A protective role for the embryo surrounding region of the maizeendosperm, as evidenced by the characterisation of ZmESR-6,a defensin gene specifically expressed in this region

Maite Balandın1, Joaquın Royo1, Elisa Gomez1, Luis M. Muniz1, Antonio Molina2 andGregorio Hueros1,*1Departamento de Biologıa Celular y Genetica, Universidad de Alcala, E-28871, Alcala de Henares (Madrid),Spain (*author for correspondence: e-mail gregorio.hueros@uah.es); 2Departamento de Biotecnologıa,Escuela Tecnica Superior de Ingenieros Agronomos, Universidad Politecnica, E-28040, Madrid, Spain

Received 31 December 2004; accepted in revised form 9 March 2005

Key words: antimicrobial peptides, defensin, embryo surrounding region, endosperm development, maizeendosperm, plant defence

Abstract

A Zea mays cDNA clone, ZmESR-6, was isolated as a gene specifically expressed at the basal region ofimmature kernels. ZmESR-6 cDNA encoded for a small (11.1 kDa) protein homologous to plant defensins.As for other defensins, the protein contained an N-terminal signal peptide signature and a C-terminal acidicpeptide, the mature peptide has a molecular mass of 5.5 kDa. ZmESR-6 was highly expressed in developingkernels but the transcript could not be detected in any other maize tissue. The recombinant ZmESR-6protein, purified from E. coli, showed strong in vitro inhibitory activity against bacterial and fungal plantpathogens, suggesting a role for ZmESR-6 in plant defence. The distribution of the transcripts wasrestricted to the embryo surrounding region (ESR) of the kernel. Immunolocalisation experiments revealed,however, that at the grain filling phase ZmESR-6 was accumulated in the placentochalaza-cells, rather thanin the ESR cells that produce it. Our results suggest that the ESR has a role in protecting the embryo at thevery early stages of seed development, whilst contributes to the general defence mechanism of the kernel atlater developmental stages.

Introduction

Cereal seed filling is a critical process subjectedto different variables influencing its efficiency.Appropriated nutrient supply, and developmentof the grain in an environment free of pathoge-nic microorganisms are two of the main factorsaffecting this process.

In maize kernels, nutrient supply is provided bythe maternal pedicel, solutes are unloaded into thisregion from the phloem terminals. Nutrients arethen transported through the placentochalazacells to reach the storage organs of the grain.

The placentochalaza layer consists of rectangu-lar, thick-walled cells that lose their nuclei andcytoplasm early in the development. It has beensuggested that the strong suberisation of these cellwalls might play a role in pathogen resistance(Santandrea et al., 2002).

Maize endosperm is a highly differentiated tissueundergoing complex patterns of development. Inthe basal endosperm, two cell types, the transferlayer, and the embryo-surrounding region (ESR)are supposed to be involved in nutrient exchange(Schel et al., 1984;Thompson et al., 2001). Thetransfer cell layer consists of highly specialised cells

Plant Molecular Biology (2005) 58:269–282 � Springer 2005DOI 10.1007/s11103-005-3479-1

forming a cup-shaped layer at the base of theendosperm, these cells are characterised by thepresence of ingrowths of their cell wall and plasmamembrane. Transfer cells are thought to be involvedin solute exchange between thematernal pedicel andthe basal endosperm (Davis et al., 1990; Thompsonet al., 2001). Cells from the ESR are small in size,with a dense cytoplasm rich in rough endoplasmicreticulum (Schel et al., 1984).At the earliest stages ofseed development (before 6 days after pollination,DAP), these cells completely surround the embryoand are positioned on one side of the endosperm.Later on they became basal and form a continuumwith the transfer cell layer at the embryo pole. Atthis stage, ESR cells are positioned right at the baseof the suspensor.

Several ESR-specific genes have been described(Opsahl-Ferstad et al., 1997; Bonello et al., 2002).The ESR function is mostly unknown, although ithas been suggested to play a role in different typesof exchanges between embryo and endosperm(Opsahl-Ferstad et al., 1997). The recent charac-terisation of an apoplastic invertase inhibitor,ZmINVINH1, specifically expressed at the ESR(Bate et al., 2004) suggest a role for the ESR inmodulating local activity of the apoplastic invertaseat the earliest stages of seed development. In thisway endosperm and embryo would be exposed todifferent concentrations of glucose and would thusfollow different rates of cell division and distinctdevelopmental programmes.

The solute transport is apoplastic betweenmaternal and zygotic cells, as there are noplasmodesmatal connections between them(Thorne, 1985), a logical consequence of the factthat these tissues belong to different organisms. Ithas been suggested that this lack of symplasticconnexion might contribute to protect the devel-oping grain against the attack of pathogenicmicro-organisms. In fact, plant viruses are knownto use plasmodesmata as gateway to spreadthrough different tissues of the infected plant (fora review see Pickard and Beachy, 1999).

Higher plants are equipped with a set of mech-anisms protecting them from diseases caused bypathogen bacteria, fungi and viruses. Among themis the production of an arsenal of antibioticproteins that ultimately will kill the pathogensor impair their growth. Several families ofantimicrobial peptides have been described up to

now constituting, in most of the cases, the primarydefence barrier in either plants and animals(Broekaert et al., 1995; Hancock and Lehrer,1998; Garcıa Olmedo et al., 1998). Defensins are afamily of antimicrobial peptides well representedin plant and animal systems. They were firstreported in wheat and barley grains (Colilla et al.,1990; Mendez et al., 1990) and originally calledc-thionins, although structurally unrelated tocanonical a- and b-thionins (Terras et al., 1992;Bruix et al., 1993). Defensins are small (around5 kDa), and cationic, with a typical three-dimen-sional structure stabilised by eight disulfide-linkedcysteines (for review see Broekaert et al., 1995,1997; Thomma et al., 2002).

Plant defensins were initially purified fromseeds (for review see Thomma et al., 2002), butgene expression for these proteins has also beenreported in other tissues, like in pepper fruits(Meyer et al., 1996 ); different floral tissues oftomato (Milligan and Gasser, 1995), tobacco (Guet al., 1992) and potato (Moreno et al., 1994); orin the maize female gametophyte (Cordts et al.,2001). In some cases defensin gene expression isdevelopmentally regulated, as in leaves and stemsof pea (Lay et al., 2002), or in radish seeds(Terras et al., 1995) whilst in other cases, such asthe tobacco FST (Gu et al., 1992), radish Rs-AFP(Terras et al., 1995) and Arabidopsis Pdf1.2(Epple et al., 1997), defensin genes are inducedafter pathogen infection. Most of the plantdefensins tested have antifungal activity in vitroand their overexpression in transgenic plants haveled, in some cases, to increased resistance topathogen attack. Plant defensins are thought toinduce membrane permeabilisation through spe-cific interaction with high affinity binding sites onfungal cells (for a review see, Thomma et al.,2002).

We report here on the isolation and charac-terisation of a defensin-like maize gene, ZmESR-6.ZmESR-6 is specifically expressed at the endo-sperm embryo surrounding region (ESR) duringkernel development, and the protein accumulatesin the placentochalaza layer. RecombinantZmESR-6 protein showed in vitro antibacterialand antifungal activity supporting a role in plantdefence for the ESR, a discrete endosperm domainfor which no biological function had previouslybeen reported.

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Results

Isolation of a new defensin-like gene from maizekernels

ZmESR-6 was identified in a differential screeningaimed to isolate basal–kernel-specific genes. Theprocedure used was based on that of Sokolov andProckop (1994). Briefly, young maize seed (10DAP) were dissected to separate the bottom (basalpart of the kernel) and top (upper part) halves.mRNA was extracted and reverse transcribed intobottom and top cDNAs. These cDNAs were usedas templates for polymerase chain reaction (PCR)using pairs of 10-mer oligonucleotides of arbitrarysequence as PCR primers. Comparison of theband patterns produced from either top or bot-tom cDNAs allowed the identification of bottom-specific PCR bands. These were cloned andsequenced. RNA gel blot analyses using thesebands as probes confirmed that one was amplifiedfrom a basal kernel-specific transcript (data notshown). This band was subsequently used toscreen a 10 DAP endosperm cDNA library(Hueros et al., 1995). The longest (550 bp) hybri-dising clone, named ZmESR-6, contained an openreading frame for a protein of 108 amino acids, a147 bp 5 untranslated sequence and a 79 bp 3¢untranslated sequence, in addition of a poly (A)+

tail. The predicted aminoacid sequence (Figure 1)shows significant similarity to the family of anti-microbial peptides known as defensins (Figure 2).The complete ZmESR-6 protein would have amolecular mass of about 11.1 kDa, containingthree putative domains. First, a 27 aminoacidssignal peptide (underlined in Figure 1) mostlyhydrophobic and with a predicted cleavage sitebetween A-27 and K-28, as deduced by theSignalP program. The presence of this signalpeptide suggests that the protein is secreted. Asecond domain would consist of an acidicC-terminal domain tail 29 aminoacids long (initalics in Figure 1), with a predicted cleavage sitelocated by comparison to other defensins betweenC-79 and A-80 (Gu et al., 1992). Acidic domainsare present in many of the characterised defensins,and thionins although there is little sequencesimilarity among them within these regions (Guet al., 1992). The third and central domain (shownin bold characters in Figure 1) would be themature defensin protein, is 52 aminoacids long,with a Mw of 5.5 kDa, a pI 8.73 and a net chargeat pH 7 of 3.68.

A typical feature of the defensin family ofproteins is the low degree of overall sequenceconservation among their members. In fact, thecentral domain of ZmESR-6 shares only a 28.8%sequence identity with c-purothionin, its closest

Figure 1. Nucleotide sequence of ZmEsr6 and predicted aminoacid sequence. The ATG translation start and the TGA termination

codons are located at position 148 and 469 bp respectively. The open reading frame is indicated in capital letters. The predicted

signal peptide is underlined, mature protein and acidic prodomain are in bold and italic characters, respectively.

q

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known relative. Nevertheless, all defensins haveeight Cys residues in conserved positions, a featurethought to be important for stabilisation of theirspatial structure, and this feature is present in thesequence of ZmESR-6. A number of discrepanciesare noted, however, when analyzing the ZmESR-6structure, the spacing among the first three Cys isslightly different to that commonly seen in defen-sins, including the other maize members of thegroup reported up to now (Kusmerich et al., 1998;Cordts et al., 2001). In ZmESR-6, the first Cys, atposition 3, is separated from the second by 8aminoacids in contrast to the 10–11 aminoacidspresent in most defensins. The second and thirdCys are separated by 11 aminoacids in ZmESR-6,but only for 5 in the other characterised defensins.Despite these changes, secondary structure analy-sis of mature ZmESR-6, performed with PHDsec,predicts the presence of an a-helix in the centralregion, and two b-sheets in similar positions as ithas been described for other plant defensins(data not shown). Moreover, the four disulphidebridges present in all defensins (Bruix et al., 1993;Fant et al., 1998; Lay et al., 2003) are predicted(DISULFIND program) to be formed betweenCys residues located in equivalent positions inZmESR-6. In this model, two Cys located in thea-helix form two disulfide bridges with the Cys-X-Cys segment in the third b-strand, a structuralmotif known as CSab. We have used Swiss Modelto predict the three-dimensional structure ofZmESR-6. When this program was run usingc-1-P thionin as a template, the predicted struc-tural similarity between ZmESR-6 and c-1-Pthionin extended over the last 37 aminoacids ofthe mature ZmESR-6 (from Ile-16 to Cys-52),whilst the programme failed to align the 15aminoacids at the N-terminus.

Genomic organisation and mapping of the ZmESR-6 locus

Genomic DNA gel blot analyses in the inbred lineA69Y demonstrated that ZmESR-6 is probably asingle-copy gene in maize. The position ofZmESR-6 in the maize genome was determinedby restriction fragment length polymorphismanalysis using a population of immortalised F2lines (Tx303/CO159IF2 1995–1998) (Burr et al.,1994). Genomic DNA from the parental lines wasdigested with a collection of 16 restriction enzymes.One of those enzymes producing polymorphism,BglII, was subsequently used for mapping (datanot shown). The gene mapped on chromosome 4 ofmaize, between csu 704 and csu 201. To ourknowledge, no gene influencing seed phenotype hasbeen mapped to this region.

ZmESR-6 gene is developmentally regulated

The expression pattern of ZmESR-6 in differentmaize tissues and at several developmental stagesof the seed was studied by Northern blot analyses(Figure 3). The transcript was detected only inimmature kernels, being undetectable in the othersamples of vegetative or reproductive tissues anal-ysed (Figure 3A). Within the seed (Figure 3B),expression was first detected at 5 DAP, transcriptaccumulation reaches a maximum around 11 DAPand declines afterwards, being undetectable inmature seeds. RNAs from upper and lower halvesof the kernels were separately analysed in theNorthern blot shown in Figure 3B for kernels olderthan 8 DAP (T means upper and B means lower),the results show that the transcript is only found inthe lower half of the kernels. The expression ofZmESR-6 could not be induced, in either young or

Figure 2. Aminoacid alignments of the deduced ZmEsr6 mature protein with similar plant defensin molecules from radish

(Rs-AFP2, Acc P30230), sunflower (Ha defensin, Acc. AF364865) tobacco flowers (FST, Acc. P32026), maize c1-zeathionin (gam-

ma1-ZT, Acc. P81008), maize c2-zeathionin (gamma1-ZT, Acc. P81009) and barley c1-hordothionin (gamma?-HT Acc. P20230).

Two proteins classified as protease inhibitors, from Arabidopsis (PDF 2.3, Acc. NP 178321) and potato (P322, Acc. P20346) are

also included. Identical residues in all the sequences are shadowed in black.

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mature maize leaves, by treatments that have beenshown to induce other defence related genes, suchas wounding, salicylic acid or jasmonic acid (datanot shown).

In situ localisation of ZmESR-6 transcripts

In order to determine the precise localisation ofthe ZmESR-6 transcripts, in situ hybridisationanalyses were performed in longitudinal sectionsof maize kernels at different stages of development.Figure 4 shows that ZmESR-6 antisense probeshybridised specifically to cells of the ESR. Thesignal was detected already at 5 DAP (Figure 4A),and remained very strong until 16 DAP (Fig-ure 4D–G), the results observed by Northern blotanalyses (Figure 3B) should thus be corrected toaccount for the dilution factor introduced by theincrease in the accumulation of storage productsrelated transcripts occurring between 12 and18 DAP, as well as to account for the relativedecrease in seed volume occupied by the ESR. At 5DAP (Figure 4A) the ESR cells completely sur-round the developing embryo (not shown in thisparticular section) formed at this stage by a smallnumber of cells at one side of the endosperm (thisregion is shown at higher magnification in Fig-ure 4B). At 11 DAP (Figure 4C) the embryo has

emerged from the ESR area and the ESR cellsbecame located at the base of the suspensor where,along with the transfer cell layer, form the borderbetween maternal and filial tissues at the base ofthe kernel. Figure 4D shows the situation at 16DAP, squared areas labelled 1, 2 and 3 are shownat higher magnification in Figure 4E–G respec-tively. The ZmESR-6 transcript is restricted to thecubical, densely cytoplasmic, cells of the ESRregion, whilst no signal was detected in adjacenttissues such as transfer cells, embryo or pedicel(Figure 4D and G). At 16 DAP we have con-sistently observed ZmESR-6 hybridisation signalat the abgerminal side of the transfer cell layer(Figure 4D, square 1 and E) as well as a weaksignal in one or two cells in the centre of thetransfer cell layer (Figure 4D, square 2 and F).

Localisation of the ZmESR-6 protein

Western blot analyses using ZmESR-6 anti-sera showed very poor reaction with the maizeZmESR-6 protein in crude extracts (data notshown). The protein was readily localysed, how-ever, in concentrated extracellular protein extracts,prepared from ground immature maize kernelsextracted with Tris-ClH pH 7.5, in the absence ofdetergents (Figure 5). The reacting protein had the

Figure 3. Expression analyses of ZmEsr6. The Northern blots were hybridised with the ZmESR-6 cDNA. Ten microgram total

RNA were loaded per lane. A. Analysis of the expression of ZmEsr6 in different tissues. U, unpollinated female flowers; Seed, 20

DAP seeds; C, coleoptyles; L, leaves; R, roots; S, silks; T, tassels. B. Accumulation of ZmEsr6 transcripts at different stages of the

development of the seed. Numbers above the figure indicate days after pollination of the kernels used to extract the RNA. T,

means the upper half of the seed, B, means the lower half.

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expected electrophoretic mobility for the matureZmESR-6 (5.5 kDa), and as expected, the proteinwas smaller than the purified recombinant protein(with a predicted Mw of 6.8 kD) used to raise theantibody.

The maize ZmESR-6 protein was localisedin wax-embedded sections from 11 and 16 DAPkernels (Figure 6). The protein was detected(brown colour) in the ESR cells but the strongestsignal was found in the pedicel region adjacent tothe ESR, at the placentochalazal region. Controlexperiments with pre-immune serum did not revealany signal (not shown). This result indicates thatthe ZmESR-6 protein, albeit produced in the ESRcells, is released into the apoplast and accumulates

in the placentochalazal layer, preferentially at theembryo pole of the pedicel. Accumulation ofthe ZmESR-6 protein at the opposite pole, theabgerminal side, of the pedicel was also observedbut only in 16 DAP sections (Figure 6B), inagreement with the results observed for the tran-scription of the gene (Figure 4E). The gradient ofsignal intensity from both sides of the pediceltowards the centre, further confirm that theprotein is diffusing through the apoplastic spacein the placentochalaza, from the endosperm cellsin which it is produced. Interestingly, the patternof accumulation of ZmESR-6 in the placentocha-laza is highly complementary to that observed fortransfer cell-produced peptides also accumulated

( ) ( )

Figure 4. In situ hybridisation of the ZmESR-6 transcripts. Sagital sections of 5 (A, B), 11 (C) and 16 DAP (D–G) kernels were

hybridised with antisense S35 labelled ZmESR-6 probes. Control sense probes produced no detectable signal and are not shown.

(B) is a magnification of the region squared in (A). (E), (F) and (G) are magnifications of the regions squared in Figure (D) and

labelled 1, 2 and 3 respectively. En, endosperm, ESR, embryo surrounding region, TC, transfer cells, Em, embryo, P-C, placento-

chalaza. Bars = 500 lm in (A), (C) and (D), and 250 lm in (B), (E)–(G).

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in that region, section in Figure 6E was reactedwith an antibody specific for BAP-2 (Serna et al.,2001).

Antimicrobial activity of ZmESR-6 protein

The in vitro growth inhibitory activity of therecombinant ZmESR-6 was tested against bacte-rial and fungal plant pathogens. As shown inTable 1, ZmESR-6 was active (IC50 ranging from0.2 to 15 lM) against all the pathogens tested, thatincluded the Gram positive bacterium Clavibactermichiganensis, two Gram negative bacteria(Xanthomonas campestris and Rhizobium meliloti)and three fungal pathogens (two Fusarium oxyo-sporum subspecies and Plectosphaerella cucumeri-na). The IC50 values of ZmESR-6 were similar tothose of the wheat thionin, an antimicrobialpeptide purified from endosperm (Molina et al.,1993). In contrast the recombinant ZmCKS(CMP-KDO synthetase, Royo et al., 2000), usedin these experiments as a negative control, had noeffect on the growth of the bacteria and fungianalysed, even at the highest concentration tested

(20 lM; data not shown). Since this protein waspurified following the same protocol and from thesame E. coli strain as ZmESR-6, this negativecontrol excludes the possibility of contaminationof the recombinant protein with growth inhibitorycompounds.

Discussion

ZmESR-6 is a novel defensin gene with in vitroantipathogenic activity

In a search for genes expressed in developingmaize kernels, we have identified a cDNA clone,designated ZmESR-6, encoding for protein str-ucturally related to the plant defensin family.ZmESR-6 has the tripartite structure of defensinswith a central mature protein domain flanked by aN-terminal signal peptide and a C-terminal acidictail (Figure 1). The presence of a signal peptide atits N-terminus, suggests that ZmESR-6 is synthes-ised at the RER as a pre-protein and secreted ashas been shown for other antimicrobial peptidesexpressed in developing maize kernels (Serna et al.,2001). The predicted mature protein domaincontains eight Cys located in equivalent positionsto those of other members of the family (Fig-ure 2) and there is a CSab motif in the predictedthree-dimensional structure of ZmESR-6. Thismotif is present in the members of a superfamilyof proteins that include not only plant but alsoinvertebrate defensins (like drosomycin, and somescorpion neurotoxins) (Bart et al., 2002). Theseproteins have diverse activities like antifungal,antibacterial, inhibitors of insect gut a-amylases,inhibitors of protein synthesis, and blockers of ionchannels. It has been suggested that the CSabmotif represent a robust and versatile scaffold thatcan incorporate a broad range of functionalactivities (Lay et al., 2003). It is interesting topoint out that the first three Cys residues inZmESR-6 are positioned in a slightly different wayas compared to the organisation observed in othermembers of the defensin family, because of thepresence of a higher number of aminoacids amongthe Cys residues in ZmESR-6 (20 vs. 16 in the restof the defensins). In fact, the Swiss Model programcannot predict overlaping of structure betweenZmESR-6 and c-1-P thionin over the first 15aminoacids of the mature ZmESR-6. The possible

Figure 5. Western blot analysis using the anti-ZmESR6 anti-

serum. Lane 1, 10 ng purified recombinant E. coli Zm-ESR6,

Lane 2, 20 lg proteins extracted from cell walls of the basal

part of maize kernels as described in Methods. Molecular

weight markers (Broad range, New England Biolabs) are indi-

cated on the left. An asterisk on the right marks an unrelated

polypeptide that cross-reacting with the antibody.

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effects of these changes on the biological activityof ZmESR-6 as compared with that of otherdefensins have to be further investigated.

We have raised an antibody against therecombinant ZmESR-6, in western blot experi-ments with maize kernel protein extracts (Fig-ure 5) this antibody recognizes a basal kernel-specific peptide with the predicted size for themature ZmESR-6, very similar to that of thepurified recombinant ZmESR-6 expressed in E.coli. Since the recombinant protein was con-structed in base to the sequence of the putativemature protein, eliminating sequences correspond-ing to the signal peptide at the N-terminus and tothe acidic propeptide at the C-terminus, theseresults indicate that ZmESR-6 processing involvescutting away those regions and that its unpro-cessed and intermediary forms do not accumulatein planta. The recombinant mature ZmESR-6 canthus be used for the analyses of the in vitroantipathogenic activity. The acidic propeptide ofdefensins has been proposed to neutralize thetoxicity of the basic central domain and thus

mitigate its effects in the cells that are synthesizingit (Garcıa-Olmedo, 1992; Gu et al., 1992), but itshould be eliminated to get an active protein in itsaccumulation site.

The antibody recognizes additional bands inseed protein extracts (Figure 5, lane 2, asterisk)but they correspond to unrelated peptides, sincethese high molecular weight bands are present inprotein extracts from tissues in which ZmESR-6 isnot expressed, such as the upper part of thedeveloping seeds (not shown). More importantly,the unspecific reactivity of the antibody does notinterfere with the results of the immunolocalisa-tion experiments shown in Figure 6, since in theconditions used for the immunolocalisation exper-iments, no signal was detected in the upper halvesof the seed (see next section). The faint bandsobserved in lane 2 may correspond to the unpro-cessed forms of ESR-6 but further investigationneeds to be done to confirm this hypothesis. In anycase, the prominence of the band corresponding tothe mature peptide points to the existence of arapid processing mechanism for ZmESR-6.

Figure 6. Inmunolocalisation of ZmESR-6 protein. Sagital sections of 11 DAP (A and E) and 16 DAP (B– D) kernels were reacted

with the anti-ZmESR6 (A–D) or the anti-BAP2 (E) antibodies. Control experiments using pre-immune serum produced no detect-

able signal and are not shown. (C) and (D) are magnifications of the regions squared in (B) and labelled 1 and 2, respectively.

Arrows in (A), (B) and (E) point to the brown DAB precipitate that indicates positive reaction of the antibodies. ESR, embryo

surrounding region, TC, transfer cells, Em, embryo, P-C, placentochalaza. Bars mean 500 lm in (A), (B) and (E) and 250 lm in

(C) and (D).

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The protein extraction procedure used in theexperiment shown in Figure 5 also suggested thatthe mature ZmESR-6 is located in the extracellularcompartment of the cells. The mature protein wasreadily extracted from ground tissue in the absenceof any detergent, the protein extract containedtherefore minimal amounts of intracellular ormembrane bound peptides.

Most of the defensins exhibit an inhibitoryactivity against fungi and a few of them alsoagainst bacteria (Garcıa-Olmedo et al., 1998;Thomma et al., 2002). Our data (Table 1)suggest that ZmESR-6 has a typical defensinactivity. It efficiently inhibits growth of fungi atthe micromolar range, with similar IC50 valuesas the antibiotic wheat thionin (Molina et al.,1993) and of the same order than thosedescribed for other plant defensins (Moreno etal., 1994; Osborn et al., 1995; Segura et al.,1998). As previously described for other plantdefensins, ZmESR-6 was more active againstfungi than Gram-negative bacteria, and likepotato defensin StPTH and some spinach defen-sins (Moreno et al., 1994; Segura et al., 1998), itinhibited the growth of the Gram-positive bac-teria Clavibacter michiganensis, a known maizepathogen. Interestingly, the effective concentra-tions of thionin causing 50% microbial inhibi-tion are below those present in the tissues wherethese peptides are expressed (Molina et al.,1993). In the case of ZmESR-6, although theconcentration of the peptide at the whole seedlevel is not as high as that of thionin, theconcentration at the sites of protein accumula-tion (as judged from the immunolocalisationexperiments, Figure 6) seems to be very high.

The protective role of the ESR of the maizeendosperm, as illustrated by the expressionof ZmESR-6 during kernel development

ZmESR-6 mRNA preferentially accumulates atthe early stages of seed development (Figure 3B)and is only found at the ESR of the maize kernel(Figures 3A and 4). Seed-specific expression hasbeen reported for many of the plant defensinsidentified to date , for instance c-thionins fromwheat (Colilla et al., 1990) and barley (Mendezet al., 1990). In most cases, however, these peptideswere shown to be accumulated during seed devel-opment until maturity, where are thought toprovide protection against pathogens to the ger-minating kernel. The expression pattern ofZmESR-6 suggest for this gene a role in protectingkernel structures during seed maturation. Consis-tently with this specialised role, the expression ofZmESR-6 could not be induced in leaves afterwounding, infection or treatment with salicylate orjasmonate (data not shown), treatments that havebeen shown to be effective with other defensins(Chiang and Hadwiger 1991; Terras et al., 1995;Epple et al., 1997; Thomma et al., 1998, 2002).

Next question was to understand the structureor structures that ZmESR-6 contributes to protectin developing kernels. The information obtainedfrom in situ hybridisation (Figure 4) and immu-nolocalisation experiments (Figure 6) providedsupport for a model in which ZmESR-6 and, byextension, the ESR cells contribute to the protec-tion of the seed in two different ways:

At 5 DAP the ESR consists of a small group ofcells enclosing the tiny embryo (Schel et al., 1984),these cells express already high levels of ZmESR-6mRNA (Figure 4A and B), a role for the ESR andgenes such as ZmESR-6 in the protection of thedeveloping embryo is at this stage both obvious andbiologically meaningful. A similar role, although ata much earlier stage of development, could have themaize defensin-like gene ZmES1-4, which isexpressed in the female gametophyte,most probablyto protect it against pathogens or fungi sporesaccompanying the pollen tube (Cordts et al., 2001).In fact, Fusarium infection of cereal ears occursprimarily at flowering (Kang and Buchenauer,2000).

As the embryo and endosperm developmentproceeds the ESR became restricted to a smallarea at the base of the embryo suspensor

Table 1. Antimicrobial in vitro assays of ZmESR-6 and apositive control, the wheat thionin. The micromolar con-centration of protein that gave an IC value of 50 is indicatedfor each pathogen.

Bacteria Protein IC50 (lM)

ZmESR-6 Thionin

Clavibacter michiganensis 0.2 0.2

Xanthomonas campestris 15 10

Rhizobium melilote 5 2

Fungi

Fusariumk oxyosporium f.sp. 3 ± 1 3

Conglutinans

Fusarium oxyosporum f.sp.lycopersici 3 ± 1 1

Plectosphaerella cucumerina 2 NT

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(Opsahl-Ferstad et al., 1997). ZmESR-6 expres-sion is also localised at the base of the embryosuspensor showing very strong levels of transcriptaccumulation in the ESR cells (Figure 4C and D).Intriguingly, at 16 DAP additional focuses ofZmESR-6 expression were observed in other partsof the basal cell layers, namely in the transfer celllayer (Figure 4E and F). The reasons for theseadditional foci of expression need to be furtherinvestigated but do not seem to involve change toESR identity for some cells in the transfer celllayer, since they express transfer cells markers(Hueros et al., 1995, 1999) and fail to express otherESR markers (Opsahl-Ferstad et al., 1997). Theconnection between localisation of the ZmESR-6expression and its role in embryo defence is nowless evident than at 5 DAP, immunolocalisation ofthe ZmESR-6 product (Figure 6) provided, how-ever, a functional explanation for this expressionpattern. The protein is released into the placento-chalaza region from both edges of the kernel basallayer (Figures 6A–C), the gradient observed inprotein concentration, from the cells in which it isproduced towards the centre of the placentocha-laza, provides additional support for a model inwhich the protein would diffuse from the ESRcells, where it is synthesised (Figure 6C), to theplacentochalaza region where would be accumu-lated in its active form. Interestingly, the pattern ofaccumulation of ZmESR-6 is highly complemen-tary to that of the antifungal protein BAP2(Figure 6E and Serna et al., 2001), BAP2 issynthesised in the transfer cells and accumulatesin its mature form in the placentochalaza cells.Similar roles in defence have been proposed forother basal–kernel-specific genes, like BETL1, 3and 4 (Hueros et al., 1999), which have also beenfound to be secreted and accumulate in the cellwalls of the transfer cell layer (Cai et al., 2002).

The placentochalaza is a layer of cells withengrossed cell walls, forming a barrier between filialandmother tissues plant, andhave beenproposed tohave a role in defence, transport of nutrients, andseed abscission (Santandrea et al., 2002). Theaccumulation of the antimicrobial ZmESR6 (thiswork) and BAP2 (Serna et al., 2001) peptides in theplacentochalaza cell walls (a dead tissue by the timeat which the proteins accumulate significantly)would avoid damage of the plasma membrane ofthe living cells in the pedicel while preventingmicrobial growth. This is an important feature

because the pedicel potentially provides a very richenvironment for microbial growth due to the highflux of solutes that cross it in their way into the seedafter being unloaded from the terminals of phloem.In addition, the accumulation of defensive systems,promoted by filial tissues at the entrance of the seed,would prevent the passing of pathogens into thenext generation. Several Fusarium species are endo-phytic and spread in plantamoving through phloemvessels (for a review see Azevedo et al., 2000).Fusarium moniliforme the most frequently fungusfound in maize seeds, is transmitted not onlyhorizontally but also vertically to the next genera-tion via infection of the seeds. The accumulation ofbasal endosperm-produced peptides in the pedicel,but not in other regions of the endosperm, indicatesthat an active transport system operates at the baseof the endosperm to promote polarised secretioninto the maternal tissues. Furthermore, this trans-port system has to operate against the flux ofnutrients that are being uptaked by the endospermfrom the maternal tissues.

Defensins are present not only in plants butalso in animals, including humans. In all of themare mainly present in peripheral cell layers, whichis consistent with a role in the first line of defenceagainst pathogens (for a review see Hancock andLehrer 1998; Bart et al., 2002). In fact, a similarsituation to that described here, embryos display-ing mechanisms to protect themselves by releasingantimicrobial compounds at the maternal–filialinterphase, operates at the developing humanfoetus, in which the expression of defensins 1 and4 has been detected at the human placenta(Svinarich et al., 1997).

In view of the expression pattern and in vitroactivity found for ZmESR-6, we propose thatdefence is one of the functions for the ESR, protect-ing the embryo at the earliest stages of developmentand cooperatingwith the transfer cell layer, latter on,to create a barrier against pathogens ingression intothe filial cells (embryo and endosperm) from thematernal tissues. Obviously this does not excludeadditional functions for this tissue, in this context therecently characterised ESR-specific gene ZmIN-VINH1 (Bate et al., 2004) encodes a cell wallinvertase inhibitor that may serve as a barrier toprotect the slower developing embryo from hexosessupplied from an apoplastic invertase early post-pollination. Thus allowing differentiated develop-mental rates for embryo and endosperm.

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Materials and Methods

Plant material

Maize plants (Zea mays, line A69Y) were grown ina greenhouse under a 16 h-light, 8 h-dark photo-period, with supplemented illumination and atemperature of 20–25 �C.

The bacteria, Clavibacter michiganensis subsp.sepedonicus, strain C5, Rhizobium meliloti andXanthomonas campestris, as well the fungi Botrytiscinerea, Fusarium oxysporum f. sp. lycopersici,F. oxysporum f. sp. conglutinans and Plectosphae-rella cucumerina were from the Escuela TecnicaSuperior de Ingenieros Agronomos collection(Madrid, Spain).

Molecular biology methods

Standard DNA and RNA methods were carriedfollowing Sambrock et al., (1989) and as previ-ously described (Hueros et al., 1995).

Sequence analysis

Nucleotide sequence was compared to EMBL andGenebank databases using the BLAST algorithm.Prediction of protein localisation site was per-formed online using PSORT http://psort.nib-b.ac.jp/ and the signal peptide cleavage site wasidentified using the SignalP V.2.0.b2 server athttp://www.cbs.dtu.dk/. Secondary structure pre-diction of the protein was performed using PHD-sec program, disulfide bridges formation wasanalysed using the DISULFIND program, bothat the PredictProtein server http://www.embl-hei-delberg.de/predictprotein/. Three-dimensional str-ucture prediction was done using the Swiss ModelFirst Approach at http://swissmodel.expasy.org/incorporating the sequence of c-1-P thionin(1-GPS) as template. Biochemichal parametersfor protein were calculated using the Vector NTIsoftware (Informax, Bethesda, USA).

Bacterial expression, Purification of the ZmESR-6protein and preparation of the antiserum

The mature ZmESR-6 coding sequence (shown inbold in Figure 1) was generated by PCR usingprimers which added suitable restriction sites. ThePCR product was digested with the restriction

enzymes Bam HI and BglII and cloned into thepQE60 expression vector (Qiagen GmbH) to yieldpQE60-Esr6. The pQE60 vector provides a startcodon followed by three aminoacids, which resultsin the addition at the N-terminus of four amino-acids (MGGS) to the first residue of the matureZmESR-6 protein. At the C-terminus, an exten-sion of eight aminoacids (RSHHHHHH) are fusedto the last aminoacid of the mature protein.Protein overexpresion and purification was donefollowing the manufacturer�s instructions (TheQIAexpressionist, Qiagen, Germany).

The fusion protein was purified under denatur-ing conditions to obtain polyclonal antibodies,and under native conditions for biological activityassays. The protein was purified to almost homo-geneity as seen after SDS-PAGE electrophoresisand Coomassie blue staining. The purified proteinwas then desalted by dialysis in 0.1 M acetic acid,lyophilised and dissolved in distilled water. TheZmCKS protein (Royo et al., 2000) was preparedunder the same conditions as ZmESR-6, to be usedas negative control.

In situ hybridisation, Inmunolocalisation

Maize seeds were collected at different DAP andfixed in 4% paraformaldehyde, 0.1% glutaralde-hyde in 0.1 M sodium phosphate buffer pH 7,2 for12–24 h depending on the tissue volume. Sampleswere dehydrated and embedded in wax (Paraplast,Sigma, USA) using xylol as solvent. Sections10 lm thick were affixed to glass slides treatedwith 3-aminopropyltriethoxylane. Sections weredeparaffinised in xylol and rehydrated through anethanol series.

For the in situ hybridisation, 35S-labelled anti-sense and sense probes were synthesised usingT3 and T7 polymerases (Boehringher–Manheim,Germany) from linearised pBluescript SK+ plas-mid containing the ZmEsr6 cDNA. Probes werepartially hydrolysed with sodium carbonate. Sec-tions were hybridised as previously described(Hueros et al., 1995), following the method ofCox and Goldberg (1988).

For the immunolocalisation experiments, inhi-bition of endogenous peroxidase was carried outby incubating the sections in 0.3% v/v H2O2 in0.5% methanol for 10 min. Tissue was thenwashed in PBS and blocked with 10% normaldonkey serum (Chemicon International, USA) in

279

PBS for 30 min at room temperature. Sectionswere incubated with anti-ESR6 serum or pre-immune serum diluted 1:100 in 1% donkey serumfor 2 h at room temperature and then overnight at4 C. The immunoreaction was detected usinghorseradish peroxidase-coupled secondary anti-body (Sigma, USA) diluted 1:500, and 3¢-diam-inobenzidine (Roche, Germany).

Protein gel electrophoresis and inmunoblotting

For protein extraction, the lower halves of 21 DAPmaize seeds were grinded in liquid nitrogen with apestle and a mortar. The powder was thenextracted in four volumes of 0.1 M Tris-ClH, pH7.5, under agitation and at 4 �C. Cell debris wereremoved by centrifugation at 10 000 rpm for10 min and the supernatant was dialysed in thecold room for 24 h against 0.1 M acetic acid. Thesupernatant was cleared again by centrifugationand lyophilised. Proteins were finally re-suspendedin water.

Protein samples were fractionated in 15%denaturing SDS-PAGE gels under standard meth-ods and electroblotted onto Inmobilon-P mem-branes (Millipore, USA). The membrane wastreated with a 1:500 dilution of the inmunopurifiedanti-ZmESR6 and detected with horseradish per-oxidase-coupled second antibody (Sigma, USA) ata 1:5000 dilution and with the Supersignal West-Pico Chemiluminiscent reagent (Pierce, USA).

In vitro inhibition tests

Antimicrobial activity of protein samples againstbacteria and fungi was assayed spectrophotomet-rically in 96-well microtiter plates as previouslydescribed (Berrocal-Lobo et al., 2002). Briefly,33 ll of a bacterial suspension (final concentration104 cfu ml)1) in nutrient broth media, or a fungispore suspension (final concentration 104 sporesml)1) in potato dextrose broth, were mixed withdifferent amounts of the corresponding proteinsdissolved in 67 ll of sterile water. The plates wereincubated at 28 �C and growth was recorded after16–48 h, by measuring A490 in an ELISA platereader. IC50 values (i.e., the concentration of theprotein required to inhibit 50% of the growth ofthe microorganism) were calculated from dose-response curves. Purified thionin from wheatendosperm (Molina et al., 1993), and recombinant

ZmCKS (Royo et al., 2000) were used as positiveand negative controls, respectively. Proteins werequantified using the Bradford assay, according tothe manufacturer�s protocol (Sigma, USA), usingbovine serum albumin as a standard.

Acknowledgements

The immortalised maize F2 lines used for map-ping the ZmESR-6 locus were kindly providedby Dr Ed H. Coe. This work was supported bygrants 07G/0030/00 and 07B/0050/02 to G.H and07B/0002/2002 to A.M., from the Comunidad deMadrid (Spain). We thank technical support toYolanda Sanz and Gemma Lopez.

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