize the role of ZmMADS1and ZmMADS3 during the earliest events of fertili-zation and embryo development.

MATERIAL AND METHODS

Screening of cDNA Libraries, Sequence Analyses, andGene Mapping

cDNA libraries of maize (Zea mays) ECs (Dresselhaus etal., 1994) and mature pollen (Heuer et al., 2000) werescreened with the MADS box region of maize MADS boxgenes as described by Heuer et al. (2000). cDNA isolationand FASTA homology search with ZmMADS1 and Zm-MADS3 full-length cDNA sequences were performed asdescribed therein. Alignment of ZmMADS1 and Zm-MADS3 homologous MADS box genes, MIKC-type maizeMADS box genes, and representatives of MADS box genesubfamilies subsequently were performed at the proteinlevel with ClustalX version 1.8 (Thompson et al., 1997) andgraphically illustrated with TREEVIEW (Page, 1996). Gen-Bank and EMBL accession nos. of proteins aligned withZmMADS1 (accession no. AF112148) and ZmMADS3 (ac-cession 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; Lt-MADS2, 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,




L46400; ZMM1, X81199; ZMM2, X81200; and ZmMADS2,AF112149. The 3�-untranslated region of ZmMADS3 wasamplified by PCR using the primers below and used as aprobe in DNA gel blots to identify RFLPs between theparents of the inbred mapping populations CO159 �TX303 and CM37 � T232A (Burr and Burr, 1991). Theresulting polymorhisms were scored within the corre-sponding loci placed on the Brookhaven National Labora-tory map using the Map-Maker program.

Northern-Blot and Single-Cell RT-PCR Analyses

Plant material for northern-blot analyses was collected inthe greenhouse from maize inbred line A188. Node sam-ples include the complete node section plus approximately0.5-cm apical and basal adjacent internode regions. Imma-ture tassels were approximately 1 to 2 cm in size. Root tipswere isolated from seedlings cultivated under sterile con-ditions in a growth chamber. RNA was isolated withTRIzol (Gibco-BRL, Karlsruhe, Germany) according to themanufacturer’s specification. Northern-blot analyses wereperformed according to Heuer et al. (2000) with probesspecific for the 3�-end of ZmMADS1 and ZmMADS3, re-spectively. Filters subsequently were stripped before hy-bridization with an 18S-rRNA probe. Relative RNAamounts were quantified with a bio-imager system (BAS-1000, Fuji, Tokyo).

ECs, SYs, CCs, APs, and in vitro zygotes were isolatedfrom maize inbred line A188 (Green and Phillips, 1975)according to the protocols of Kranz et al. (1991, 1995). Invivo zygotes were isolated as described by Cordts et al.(2000). Multiplex RT-PCR analyses of individual cells wereperformed with specific primers for the 3� end of Zm-MADS1 (5�-GAAGGACGACGGGATGGA-3�; 5�-CACAC-AACGCGATATCACAT-3�) and intron-spanning primersspecific for the 3� end of ZmMADS3 (5�-CTGAAGCACATCAGATCAAGA-3� and 5�-AGAGGTTTTATTCATG-CATCC-3�) as described by Cordts et al. (2000). Specific am-plification of Zmcdc2 served as control for successfulRT-PCR (Cordts et al., 2000).

In Vitro Culture Systems

For the induction of type I callus (low embryogenicpotential), zygotic maize embryos derived from crosses ofmaize inbred lines H99 (D’Halluin et al., 1992) and A188were isolated 11 to 13 DAP and cultivated on modifiedN6� medium according to Brettschneider et al. (1997). Toobtain competent type II callus, immature embryos (11–13DAP) were isolated from inbred line B73 (Iowa State Uni-versity, Ames), pollinated with A188 pollen, and cultivatedon N6.1.100.25 medium (Songstad et al., 1992). Calli weresub-cultivated every 2 weeks as described by Brettschnei-der et al. (1997) for 6 months. Somatic embryo developmentfrom type II callus was initiated by transferring calli toMurashige and Skoog medium without hormones. Embry-ogenic and nonembryogenic suspension cultures werestarted from competent type II callus and cultivated incallus maintenance medium (Emons and Kieft, 1991).

RNA in Situ Hybridization Experiments

Male and female flowers at various developmentalstages were collected from maize inbred line A188 and B73.The in situ hybridization procedure basically followed theprotocol provided by Dr. L. Colombo (personal communi-cation). Samples were fixed in ethanol-acetic acid-formaldehyde medium (50% [v/v] ethanol, 5% [v/v] aceticacid, and 4% [w/v] paraformaldehyde) and embedded inparaffin (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 at37°C. Further treatment and hybridization to gene-specificprobes was performed as described by Canas et al. (1994).In vitro culture tissues were embedded in butyl-methylmethacrylat (BMM) according to the protocol of Gubler etal. (1989). Material was fixed for 2 h in 4% (w/v) parafor-maldehyde 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 roomtemperature and incubated in 70% (v/v) ethanol overnightat 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 methyl-methacrylate, 250 mg of ethylbenzoine, and 10 mm dithio-threitol) infiltration was performed at room temperaturewith 5:1, 3:1, 1:1, 1:3 ethanol:BMM (v/v) for 2 h each stepand in 100% BMM overnight before probes were trans-ferred to Beem capsules with fresh BMM solution. BMMpolymerization was performed at �20°C under long-waveUV light (8 W, TW6; N.V. Philips, Eindhoven, The Neth-erlands; at �15-cm distance) for 48 h. Sections (7–9 �m) ofBMM-embedded material were transferred to Super-Frost-Plus slides and BMM was removed with acetone (10 min100% [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. Reactionswere stopped with cold water and probes were washedthree times with water and dehydrated in 70% and 100%(v/v) ethanol before hydridization to gene-specific probesas described above. Digoxigenin-labeled RNA probes weresynthesized from ZmMADS1 and ZmMADS3 gene-specific3� ends cloned into pGEM-T-vector (Promega, Mannheim,Germany). Probes were synthesized from 1 �g of plasmidat 37°C for 3 to 4 h in 40-�L assays (40 units of T7 or Sp6RNA polymerase, Roche), 4 �L of NTP labeling mix(Roche), and 20 units of RNasin (Promega) according to themanufacturer’s protocol (Roche).

Biolistic Transformation and Analyses of TransgenicMaize Plants

Full-length ZmMADS3 cDNA was cloned in sense ori-entation into the SmaI and KpnI restriction sites in thepolylinker of the pAct1.cas vector (McElroy et al., 1995).Immature embryos from maize inbred line A188 were iso-lated 12 d after hand pollination and cobombarded withpAct1::ZmMADS3::nosT and p35S::pat::35ST (P. Eckes, un-

Heuer et al.



published data; Aventis, Frankfurt) containing phosphi-notrycin-acetyl-transferase as the selection marker. Exper-imental procedures followed the protocol of Brettschneideret al. (1997), except that embryos were bombarded twicewith 28 Hg inch vacuum and 36.46 ng of each plasmid.Cultivation and plant regeneration was carried out as de-scribed by Brettschneider et al. (1997). Sections of malespikelets for microscopic analyses were prepared as fol-lows: after prefixation in 0.5% (v/v) glutaraldehyde in 0.1m cacodylate buffer at pH 7.1 overnight at 4°C, spikeletswere fixed in 2.5% (v/v) glutaraldehyde in 0.1 m cacody-late 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 fourbuffer rinses, dehydrated in an acetone series, and embed-ded in Spurr resin. Semithin sections of 2 �m were stainedwith 0.1% (w/v) Toluidine blue in 2% (w/v) sodium tet-raborate buffer.

ACKNOWLEDGMENTS

We wish to thank Lucia Colombo and Peter Wittich andcoworkers for their help with the in situ hybridizationexperiments as well as Gislind Bracker for technical assis-tance. We acknowledge Irmhild Wachholz for her helpwith tissue preparation for microscopic analyses, BenjaminBurr for providing his RI lines and the analysis of the RFLPdata, and two unknown reviewers for many helpful sug-gestions to improve the manuscript.

Received January 12, 2001; returned for revision March 21,2001; accepted May 22, 2001.

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